Improved Taxonomy of the Genus Streptomyces

UNIVERSITEIT GENT Faculteit Wetenschappen Vakgroep Biochemie, Fysiologie & Microbiologie Laboratorium voor Microbiologie

Improved taxonomy of the genus Streptomyces

Benjamin LANOOT

Scriptie voorgelegd tot het behalen van de graad van Doctor in de Wetenschappen (Biochemie)

Promotor: Prof. Dr. ir. J. Swings Co-promotor: Dr. M. Vancanneyt

Academiejaar 2004-2005

FACULTY OF SCIENCES ______

DEPARTMENT OF BIOCHEMISTRY, PHYSIOLOGY AND MICROBIOLOGY UNIVERSITEIT LABORATORY OF MICROBIOLOGY GENT

IMPROVED TAXONOMY OF THE GENUS STREPTOMYCES

DISSERTATION

Submitted in fulfilment of the requirements for the degree of Doctor (Ph D) in Sciences, Biochemistry

December 2004

Benjamin LANOOT

Promotor: Prof. Dr. ir. J. SWINGS Co-promotor: Dr. M. VANCANNEYT

1: Aerial mycelium of a Streptomyces sp. © Michel Cavatta, Academy de Lyon, France

1 2 2: Streptomyces coelicolor colonies © John Innes Centre

3: Blue haloes surrounding Streptomyces coelicolor colonies are secreted 3 4 actinorhodin (an antibiotic) © John Innes Centre

4: Antibiotic droplet secreted by Streptomyces coelicolor © John Innes Centre

PhD thesis, Faculty of Sciences, Ghent University, Ghent, Belgium. Publicly defended in Ghent, December 9th, 2004.

Examination Commission

PROF. DR. J. VAN BEEUMEN (ACTING CHAIRMAN) Faculty of Sciences, University of Ghent

PROF. DR. IR. J. SWINGS (PROMOTOR) Faculty of Sciences, University of Ghent

DR. M. VANCANNEYT (CO-PROMOTOR) Faculty of Sciences, University of Ghent

PROF. DR. M. GOODFELLOW Department of Agricultural & Environmental Science University of Newcastle, UK

PROF. Z. LIU Institute of Microbiology Chinese Academy of Sciences, Beijing, P.R. China

DR. D. LABEDA United States Department of Agriculture National Center for Agricultural Utilization Research Peoria, IL, USA

PROF. DR. R.M. KROPPENSTEDT Deutsche Sammlung von Mikroorganismen & Zellkulturen (DSMZ) Braunschweig, Germany

DR. F. MARINELLI Vicuron Pharmaceuticals, Italy

PROF. DR. F. GOSSELE Visiting Professor in Microbiology Phibro Animal Health, Belgium

DR. G. HUYS Faculty of Sciences, University of Ghent

PROF. DR. E. VANDAMME Faculty of Applied Biological Sciences, University of Ghent

6 Dankwoord - Acknowledgements

Hierbij wens ik een aantal personen in het zonnetje te plaatsen die elk hun steentje hebben bijgedragen tot het succesvol beëindigen van deze thesis.

Tijdens mijn studentenjaren maakten Moniek G en Joris M me wegwijs in de wondere wereld van de bacteriën in het practicum Microbiologie en konden me overtuigen op de WE10V-trein te stappen voor mijn licentiaatsthesis. Een samenwerking met het International Rice Research Institute te Manilla (Filippijnen) deed me echter finaal kiezen voor doctoraal onderzoek. Dankzij Karel K, Danny J, Philippe D en Marc V werd een Belgo-Chinese samenwerking afgesloten voor initieel 2 jaar in 1998, dat 2 maal verlengd zou worden met 2 jaar. Moniek D van het Federaal Wetenschapsbeleid zorgde voor de financiering van dit project. Zonder jullie inzet en (federale) centen zou dit boekje er niet geweest zijn !

Jean, je gaf me als promotor de nodige ruimte om me te verdiepen in dit onderzoek. Ik wil je tevens bedanken voor je eindeloze steun en vertrouwen, de talloze kritische opmerkingen en verbeteringen. Kortom je was de noodzakelijke, krachtige locomotief die de zware “Streptomyces-project trein” gedurende 6 jaar door soms krappe bochten sleurde naar zijn finale bestemming.

Marc V, je was een uitstekend co-promotor die me van begin tot einde telkenmale overstelpte met inspirerende inzichten, soms zeer ontnuchterende maar terechte opmerkingen (krijg ik het slachtbankje mee naar huis?) maar die steeds voldoende energie kon overbrengen om niet de moed op te geven. Zonder jouw hulp zou dit werk moeilijk verlopen zijn. Hartelijk dank hiervoor Marc !

Verder wil ik ook alle collega’s van het Laboratorium voor Microbiologie inclusief Moudjahid, Maja (mijn chaperon), Medana, Pavel en Teresa bedanken voor de aangename werksfeer. In het bijzonder wil ik mijn “Streptomyces-reisgezellen” Margo, Ann VS, en de thesisstudentes Annick P, Nancy en Lieve bedanken voor hun inspanningen bij het screenen van de honderden isolaten en referentiestammen. Karen L, Cindy, Fabiano, Renata, Liesje, Ilse C, Evie en An B voor hun raad bij DNA-bereidingen, DNA-DNA hybridisaties en % GC bepalingen; Vinie en Anne W voor de hulp tijdens de tDNA-PCR experimenten, Roberto om het Italiaanse temperament binnengebracht te hebben in ons labo; Antoine voor het oplossen van de technische probleempjes; Paul S voor de vele schouderklopjes en de leuke antiquiteiten; Urbain voor je opbeurende gesprekken en sympathie; Stefanie V voor het frivole pasta-diner (...) tijdens een congres in Parijs; Dirk D voor je uitstekende hulp bij eiwitprofilering, me de knepen van de fascinerende fotografiewereld bijgebracht te hebben en je levenswijsheid; Annemie en “baron” Fernand voor de administratieve steun; snelle Jeanine voor het leveren van allerhande producten; Peter D voor de geapprecieerde hulp bij de numerieke verwerking van de massa’s data en Geert K voor het oplossen van de onvermijdelijke computerproblemen, zijn talloze mopjes en het mede sponsoren van de jaarlijks georganiseerde mattetaartenslag (het waren echte Geraardsbergse !). Last but not least dien ik de collega’s van het LMG zelf te bedanken voor het verzamelen van de referentiestammen (dank u Claudine), lyofiliseren, etc van de toch moeilijk handelbare streptomyceten. Er zijn er ongetwijfeld die grijze haren gekregen hebben...

This work was performed in collaboration with the Institute of Microbiology of the Chinese Academy of Sciences in Beijing (P.R. China). I would like to thank Prof Liu Zhiheng, Li Wei, Jianli Zhang, Liming Wang, Huang Ying and Miss Zhang to help constructing the reference frameworks, the isolation of hundreds of strains from different soil samples

7 collected around China and to communicate all their knowledge on traditional streptomycete systematics which is needed to understand properly the classification of these soil bacteria. Besides the professional side, I will not forget the hospitality shown by the whole group, the many nice dinners, the efforts made to get a European boy acquainted with the fascinating old Chinese culture and to always provide me with a guide when visiting Beijing… It has been an unforgettable experience despite the sometimes-encountered language frontier. I am also very grateful to the group of Michael Goodfellow (Newcastle, UK) in helping to interpret our data and how these might fit the overall picture. The members of the examination committee are acknowledged for their critical reading and comments.

Deel van dit werk kwam tot stand dmv een samenwerking met de industrie namelijk PHIBRO Animal Health te Rixensart. In het bijzonder Francis en Martine wens ik te bedanken voor hun kritische commentaren en inzichten.

Bedankt beste ouders, broertje en zusje voor jullie onvoorwaardelijke steun en de nodige ontspanning die jullie boden. Dit is werkelijk onbetaalbaar !

Ten slotte zijn er nog de de enthousiaste supporters Marie-Jeanne VDB (Europacollege), Hans S, Jean Pierre L, Geert M, Rudy DB, Arlette DD en het personeel van FGRA.

Heidi, kusjes om me steeds op de sporen gehouden te hebben tijdens de laatste maanden van het schrijven.

Gent, 9 december 2004

8 CONTENTS

Dankwoord - Acknowledgements Contents List of non-standard abbreviations List of Figures List of Tables

PART 1: Literature review

Chapter 1: The genus Streptomyces 23 1.1 Introduction 1.2 Circumscription 1.3 Phylogenetic allocation 1.4 Cultivation 1.5 Occurrence 1.6 Importance 1.6.1 Human pathogens 1.6.2 Plant pathogens 1.6.3 Humification and biodegradation 1.6.4 Biodeterioration 1.6.5 Bioremediation 1.6.6 Production of enzymes 1.6.7 Production of natural products 1.6.8 Bioconversion of chemical compounds

Chapter 2: Taxonomy of the genus Streptomyces 51 2.1 Introduction 2.2 The early chaotic years 2.3 Numerical taxonomy 2.4 Chemotaxonomy 2.5 DNA-DNA hybridisations 2.6 Genotyping screening methods 2.7 Phylogeny 2.7.1 16S rDNA sequence analysis 2.7.2 Analysis of other stretches DNA 2.7.2.1 Sequencing of protein coding genes 2.7.2.2 16S rDNA – 23S rDNA ITS analysis 2.7.3 Whole genome sequencing and analysis

Chapter 3: Aims & Conceptual framework 85 3.1 General aims 3.2 Specific aims

9 PART 2: Experimental work

Chapter 4: Detection and reclassification of synonymous species 91

4.1 The search for synonyms among streptomycetes using SDS-PAGE of whole-cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violatus.

Lanoot B, Vancanneyt M, Cleenwerck I, Wang L, Li W, Liu Z & Swings J. (2002). Int J Syst Evol Microbiol 52, 823-829.

4.2 BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus.

Lanoot B, Vancanneyt M, Dawyndt P, Cnockaert MC, Zhang J, Huang Y, Liu Z & Swings J. (2004). System Appl Microbiol 27, 84-92.

4.3 Reclassification of Streptomyces nigrifaciens as a later synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as later synonyms of Streptomyces anulatus; Streptomyces chibaensis as later synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as later synonym of S. minutiscleroticus; Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as later synonyms of Streptomyces microflavus.

Lanoot B, Vancanneyt M, Van Schoor A, Liu Z & Swings J. (2005a). Int J Syst Evol Microbiol, in press.

Chapter 5: Intra-species genotypic relatedness of S. virginiae strains and correlation with phenotypic traits 135

Phenotypic and genotypic characterisation of variants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae .

Lanoot B, Vancanneyt M , Hoste B , Cnockaert MC, Piecq M, Gosselé F & Swings J. (2005b). System Appl Microbiol, in press.

10 Chapter 6: Phylogenetic grouping using 16S-ITS RFLP fingerprinting as a valuable alternative for 16S rRNA gene sequencing 153

6.1 Preliminary study 6.2 Phylogenetic grouping of streptomycetes using 16S-ITS RFLP fingerprinting.

Lanoot B, Vancanneyt M, Hoste B, Vandemeulebroecke K, Cnockaert MC, Dawyndt P, Liu Z & Swings J. Research in Microbiology, submitted.

Chapter 7: Identification of isolates and description of new Streptomyces species 171

7.1 Screening of large numbers of isolates 7.2 Description of new taxa 7.2.1 Streptomyces glauciniger sp. nov., a novel mesophilic streptomycete isolated from soil in South China.

Huang Y, Li W, Wang L, Lanoot B, Vancanneyt M, Rodriguez Z, Liu Z & Swings J. (2004). Int J Syst Evol Microbiol 54, 2085-2089.

7.2.2 Streptomyces jietaisiensis sp. nov., isolated from soil in North China.

He L, Wang L, Li W, Liu Z, Lanoot B, Vancanneyt M, Swings J & Huang Y. Int J Syst Evol Microbiol, submitted.

7.2.3 Streptomyces scopiformis sp. nov., a novel streptomycete with fastigiate spore chains.

Li W, Lanoot B, Zhang Y, Vancanneyt M, Swings J & Liu Z. (2002). Int J Syst Evol Microbiol 52, 1629 – 1633.

PART 3: Conclusions & perspectives 211

Chapter 8: 8.1 Detection and reclassification of synonymous species 8.1.1 Evaluation of a fast and reliable screening method 8.1.2 Proposed reclassifications 8.2 Intra-species genotypic relatedness of S. virginiae strains and correlation with phenotypic traits 8.3 Phylogenetic grouping using 16S-ITS RFLP fingerprinting as a valuable alternative for 16S rRNA gene sequencing 8.4 Identification of streptomycetes from Chinese and Moroccan soil and description of new taxa 8.5 Description of new species 8.6 General conclusions

Summary - samenvatting 223 Bibliography 231

11 ANNEXES 279

Annex I- List of standard media Annex II- List of reference strains used in this study Annex III- List of isolates used in this study Annex IV- Dendrogram of BOX-PCR patterns of reference strains Annex V-Dendrogram of 16S-ITS RFLP patterns of reference strains

Curriculum vitae

12 List of non-standard abbreviations

AFLP Amplified Fragment Length Polymorphism AL Approved lists ATCC American Type Culture Collection, Manassas, VA, USA

A2pm Diaminopimelic acid BCCM / LMG Belgian Co-ordinated Collections of Microorganisms / Laboratorium voor Microbiologie, Ghent University, Gent, Belgium CCCM / IMCAS Chinese Culture Collection for Microorganisms / Institute for Microbiology of the Chinese Academy of Sciences, Beijing, P.R. China CCMM Collections Coordonnées Marocaines de Micro-organismes, Rabat, Morocco DAB 2,4-diaminobutyric acid DDBJ DNA Database of Japan DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany EMBL European Molecular Biology Laboratory, Cambridge, UK ERIC Enterobacterial Repetitive Intergenic Consensus FFSM Faculté des Sciences Semlalia de Marrakech, Morocco G + C Guanine + Cytosine (content) GRAS Generally regarded as safe gyrA Gene coding for gyrase HPLC High-Performance Liquid Chromatography IFO Institute for Fermentation, Osaka, Japan IMRU Institute of Microbiology, Rutgers - The State University, New Jersey, USA ISP International Streptomyces Project ITS Intergenic Transcribed Spacer JCM Japanese Collection of Microorganisms, Tokyo, Japan K. Kitasatospora MAFF National Institute of Agrobiological Sciences, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan MLST Multi Locus Sequence Typing MRSA Multi Resistent Staphylococcus aureus MW Molecular Weight (Marker)

13 NRRL Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA nt Nucleotides OD Optical density PCR Polymerase Chain Reaction prcA Gene coding for the 20S proteosome α subunit r Ribosomal r Regression coefficient R-number Research number RDP Ribosomal Database Project recA Gene coding for recombinase protein REP Repetitive Extragenic Palindrome RFLP Restriction Fragment Length Polymorphism RISSC Ribosomal Intergenic Spacer Sequence Collection rnpB Gene coding for ribonuclease P subunit B rpoB Gene coding for protein involved in protein translation S Svedberg S. Streptomyces SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SSC Saline sodium citrate sp(p). (Multiple) species T Type strain UPGMA Unweighted Pair Group Method with arithmetic Averages WT Wild type “…” Invalid species

14 LIST OF FIGURES

List of Figures

Chapter 1 Fig. 1: Explosive increase in number of validly described Streptomyces species between mid 1950s and mid 1960s. Fig. 2: The Streptomyces life cycle. Fig. 3: Configuration of spore chains (A) and spore surface ornamentation (B) found among streptomycetes. Fig. 4: Phylogenetic position of the family Streptomycetaceae based on 16S rRNA gene sequence analysis. Fig. 5: Relative abundance of streptomycetes in a compost bin. Fig. 6: Potato suffering from scab formation. Fig. 7: Production of secondary metabolites in life cycle.

Chapter 2 Fig. 8: Simplified dendrogram depicting the taxonomic clusters within the genus Streptomyces by numerical taxonomy (Williams et al., 1983a). Fig. 9: Taxonomic discriminative power of genomic fingerprint techniques (Vandamme et al., 1996). Fig. 10: Phylogenetic relationships between members of the genera Streptomyces, Kitasatospora and Streptacidiphilus based on almost complete 16S rRNA gene sequences. Fig. 11: Genotypic analysis of the major streptomycete clusters, as defined by Williams et al. (1983a) through comparison of the 16S rDNA from nucleotides 158-276 including the γ region. Fig. 12: Graphical representation of polymorphisms of 16S rDNA, gyrA, prcA, recA, rnpB and rpoB of representative strains of the S. violaceoruber and S. albidoflavus species groups and closely related species.

Chapter 3

Fig. 13: Flowchart

Chapter 4

Fig. 14: Dendrogram derived from unweighted pair group average linkage of r values (expressed as percentage) of protein profiles. Fig. 15: Principle of SDS-PAGE of whole-cell proteins. Fig. 16: Fluorescent microplate method of Ezaki et al. (1989).

15 Fig. 17: All strains found in this study possessing visually identical protein profiles, except for cluster 22, encompassing strains with similar protein profiles. A correlation is made with the numerical phenotypic studies of Williams et al. (1989) and Kämpfer et al. (1991). Fig. 18: Principle of the rep-PCR fingerprint technique (Versalovic et al., 1991). Fig. 19: Dendrogram showing 30 clusters containing strains with nearly identical rep patterns. Fig. 20: Selection of BOX patterns of strains with uncertain affiliation and corresponding DNA-DNA homology values.

Chapter 5

Fig. 21: Genealogical tree of S. virginiae strain 899 and its variants. Fig. 22: Simplified overview of the AFLP fingerprint technique. Fig. 23: Differences in colour of substrate mycelium of the type strain of S. virginiae LMG 20534T and S. virginiae 899 derived strains. Fig. 24: In silico matching of the BOXA1R primer to the whole-genome sequence of S. coelicolor A3(2). Fig. 25: Dendrogram showing BOX-PCR fingerprints of strain 899, 19 variants derived from 899, 5 S. virginiae strains and 2 functionally related species. Fig. 26: Discriminative AFLP fingerprints of strain 899 and its deleted mutant PD 170.

Chapter 6 Fig. 27: Principle of the 16S rRNA gene RFLP technique. Fig. 28: Dendrogram showing a selection of combined 16S RFLP fingerprints of 14 Streptomyces reference strains obtained with restriction enzymes BstUI, RsaI, TaqI and NIaIII. Fig. 29: Principle of 16S rDNA-ITS RFLP fingerprinting. Fig. 30: Neighbour Joining tree based on almost complete 16S rDNA sequences of 158 Streptomyces and Kitasatospora type strains in correlation with corresponding 16S- ITS RFLP fingerprinting data.

Chapter 7

Fig. 31: Correlation between produced bioactive compound and strain relatedness using protein profiling. Fig. 32: Unrooted neighbour-joining tree (Saitou & Nei, 1987) based on almost complete 16S rDNA sequences showing the position of strain FXJ14T in the Streptomyces tree. Fig. 33: Closest neighbours of FXJ-14T using the constructed reference frameworks in BOX-PCR and 16S-ITS RFLP fingerprinting.

16 LIST OF FIGURES

Fig. 34: Partial neighbour-joining tree (Saitou & Nei, 1987) based on the 120 nt γ -region of 16S rDNA showing the relationships between strain FXJ46T and other Streptomyces species. Fig. 35: Unrooted neighbour-joining tree (Saitou & Nei, 1987) based on almost complete 16S rRNA gene sequences showing the phylogenetic relationships between strain FXJ46T and related Streptomyces species. Fig. 36. Scanning electron micrograph of strain A25T grown on Bennett’s agar for 14-day at 28 °C. The scale bar indicates 6 µm. Fig. 37: Neighbour-Joining tree based on nearly complete 16S rDNA sequences of 45 streptomycetes.

17 List of Tables

Chapter 1

Table 1: Discriminative features for the genera Streptomyces, Kitasatospora and Strepacidiphilus, belonging to the family Streptomycetaceae. Table 2: Role of Streptomyces spp. in the degradation of important recalcitrant natural compounds. Table 3: Role of Streptomyces spp. in the biodegradation of industrial chemical compounds and in the decontamination of heavy metal polluted soils and water. Table 4: Streptomyces spp. as producers of industrially important enzymes. Table 5: Streptomyces spp. as producer of non-antibiotic natural compounds.

Chapter 2 Table 6: Proposed subjective synonyms and allied species as defined by Williams et al. (1983a). Table 7: Predominant cellular fatty acids from Streptomyces spp. Table 8: DNA hybridization between several Streptomyces taxa.

Chapter 4 Table 9: Strains possessing unique patterns Table 10: Numerical analysis of BOX-PCR patterns of 473 Streptomyces type strains with delineation of 30 clusters containing strains with nearly identical patterns. Table 11: DNA-DNA reassociation values among Streptomyces species of 5 delineated BOX-PCR clusters Table 12: Phenotypic characteristics of the reclassified species S. anulatus, S. corchorusii, S. minutiscleroticus, S. flavovirens and S. microflavus and later synonym(s).

Chapter 5 Table 13: Strains examined

Chapter 6 Table 14: Delineation of 17 groups of closely related Streptomyces species based on 16S RFLP fingerprinting using 4 restriction enzymes. Table 15: Delineation of clusters with closely related species among 463 Streptomyces and Kitasatospora type strains using 16S-ITS RFLP fingerprinting.

Chapter 7 Table 16: Growth and cultural characteristics of strain FXJ14T Table 17: Phenotypic properties separating strain FXJ14T from related Streptomyces species Table 18: Comparison of cultural characteristics of strain FXJ46T and Streptomyces griseoaurantiacus DSM 40430T

18 LIST OF TABLES

Table 19: Phenotypic properties of strain FXJ46T and related Streptomyces species. Table 20: Diagnostic characteristics for strain A25T and related strains.

PART 1

Literature review

22 1. LITERATURE REVIEW

The genus Streptomyces

1.1 Introduction

The genus Streptomyces comprises a large group of microorganisms with some characteristic features compared to most other bacteria such as their complex Fungi- like life cycle and earthy odor. Furthermore, they are ubiquitous in nature and show a higher diversity in color of colonies, secreted pigments, etc. compared to other bacteria. The morphological heterogeneity has always been used for classification since the creation of the genus in 1943 by Waksman & Henrici until present. However in this early period little was known about their real taxonomic status. Slight differences in morphological and cultural properties were enough for describing new species. After the discovery of another (economically important) feature of streptomycetes (=members of the genus Streptomyces), namely production of bioactive compounds, the number of poorly described species exploded in the 1950s, 60s and 70s. Between 1955 and 1962, over 300 new species were proposed (Fig. 1).

23 Fig. 1: Explosive increase in number of validly described Streptomyces species between mid 1950s and mid 1960s.

The introduction of biochemical, chemotaxonomical and genotypical data in bacterial systematics during the last decades, slowly but gradually improved the phenotypy dominated taxonomy of Streptomyces. Despite the latter efforts made, the genus Streptomyces still contains over 500 validly described species (List Valid names, 07/2004, DSMZ). Classification and identification of potentially new bioactive species should become more accessible for taxonomists through the provision of a transparent and less time-consuming classification alternative than the currently applied approach. By this, the frequent re-isolation of species producing known antibiotic structures may be avoided and screening efforts significantly improved for searching novel species, producing novel antibiotics, in answer to the emerging worldwide resistance of bacteria against antibiotics (Bax et al., 1998; Projan, 2003).

24 1. LITERATURE REVIEW

1.2 Circumscription

Early reports classified ‘streptomycete’ species in several genera e.g. Oospora (Thaxter, 1872) and Actinomyces (Bergey 1923). In 1943, the genus Streptomyces (Strep.to.my’ces. Gr. adj. Streptos pli-ant, bent; Gr.n.: myces fungus; M.L. masc. N.: Streptomyces pliant or bent fungus) was proposed by Waksman & Henrici. It was lifted from the genus Actinomyces and re-classified in the family Streptomycetaceae (see Section 1.3) as aerobic spore-forming actinomycetes to avoid confusion with pathogenic microaerophilic organisms. The type species is Streptomyces albus (Rossi-Doria) Waksman & Henrici 1943, 339.

Colonies are discrete and lichenoid, leathery or butyrous. Initially smooth surfaced but develop later a weft of aerial mycelium that may appear floccose, cottony, granular, powdery or velvety. Cells stain Gram positive and are non-acid- alcohol fast.

Streptomycetes have, compared to other bacteria, an atypical lifecycle (Fig. 2) resembling the filamentous cycle of Fungi. The cycle begins with a spore, also called arthrospore or conidium. Germination of these spores starts under favourable environmental conditions e.g. temperature, presence of exogenous nutrients like fragments of carrots or mycelia of Fungi (Goodfellow et al., 1983), aerobic conditions and lack of inhibitory effects from other microorganisms (Kutzner, 1981) to give rise to an extensive branching network of multinucleoid hyphae known as the substrate (primary) mycelium. These are approximately 0.5-1.0 µm in diameter, often lack cross-walls and grow at the apices. Differentiation starts with the formation of specialized aerial hyphae (secondary mycelium), which grow in to the air away from the surface of the colony. Later these aerial hyphae undergo septation into uninucleoid compartments and metamorphose into chains of spores (sporophores) that rarely fragments. The number of spores in a chain varies (3 to many). For some species e.g. former Chainia spp. (Goodfellow et al., 1986b), sclerotia may be formed, reaching up to 100 µm in diameter and consisting of dense masses of enlarged, branching hyphae. Finally, non-motile spores are released into the environment.

25 The onset of differentiation is frequently associated with the production of pigments, antibiotics1 and other secondary metabolites (see Section 1.6.7).

Germination

Free spore Spore maturation Germ tube

First phase of growth

Substrate mycelium Chains of uninucleate cells

Second phase Aerial mycelium of growth

Antibiotic production

Fig. 2: The Streptomyces life cycle Obtained from http://homepage.smc.edu/ishida_carolyn

The genus Streptomyces is morphologically highly diverse. Color of substrate and aerial mycelium, configuration of spore chains and spore ornamentation are used as taxonomic markers. All of them are determined using cultivation on standard media and fixed incubation times (Shirling & Gottlieb, 1966). A system of color wheels has been devised for accurate determination of the color of mycelium (Tresner & Backus, 1963). Configuration of spore chains is determined by electron microscopy and is assigned to one of the following categories: straight, flexuous, spirales and verticilliate. Variation within each of these categories may be observed as shown in Fig. 3. Ideally, one configuration is found within a strain although a combination of several configurations has also been described e.g. S. griseoruber possessing the configurations spirals and straight (Shirling & Gottlieb, 1968b). Spore ornamentation is either smooth, spiny, rugose or hairy (Fig. 3). Occasionally, assignment to one of these ornamentations is difficult as a result of immaturity of the

1 Are defined as chemical substances either produced by microorganisms or chemically synthetised able to inhibit growth or kill other microorganisms (Waksman).

26 1. LITERATURE REVIEW

strain or strain-variability. Although sporulation-favouring media are used, in some cases, the strain fails to sporulate.

The production of soluble pigments and their change in color on addition of HCl or NaOH and production of melanin pigments on peptone yeast-extract iron or tyrosine agar (see Annex I) are taxonomic markers. Soluble pigments are considered as predictive for the production of bioactive compounds.

27 Fig. 3: Configuration of spore chains (A) and spore surface ornamentation (B) found among streptomycetes.

(A)

Madigan et al., 2003 (B)

28 1. LITERATURE REVIEW

Chemical analysis of the cell wall shows that streptomycetes, including the former genera Chainia, Elytrosporangium, Microellobosporia, Streptoverticillium, Actinopycnidium and Actinosportangium (see Section 2) have a cell wall type I sensu Lechevalier & Lechevalier (1970). This means they contain the LL isomeric form of diaminopimelic acid (A2pm, Hasegawa et al., 1983) and glycine but no characteristic sugars in the peptidoglycan layer. However, within Streptomyces, meso- A2pm may constitute up to 16 % of all A2pm detected and glycine in the interpeptide bridge has also been found in other Gram positive genera. The N-acyl muramyl residues of the cell wall peptidoglycans are acetyl (Uchida & Seino, 1997). Predominant isoprenologues have nine isoprene units of the hexa- and octahydrogenated 2 menaquinones (MK-9[H6, H8]) (Collins, 1985). Typical polar lipids (Minnikin et al., 1984) are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides (phospholipid type II sensu Lechevalier et al. (1977). Mycolic acids are not found. The fatty acid profile (Sasser, 1990; Kämpfer & Kroppenstedt, 1996) is mainly comprising saturated straight chain and iso- and anteiso-branched chain fatty acids (=fatty acid type 2c sensu Kroppenstedt, (1985).

The size of the genome is around 9 Mb (S. coelicolor, Bentley et al., 2002; S. avermitilis, Ikeda et al., 2003), about 2 x bigger than the genome size of the model organism Escherichia coli. DNA G + C content ranges between 66 and 78 mol%.

2 Have an analogues structure to vitamin K2 and play an important role in electron transport in the plasma membrane.

29 1.3 Phylogenetic allocation

Streptomycetes are classified within the Gram-positive bacteria and form together with the genera Kitasatospora (Ōmura et al., 1982) and Streptacidiphilus (Kim et al., 2003) the family Streptomycetaceae. The latter family constitutes a separate phylogenetic branch within the phylum Firmacutes, class Actinobacteria, order Actinomycetales, suborder Streptomycineae based on 16S rRNA gene sequence analysis (Fig. 4; Stackebrandt et al., 1997, see Section 2.7.1).

------

Fig. 4: Phylogenetic position of the family Streptomycetaceae based on 16S rRNA gene sequence analysis. Adapted from Stackebrandt et al. (1997). ------

The genus Streptomyces can be separated from its sister genera Streptacidiphilus and Kitasatospora using cultural, biochemical, chemotaxonomic (see Section 2.4) and genotypic data (see Sections 2.6 & 2.7): pH range for growth;

30 1. LITERATURE REVIEW

major amounts of LL- A2pm and traces of the meso form; the sugars in whole-cell hydrolysates are not diagnostic (Table 1).

Streptomyces Kitasatospora Streptacidiphilus

Cultural pH range for growth 3.5 - 11.5 5.5 – 9.0 3.5 – 6.0

Biochemical A2pm isomer(s) in whole- LL and trace of LL and meso in different LL cell hydrolysates meso ratios

Diagnostic sugars in None Galactose Galactose, rhamnose whole-cell hydrolysates Chemotaxonomic Resistance to phage S7 No Yes ND (DSM 49153)

Genotypic 16S rRNA gene based Separate branch* Separate branch* phylogeny

16S-23S spacer Specific nine-nucleotide sequencing signature

Table 1: Discriminative features for the genera Streptomyces, Kitasatospora and Strepacidiphilus, belonging to the family Streptomycetaceae. Data taken from Shirling & Gottlieb, 1976; Ōmura et al., 1989; Lonsdale, 1985; Williams et al., 1989; Nakagaito et al., 1992 and Kim et al., 2002). ND: not determined; *: within a phylogenetic tree comprising Streptomyces, Kitasatospora and Streptacidiphilus species (See section 2.7).

31 1.4 Cultivation

Streptomycetes are non-fastidious organisms with an obligate oxidative respiration (except S. thermoautotrophicus growing under micro-aerophylic atmosphere), and requiring quite low humidity for growth between 10 and 30% (Kutzner, 1981).

Utlization of carbon and nitrogen sources is one of the most flexible among Bacteria and is therefore used to classify streptomycetes (see Section 2.3). All have a chemo-organotrophic metabolism with exception of the species S. thermocarboxydovorans, S. thermocarboxydus (Kim et al., 1998), S. thermospinisporus (Kim & Goodfellow, 2002) and S. thermoautotrophicus (Gadkari et al., 1990) which have respectively a chemolithotrophic and autotrophic metabolism (S. thermoautotrophicus). An inorganic nitrogen source is required for their growth (Williams et al., 1989). Hence, multiple defined and undefined media serve to grow streptomycetes such as the general purpose media nutrient agar and trypticase soy broth (Difco) or Bennett’s medium and oatmeal agar (See Annex I) for standardized cultivation respectively.

Regarding acidity, the majority of streptomycetes described so far are neutrophilic and are able to grow between pH 5.0 and 9.0 with an optimum close to neutrality i.e. pH 6.5 – 8.0 (Goodfellow & Williams, 1983; Williams et al., 1989). The existence of alkalophilic and alkaline-resistant strains e.g. S. caeruleus has been reported by Taber (1959, 1960) and Mikami et al. (1982). These strains grow between pH 8.0 and 11.5 with an optimum at pH 9 to 9.5. On the other hand, the streptomycete metabolism may tolerate acid pH levels e.g. the potato pathogen S. acidiscabies (Lambert and Loria, 1989b). Recently, S. yeochonensis (Kim et al., 2004) was described, a novel neutrotolerant acidophilic streptomycete tolerating a pH level of 3.5 with optimal growth between pH 5.0 and 5.5. It was isolated from different horizons of soil from a Pinus thunbergii forest, Yeochon, Republic of Korea.

Regarding the growth temperature, the majority of streptomycetes are described as mesophilic with an optimum at 28 °C. In literature only one psychrotolerant species has been described, so far, i.e. S. beijiangensis (Li et al., 2002), with an 1. LITERATURE REVIEW

optimal growth temperature between 8 and 20 °C and a minimal growth temperature limit of 0 °C. A minority is thermophilic, preferring growth temperatures between 28 and 55 °C. These organisms inhabit non-common habitats such as arid soil types and hot springs e.g. S. thermogriseus (Xu et al., 1998). More attention is currently paid to explore these habitats to search for enzymes and bioactive molecules with an increased thermostability (see Section 1.6.6). The effect of temperature change on the microbial succession of the dominating taxa of microorganisms and total microbial activity in a compost bin was monitored recently (Ryckeboer et al., 2003). It was shown that among total microbial microflora, Streptomyces became the dominant genus during composting, surviving high temperatures above 75 °C thanks to the formation of heat-resistant arthrospores.

33 1.5 Occurrence

Streptomycetes are saprophytes, widely distributed in terrestrial and aquatic habitats. Well drained soil (sandy loam, soils covering limestone), fodder and compost appear to be primary reservoirs for their isolation. The characteristic earthy odor of soil is caused by the production of a series of streptomycete metabolites called geosmins (sesquiterpenoid compounds). Their omnipresence is due to features intrinsic for actinomycetes, including streptomycetes namely the formation and easy dispersion in nature of arthrospores and secretion of growth inhibiting metabolites. The species S. thermogriseus was for instance isolated from a hot spring (Xu et al., 1998). Secondary metabolites like antibiotics make streptomycetes very competitive in nature, especially in nutrient poor habitats like deserts. In a compost bin (Ryckeboer et al., 2003), rich in recalcitrant organic matter, 70 % of all strains isolated were Prokaryotes, dominated by the genus Streptomyces (13%). Fungi represented only 30 % of all isolates (Fig 5). In general, the genus Streptomyces represents more than 95 % of actinomycetes isolated from most soil types. Environments rich in degradable compounds e.g. the rhizosphere of plants, show higher ratios non-streptomycetes / streptomycetes, due to lower food competition levels between microorganisms (Amir & Pineau, 1998).

Data concerning their geographical distribution or more specifically to their macro-and microniches are scarce (Saintpierre, 2001). This is due to the fact that ecological studies mostly apply the dilution plate technique, which does not allow to determine whether streptomycetes in the environment are present as active mycelium or sleeping arthrospores.

34 1. LITERATURE REVIEW

------13 % 13% % Fungi

39 % 39% 18 % 18% % non-Streptomyces % Streptomyces % unidentified

Prokaryotes

30% 30 %

1 2 3 4

Fig. 5: Relative abundance of streptomycetes in a compost bin. Data compiled from Ryckeboer et al., 2003.

------

35 1.6 Importance

1.6.1 Human pathogens

From literature only one Streptomyces species i.e. S. somaliensis is known to be a human pathogen. It causes actinomycetoma, a localized chronic destructive and progressive infection of skin, subcutaneous tissue and eventually bone. The disease in endemic in tropical and subtropical climates e.g. Kenya, Mali (Mahe et al., 1996), Saudi Arabia (Bendl et al., 1987), Somalia, South India (Klokke et al., 1968) and Sudan. It leads frequently to deformities and amputation of infected extremeties. Non-streptomycetes also cause this disease, notably i.e. Actinomadura madurae, Actinomadura pelletieri, Nocardia otitidiscaviarum and Nocardia transvalensis. Recent work of Quintana et al. (2003) showed that the causal agents of actinomycetoma are underspeciated and may represent several Streptomyces and Actinomadura species. Other Streptomyces spp. isolated from humans (Trujilo & Goodfellow, 2003; Mossad et al., 1995; Unaogu & Gugnani 1990) include S. violaceoruber, S. coelicolor Müller, S. griseus, S. albus and S. candidus from pulmonary streptotrichosis patients, dental caries, blood, tonsils, skin, sputum and bone. For each of these organisms, the level of pathogenicity still needs to be unraveled. S. albus has been reported as causative agent of allergic alveolitis (Dutkiewicz et al., 2001).

1.6.2 Plant pathogens

Until present, 12 species are regarded as highly virulent for plants: S. scabiei (Thaxter, 1892), S. acidiscabies (Lambert & Loria, 1989a), S. turgidiscabies (Miyajima et al., 1998), S. europascabiei (Bouchek-Mechiche et al., 2000), S. stelliscabiei (Bouchek-Mechiche et al., 2000), S. reticuliscabiei (Bouchek-Mechiche et al., 2000), S. caviscabies (Goyer et al., 1996), S. luridiscabiei (Park et al., 2003), S. puniscabiei (Park et al., 2003), S. niveiscabiei (Park et al., 2003) and S. cheleniumii (Natsume et al., 2003) causing scab formation on potatoes (Fig. 6); S. ipomoea, causing soft rot of sweet potatoes. Seven species have been described as less virulent for potatoes i.e. S. aureofaciens, S. griseus, S. rochei, S. setonii, S. tendae and S. venezulae (Corbaz, 1964; Doering-Saad et al., 1992; Gordon & Horan, 1968).

36 1. LITERATURE REVIEW

Only a small number of Streptomyces species cause diseases of diverse root crops such as potato, radish, turnip, beet, carrot and sweet potato (Goyer & Beaulieu, 1997; Labeda & Lyons, 1992; Bouchek-Mechiche et al., 2000).

Almost all work done on plant pathogenic streptomycetes refers to scab formation on potato (Solanum tuberosum cultivars) because of the significant economical impact (Loria et al., 1997). Current conservative estimates of losses due to this disease in Tasmania alone are in excess of € 2.1 million per year (Wilson et al., 2003). The first report on this disease (Thaxter, 1892) pointed Oospora scabies (reclassified as S. scabiei by Waksman & Henrici) as causal organism. This worldwide distributed saprophytic organism predominates in sandy, well-drained soil of neutral to light alkaline pH and in dry years. However, an indistinguishable form of the disease may also be found in soils with pH values as low as 4.5, caused by S. acidiscabies (Lambert & Loria, 1989b). Both bacteria are unable to penetrate the intact skin of the potato tuber but invades young tubers through lenticels (Lapwood, 1973). Phytotoxins secreted by the bacterium (thaxotomins; concanamycin A and B; Lawrence et al., 1990; Natsume et al., 2003) induce the formation of wound periderm by the plant. As the pathogen continues to colonize the tuber, slightly raised lesions are developed composed of rough corky tissue (Kutzner, 1981). As a result, shallow- or deep-pitted lesions occur (Archuleta et al., 1981, Doering-Saad et al., 1992; Faucher et al., 1995). Although this disease has little effect on the total yield, the value of affected tubers is reduced, and the content and quality of the starch in the tubers decreased. Scab symptoms depend on several factors of the environment and plant itself e.g. time of infection, growth rate of the potato, potato cultivar, and virulence of the infecting streptomycete (Kutzner, 1981). Netted scab of potato has only been reported in European countries causing superficial brown lesions on the skin of the tubers and on the roots (Bang, 1979; Scholte & Labruyere, 1985) while russet scab is found in North America (Harrison, 1962; Faucher et al., 1993) and Japan (Oniki et al., 1986). 16S rDNA sequence based comparison (see Section 2.7.1) suggested that streptomycetes causing potato scab are phylogenetically not related (Takeuchi et al., 1996; Song et al., 2004). Hence it is likely that genes required for inducing

37 pathogenicity are spread among different Streptomyces species within their natural habitat (Doering-Saad et al., 1992)

Fig. 6: Potato suffering from scab formation.

A second economically important disease, found in warm climates, is caused by S. ipomoea. It causes soil rot (also named as pox) of sweet potatoes (Ipomoea batatas)and shows two symptoms (i) dwarfed plants and little or no vine growth; poorly developed root system, most of the roots being entirely rotten and many of them breaking off when the plant is lifted from the soil (ii) in mature potatoes pits or cavities with irregular jagged or roughened margins are found (Kutzner, 1981; Person and Martin, 1940). S. ipomoea is not pathogenic to Solanum tuberosum plants despite sharing analogues pathogenicity conditions to those of S. scabiei. Both bacterial species are not related to each other (Labeda & Lyons, 1992).

1.6.3 Humification and biodegradation

Plant leaves and stems consist of the important structural components cellulose, lignin and pectin. They are chemically almost inert e.g. chitin occurring in the exoskeletons of insects and fungal cell walls or keratin in nails (Madigan et al., 2003). In order to shortcut the nutrient cycle in nature, transformation and degradation of these molecules into re-usable components for the ecosystem is necessary. Besides streptomycetes (Table 2), also a limited number of other aerobic (e.g. Cytophaga) and anaerobic (e.g. Clostridium) bacteria and Fungi play a major role in it (Eriksson et al., 1992; Johansen et al., 2002; Wirth & Ulrich, 2002).

38 1. LITERATURE REVIEW

______Natural compound Streptomyces involved ______

Cellulose ---> S. drozdowiczii (Semedo et al., 2004) S. reticuli (Schlosser et al., 1999) S. albaduncus (Marchand & Singh, 1997)

Chitin ---> S. griseus (Kawase et al., 2004) S. olivaceoviridis (Saito & Schrempf, 2004) S. thermoviolaceus strain OPC-520 (Kubota et al., 2004) S. plicatus (Abd-Allah, 2001) S. viridificans (Gupta et al., 1995)

Lignin ---> S. violaceusniger strain YCED9 (Chamberlain et al., 2000) S. cyaneus (Berrocal et al., 2000) S. viridosporus (Ball, 1991) S. badius strain 252 (Magnuson et al., 1991) ______

Table 2: Role of Streptomyces spp. in the degradation of important recalcitrant natural compounds.

Degradation of biomolecules is not linked to one particular species as shown for the degradation of cellulose by S. drozdowiczii and S. reticuli (Table 2). Their coding gene clusters are located on plasmids together with other genes coding for e.g. resistance against poisonous compounds, heavy metals etc. (see Section 1.6.5). These extra-genomic elements are easily transferred among species, even in soil (Egan et al., 1998; Omura et al., 2001). Recombinatory events may take place between the plasmid and the genome of the host resulting in an incorporation of novel degradation genes.

1.6.4 Biodeterioration

Microorganisms can cause damage to non-biotic materials (Williams et al., 1985). Mural paintings for instance may be the subject of esthetical damage (overgrowth and discoloration) and structural damage (due to the utilisation of the organic compounds present) (Heyrman et al., 2001). Several bacterial genera interact together in a consortium mostly aided by Fungi. Among the microflora of biodeteriorated mural paintings in the Servilia Tomb mainly Bacillus species were found together with members of the genera Streptomyces, Arthrobacter,

39 Micrococcus, Sphingomonas and Paenibacillus (Heyrman et al., 1999, 2001). The genus Streptomyces represented less than 10 % of the total colony number and is a typical colonizer of damaged mural paintings (Ciferri, 1999; Karpovich-Tate & Rebrikova, 1990; Weirich, 1989). In other similar studies, streptomycetes were isolated as the predominant bacteria (Donato et al., 2000; Groth et al., 1999). According to Giacobini et al. (1988), they are among the first colonizers of frescoes located in tombs, crypts and grottoes.

Presence of Streptomyces species has also been reported in air and on materials in water-damaged buildings causing irritation problems in humans e.g. on mucous membranes, eye and respiratory tract (Rintala et al., 2002, 2004; Hyvarinen et al., 2001; Nevalainen et al., 1991; Peltola et al., 2001).

Streptomycetes may inhabit nutrient poor environments provided an organic substrate, either present in-situ or delivered by Fungi, is available as carbon source for their growth.

1.6.5 Bioremediation

Pollution of soil and wastewater with chemical compounds such as petroleum derivates, solvents, chlorinated compounds or herbicides is an environmental problem in industrialized countries. These soils and those contaminated with high levels of heavy metals can effect both the qualitative and the quantitative structure of microbial communities, resulting in decreased metabolic activity and biomass as well as decreased diversity (Hattori, 1992; Wuertz & Mergeay, 1997; Sandaa et al., 1999; Kozdroj & VanElsas, 2001). Problems arise as a result of pollution e.g. illness of humans either directly or via contamination of food, lower crop yields or decreased efficacy of wastewater plants. Great attention is now being paid to the microbial remediation of such poisoned sites (Diels et al., 1999) requiring detailed knowledge of bacteria resistant to metal and chemical compounds (Roane & Kellogg, 1996; Baath et al., 1998; Brim et al., 1999a, b; Sandaa et al., 1999; Mengoni et al., 2001; Ambujom, 2001). Within New Caledonian ultramafic soils for instance, rich in metals toxic to bacteria, actinomycetes constituted between 56 and 86 % of all bacteria

40 1. LITERATURE REVIEW

isolated on nutrient agar (non-selective medium). Of this number, up to 95 % belonged morphologically to the genus Streptomyces. Besides Streptomyces (Table 3), also other genera such as Pseudomonas, Alcaligenes, Ralstonia, Burkholderia and Sphingopyxis may play an active role in decontamination processes (Amoroso et al., 2000, Stoppel & Schlegel, 1995; Totevova et al., 2002, Mergeay et al., 1985; Schmidt et al., 1991; Lallai & Mura, 2004, Smejkal et al., 2001, Goris et al., 2004 and Godoy et al., 2003).

Biodegradation of Use Reference

- Methyl tertiary butyl ether (MTBE) Gasoline additive Okeke et al., 2003

- Hydrocarbon Petrol chemical industry Radwan et al., 1995

- Trichloroethylene Solvent (cytotoxic) Sukesan et al., 1998

- Phenol Carcinogenic disinfectant Ambujom, 2001

- Alachlor Herbicide Sette et al., 2004

- Bromoxynil Herbicide Neuzil et al., 1988

- Trifluralin and atrazine Herbicides Mata-Sandoval et al.,2001

- Natural rubber, synthetic poly (cis-1,4- Automobile industry and Bode et al., 2001 isoprene), and cross-linked natural medicine rubber (latex gloves)

- Nitroaromatic compounds Various e.g. explosives Glaus et al., 1992

Waste management

Use Reference - Reduction of Cr VI to Cr III Immobilization of toxic Desjardin et al., 2002 metal - Biosorptive flotation for metal Removal of metals Zouboulis et al., 2001 ions recovery

Table 3: Role of Streptomyces spp. in the biodegradation of industrial chemical compounds and in the decontamination of heavy metal polluted soils and water.

41 1.6.6 Production of enzymes

Most of the industrially used enzymes are hydrolases, mainly used in the manufacturing of detergents (37 %), food & drinks (19 %), textiles (12 %) and animal feed (6 %). They are mainly produced on large scale by Streptomyces spp. because of the non-fastidious metabolism and well-developed secretion system of this group of organisms. Indeed, analysis of whole-genome sequences of S. coelicolor genes (Bentley et al., 2002) revealed that respectively 7.8 % (614 proteins) and 10.5 % (819 proteins) were transporters and putatively secreted proteins (Weber et al., 2003). In addition, streptomycetes are generally regarded as safe (GRAS). Industrially important hydrolases produced by streptomycetes include proteases, lipases, alpha- amylases, phosphatases, xylose isomerases and xylanases (Table 4). These are used respectively for degradation of proteins (textile), fat removal (detergents), hydrolysis of starch into glucose (soft drinks, beer production, baking, detergents), release of phosphate (animal feed), conversion of glucose to fructose (soft drinks) and degradation of polysaccharides - biobleaching (animal feed, textile, paper industry). Screening of novel isolates or protein engineering allowed the selection of improved proteins possessing a higher thermo-stability (Kristijansson, 1989), alkali- tolerance (Horikoshi, 1999), higher pH-optimum or better substrate specifity. The Novo pharmaceutical group for instance reported a highly alkaline lipase from a strain of Streptomyces spp. useful in laundry and dishwashing detergents and industrial cleaners.

42 1. LITERATURE REVIEW

Enzyme Biological activity Produced by Reference (type of industry)

Alpha-amylase Production of glucose -maltose S. sp Ammar et al., 2002 (Food industry) S. megasporus SO 12 Dey & Agarwal (1999) S. lividans Nguyen et al., 1997 S. griseus Vigal et al., 1994 S. rimosus Vukelic et al., 1992 S. limosus Virolle & Bibb, 1988 S. venezulae Virolle et al., 1988 S. hygroscopicus Hoshiko et al., 1987 S. yunnanensis Zhang et al., 2003 S. scopiformis Li et al., 2002 S. thermogriseus Xu et al., 1998

Lipase Fat removal S. cinnamoneus Sommer et al., 1997 (Cleaning & Food industry) S. rimosus Vujaklija et al., 2002 S. seoulensis Chun et al., 1997

Xylanase Hydrolysis of noncellulosic S. roseiscleroticus Grabski et al., 1993 polysaccharides in wood pulp / S. galbus Kanson & Nagieb, 2004 Biobleaching S. albus Antonopoulos et al., 2001 (Animal feed & Paper industry) S. speibonae Meyers et al., 2003 S. mexicanus Petrosyan et al., 2003 S. thermocoprophilus Kim et al., 2000

Protease Degradation of proteins S. lividans TK 24 Aretz et al., 1989 (Textile)

Phosphatase Release of phosphate S. stramineus Labeda et al., 1997 (Animal feed)

Xylose Conversion of glucose to S. murinus Bandlish et al., 2002 isomerase fructose S. olivaceoviridis E-86 Kaneko et al., 2001 (Food industry) S. thermovulgaris 127 Raykovska et al., 2001 S. fradiae Thakur et al., 1988

Table 4: Streptomyces spp. as producers of industrially important enzymes.

1.6.7 Production of natural products

Streptomycetes produce secondary metabolites when they encounter a lack in nutrients for growth i.e. during the idiophase (Fig. 7).

43 ------

Fig. 7: Production of secondary metabolites in life cycle. Adapted from F. Marinelli (Vicuron). ------

The history of discovery of bioactive compounds in Streptomyces starts with the purification of the antibiotic actinomycin from S. antibioticus (Waksman et al., 1941) followed by streptothricin in 1942. It was however streptomycin, produced by S. griseus that triggered systematic screening of antibiotics from streptomycetes and other filamentous microorganisms in the 1950s, 60s and 70s. Between 1955 and 1962, about 80 % of the antibiotics mostly originated from Streptomyces (Berdy, 1974). The highest peak of discovery was in 1970. Currently, over 23000 microbial secondary metabolites are known, 42 % of which are produced by actinobacteria, 42 % by Fungi and 16 % by other bacteria (Lazzarini et al., 2000). Among the actinobacteria, the genus Streptomyces is the largest producer of antibiotics (Watve et al., 2001). Of the 12000 secondary metabolites with antibiotic activity known in 1995, 55 % were produced by streptomycetes and additional 11 % by other actinomycetes i.e. Micromonospora, Actinomadura, Amycolatopsis, Saccharopolyspora and Kibdelosporangium (Sanglier et al., 1996; Woodruff, 1999; Demain, 2002). Within Streptomyces, especially the S. albidoflavus group are known producers of antibiotics: 39 % antifungal compounds, 32 % anti-Gram-positive compounds and 10 % anti-Gram-negative compounds (Williams et al., 1983b).

44 1. LITERATURE REVIEW

Besides the field of antibiotics (see further), a wealth of other natural compounds are produced by streptomycetes (Weber et al., 2003) (Table 5).

Compound Biological activity Produced by Reference

Anthracyclines Antitumor S. galilaeus Fujii & Ebizuka, 1997 Werner et al., 1984

Cyclipostin Inhibitor of lipase S. sp. Vertesy et al., 2002 Hygromycin Immunosuppressive S. hygroscopicus Uyeda et al., 2001

Piperastatin A Inhibitor of serine S. lavendofoliae Murakami et al., 1996 carboxypeptidase

Streptozotocin Diabetogenic S. achromogenes Herr et al., 1967 Bafilomycin ATPase-inhibitor of S. griseus Frandberg et al., 2000 micro-organisms, plant S. halstedii and animal cells

Valinomycin Ionophor, toxic for pro- S. griseus Andersson et al., 1998 and eukaryotes

Table 5: Streptomyces spp. as producer of non-antibiotic natural compounds.

Antibiotics find the most important application in current medicine. They represent up to 50 % of the drug budget of hospitals (Davey et al., 1992). The overall market size for antibiotics is greater than US$ 25 billion per year (Coates et al., 2002). Antibiotics produced by Streptomyces species are:

Macrolides These antibiotics account for 11 % of all clinically used antibiotics. They consist of a large group natural members and semi-synthetic derivates, all composed of a large aglycone ring (14 or more carbon atoms) to which several sugars are attached (Anadon & Reeve-Johnson, 1999). The majority of this family of antibiotics is produced by streptomycetes e.g. spiramycin by S. ambofaciens (Abou-Zeid et al., 1980), erythromycin by S. erythraeus (Cevallos & Guerriero, 2003) and carbomycin by S. halstedii (Ashy et al., 1980). Their mode of action is inhibiting protein synthesis in the ribosomes. The spectrum of bioactivity is similar to the β-lactams and is therefore used to treat infections in patients being allergic to β-lactam antibiotics.

45 Tetracyclines Together with β-lactams, tetracycline antibiotics are the most important group of antibiotics in the medical field. The tetracyclines also find use in veterinary medicine, and are used in some countries as nutritional supplements for poultry and swine. They were some of the first broad-spectrum antibiotics, inhibiting almost all Gram- positive and Gram-negative bacteria. They act as protein synthesis inhibitors. The basic compound for semi-synthetic production of tetracyclines is secreted by S. aureofaciens (Nakano et al., 2000). Because of worldwide resistance of microorganisms towards this antibiotic, its use as therapeutic is significantly reduced.

Streptogramins Streptogramins represent a unique class of antibiotics remarkable for their antibacterial activity and their unique mechanism of action. These antibiotics are produced by a number of Streptomyces species e.g. virginiamycin secreted by S. virginiae 899 (De Somer & Van Dijck, 1955); mikamycin by S. mitakaensis (Arai et al., 1958); pristinamycin by “S. pristinaespiralis” (Preud’homme et al., 1965) and have been classified into two main structurally unrelated groups i.e. macrolactones (group A) and cyclic hexadepsipeptides (group B). Both groups bind bacterial ribosomes and inhibit protein synthesis at the elongation step. They act synergistically in vivo; the mixture of the two compounds is more powerful than the individual components and their combined action is irreversible. Streptogramins are active against Gram-positive microorganisms including those with multi-drug resistance. Moreover, the absence of cross-resistance to macrolides in many of these microorganisms and the rarity of cross-resistance between the two groups of antibiotics associated with the rapid bacterial killing are the principal features of the streptogramins, offering the possibility for treating the rising number of infections that are caused by multi-resistant Gram- positive bacteria.

Aminoglycosides This group of antibiotics contain amino sugars bonded by glycosidic linkage to other amino sugars inhibiting protein synthesis at the 30S subunit of the ribosome. Their spectrum of activity is against Gram-negative bacteria. Producers are streptomycetes e.g. S. griseus (Mansoui & Piepersberg, 1991) and S. kanamyceticus

46 1. LITERATURE REVIEW

IFO 13414 (Okami et al., 1959) secreting respectively streptomycin and kanamycin. Nowadays, aminoglycosides have a restricted use and account for only about 3 % of the total of all antibiotics produced and used.

β-lactams This group occupies the major share of the antibacterial market (54 %, Madigan et al., 2003). They have been used extensively for treatment of various bacterial infections for more than half a century. Most important β-lactams are penicillin, cephalosporin, cephamycin secreted by Penicillium chrysogenum, Acremonium chrysogenum and Nocardia lactamdurans and clavulanic acid secreted by S. clavuligerus (Thykaer & Nielsen, 2003). The mode of action of these antibiotics is at the level of the cell wall namely inhibiting peptidoglycan synthesis. The production of penicillin is one of the oldest and largest biotechnological industries with a world market of more than 60000 tons in 2000. It is probably the most cost effective drug existing although lacking activity against Gram-negative bacteria (for penicillin form G). Cephalosporin has a broader spectrum of bioactivity, as it is active against both Gram-positive and Gram-negative bacteria (Weil et al., 1995). Finally, cephamycins are described as being more active against both Gram-positive and Gram-negative bacteria compared to other β-lactams mentioned and are more resistant to Gram- negative β-lactamases.

Current novel antibiotic discoveries, remaining low in number, often have an already known chemical structural but show an improved spectrum of activity towards their target compared to structurally related compounds e.g. ripromycin (polycyclic macrolactam, antimicrobial, Bertasso et al., 2003). Only recently, a new class of an antibiotic has been reported i.e. cyclothialidine, a DNA gyrase inhibitor produced by a S. filipinensis strain (Angehrn et al., 2004). This observation is expected as, according to a mathematical modelling, 97 % of all antibacterial agents synthetized by streptomycetes still need to be discovered (Watve et al., 2001).

The amazing diversity in bioactive compounds produced by streptomycetes is due to features intrinsic to the latter group of organisms. Whole-genome sequencing of S. coelicolor strain A3(2) (Bentley et al., 2002) showed that about 5 % of the genome, corresponding to an estimated number of 23 secondary metabolite gene

47 clusters, is directed to the secondary metabolism. In S. avermitilis (Ikeda et al., 2003) even more secondary metabolite gene clusters were found i.e. 30 with a total length of 594 kb (Omura et al., 2001). The amount of genetic information coding for bioactive compounds is far in excess compared to other non-streptomycete genomes: 3 in Bacillus subtilis (Kunst et al., 1997); 4 in Pseudomonas aeruginosa PA01 (Stover et al., 2000); 2 in Ralstonia solanacearum (Salanoubat et al., 2002). Most genomes lack any natural product gene cluster (Bentley et al., 2002). Gene clusters coding for natural product synthesis are often arranged near the ends of the linear chromosome while life-threatening housekeeping genes are found more in the center (Hopwood, 1999). These termini show an intrinsic instability (Volff & Altenbuchner, 1998) with higher recombination frequencies compared to other parts of the genome. Deletions up to 25 % of the genome (2 Mb on a total genome size of 8 Mb) may occur in about 0.5 % of germinating spores (Volff & Altenbuchner, 2000). In addition, toxic substances originating from the metabolism of the bacterium itself, UV light, etc may damage the DNA leading to point mutations in genes. Hence, an array of altered (mutational origin) or re-composited (through recombinations) genes may come to expression, possibly yielding novel workable protein configurations. Finally, enrichment in genes coding for bioactive compound may occur through recombinatory events between the hosts DNA and extra-genomic stretches DNA (Mochizuki et al., 2003). These large linear elements (plasmids) are easily transferred among bacteria, even non-streptomycetes, in soil.

1.6.8 Bioconversion of chemical compounds

Natural products may need chemical manipulation to be effective or to improve their function prior to their medical application. This involves sometimes complex and expensive chemical methods employing non-environmentally compatible organic solvents. From an ecological and environmental point of view, replacement of these chemical processes by biological fermentations or enzymatic bioconversions would be desirable, as higher specificity and milder operation conditions are obtained using enzymes. This implies a thorough knowledge of the conversion pathways. The development of bioprocesses for production of semi-synthetic natural products has therefore been a high priority during the last 10-15 years (Bruggink & Roy, 2001).

48 1. LITERATURE REVIEW

Such a strategy has been successfully worked out for the production of the semi- synthetic cephalosporin and pravastatin using the enzymatic machinery of particular Streptomyces spp. Semi-synthetic cephalosporin, an important β-lactam antibiotic (see Section 1.6.7), has a low potency and cannot be directly used clinically. Chemical modification of the nucleus of the natural product cephalosporin C i.e. 7- aminocephalosporanic acid (7-ACA) results in a bioactive form (Thykaer & Nielsen, 2003). In replacement of the chemical approach to produce 7-ACA, Crawford et al. (1995) suggested the use of a recombinant Penicillium chrysogenum strain, expressing the S. clavuligerus derived expandase gene and fed with adipic acid, to produce 7-ACA with adipoyl side chains. Subsequent enzymatic removal of the latter adipoyl side chains finally results in the intermediate 7-ACA. Pravastatin is the first potent and specific inhibitor of HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. It significantly reduces plasma cholesterol levels and is thereby of great importance in the therapy of hypercholesterolemia (Arai et al., 1988; Koga et al., 1990). Pravastatin is produced by a two-step fermentation. The basic compound ML-236B is produced by the mould Penicillium citrinum followed by its hydroxylation using the cytochrome P-450sca monooxygenase system of S. carbophilus (Serizawa, 1996; Watanabe et al., 1995).

50 2. TAXONOMY

Taxonomy of the genus Streptomyces

2.1 Introduction

Bacterial taxonomy or systematics can be defined as a continuing scientific study of the diversity of organisms with the aim of characterizing and arranging them in an orderly way (Truper & Schleifer, 1991). Today its scope includes classification, nomenclature, identification and phylogeny (Vandamme et al., 1996; Goodfellow, 2000). Classification tends to arrange organisms in different taxonomic groups (also called taxons e.g. class, order, family, genus, species, strain) on basis of similarity. Ideally, such classifications should be stable, predictive, objective and highly informative (Goodfellow, 2000). The assignment of taxonomic names to the taxa, according to the International Code of Nomenclature of Bacteria (Sneath, 1992) is referred to as nomenclature. In identification, members of a taxonomic group are named on the basis of common features which distinguish them from other organisms (Schleifer & Ludwig, 1994). Finally, phylogeny tends to prove natural evolutionary relationships at different taxonomic hierarchic levels among different taxa (Vandamme et al., 1996) pointing to a common ancestor (see Section 2.7).

51 2.2 The early chaotic years

Streptomycete systematics has had a long and complex history. An overview of successive improvements is given by Anderson et al. (2001). Early descriptions of streptomycetes were based on a few characters i.e. morphological and pigmentation, ecological requirements and spore chain morphology. A first milestone in the systematics of the taxon was the proposal of the genus Streptomyces in 1943 by Waksman & Henrici. With the discovery that streptomycetes produce bioactive compounds, great interest was taken by industry leading to an array of methods for their isolation and growth. Slight differences in morphological and cultural properties justified the description of novel streptomycete species. This practise, mainly serving the industry for patenting novel antibiotics, led to a proliferation in Streptomyces species (Waksman, 1957; Kurylowicz & Gyllenberg, 1988). From mid 1950s on, their number increased almost exponentially reaching about 3000 species by 1970 though many of these so-called species were cited only in patent literature (Trejo, 1970). In order to verify the predictiveness of the tests commonly used in streptomycete systematics, two co-operative studies were started between 1958 and 1961 to tackle the classification problem and were initiated by the Subcommittee on Taxonomy of Actinomycetes of the International Committee on Bacteriological Nomenclature of the International Association of Microbiological Societies (Küster, 1959) and the Subcommittee on actinomycetes of the Committee on Taxonomy of the American Society of Microbiology (Gottlieb, 1961). From the results obtained it was concluded that streptomycete systematics were hampered by the use of variable and non-diagnostic characters, often examined under non-standardised conditions. The high reliance put on subjectively weighted phenotypic characters, the unavailability of reference type cultures of some species and difficulties to find descriptions of species reported in patent literature were also considered as major bottlenecks. Although no direct improvements were proposed, both collaborative projects led to the International Streptomyces project (ISP) in 1966 which had a great impact on Streptomyces’ systematics.

ISP was set up by the same two committees as mentioned above i.e. Subcommittee of Taxonomy of actinomycetes of the International Committee on

52 2. TAXONOMY

Bacteriological Nomenclature and the Subcommittee on actinomycetes of the Committee on the Taxonomy of the American Society of Microbiology. The aim was to provide reliable re-descriptions of existant and authentic type strains of Streptomyces and Streptoverticillium. In practice the following steps were undertaken: type and neotype strains of over 450 species were sent under code to at least three experts who had to describe them using morphological, pigmentation and carbon source utilisation properties under highly standardized conditions. Latter protocols updated species descriptions and were published (Shirling & Gottlieb, 1966; 1968a; 1968b; 1969; 1972; Gottlieb & Shirling, 1967) and type strains were deposited in a number of internationally recognized culture collections e.g. NRRL. The results from ISP studies are a milestone in Streptomyces’ systematics. Hence, they formed the basis of the classification of the genus Streptomyces in the 8th edition of Bergey’s Manual of Determinative Bacteriology (Pridham & Tresner, 1974a, 1974b). Despite the success of the project critical comments raised: (i) a species concept for the genus was not devised nor was there a search for synonymous species (ii) only a few characters were used to describe species based on the ones that were essentially those which had been intuitively selected from earlier classifications (Krainsky, 1914; Waksman & Curtis, 1916; Waksman, 1919, 1961; Jensen, 1930; Waksman & Henrici, 1948; Baldacci et al., 1954; Hesseltine et al., 1954; Gauze et al., 1957; Pridham et al., 1958; Mayama, 1959; Küster, 1961; Gottlieb, 1963) (iii) an identification scheme was not provided.

53 2.3 Numerical taxonomy

In comparison with the approach provided by ISP, numerical taxonomy involves computer-assisted calculation of similarity among a higher number of phenotypic features (units) for classification. The unit characters include traditional features used in previous studies e.g. morphology, pigmentation and data from biochemical, degradative, nutritional and tolerance tests, some of which have not previously been used in streptomycete systematics. Such polythetic classifications are thought to be more stable. Organisms in a cluster share many features in common. Nevertheless, group membership does not depend on variation in a single feature as in monothetic classifications (e.g. ISP approach). In the period 1960-1995, a total number of 21 numerical taxonomic studies were performed analyzing between 18 and 821 strains by a number of 7 to 329 features (Kim, 2004). The most important studies, comprising a sufficient number of strains, sufficient features analysed and with the greatest taxonomical impact, are the studies of Williams et al. (1983a) and Kämpfer et al. (1991). In the study of Williams et al. (1983a), 139 features of 394 Streptomyces species were studied. Species were assigned to (i) 19 major clusters, designated Cat. I, each comprising 6 to 71 strains (ii) 40 minor clusters, designated Cat. II, each comprising 2 to 5 strains (iii) 18 single-membered clusters, designated Cat. III. Defined categories were equated with species-groups (Cat. I) and species (Cat. II & III) respectively. A simplified dendrogram is given in Fig. 8. Based on these results a reduction in the number of species was proposed (Table 6): from 463 (8th edition of Bergey’s Manual) to 142. This classification approach is considered as a third milestone in Streptomyces’ systematics and thus provided the basis for the description of the genus Streptomyces in the 1989 edition of Bergey’s Manual of Systematic Bacteriology (Williams et al., 1989). The study of Williams et al. (1983a) also proved that the following actinomycete genera are synonymous and should be re-classified (Goodfellow et al., 1986a, 1986b, 1986c, 1986d; Witt & Stackebrandt, 1990) within the genus Streptomyces: genera Actinopycnidium (Krassilnikov 1962), Actinosporangium (Krassilnikov & Yuan, 1961), Chainia (Thirumalachar, 1955), Elytrosporangium (Falcäo de Morais et al., 1966), Kitasatoa (Matsumae et al., 1968) and Microellobosporia (Cross et al., 1963).

54 2. TAXONOMY

The numerical study of Williams et al. (1983a) was used to generate probabilistic schemes for the identification of unknown mesophilic streptomycetes clustering in major or minor clusters (Williams et al., 1983b; Langham et al., 1989). The proposed identification approach, based on 41 features, is more reliable than previous identification based on a few subjectively chosen features (Pridham et al., 1958, Waksman, 1961; Hütter, 1967). A similar system was devised for the identification of strains belonging to the originally described Streptoverticillium species (Locci & Schofield, 1989).

55 S. albidoflavus species group

Fig. 8: Simplified dendrogram depicting the taxonomic clusters within the genus Streptomyces by numerical taxonomy (Williams et al., 1983a). Adapted from Anderson et al. (2001).

56 2. TAXONOMY

Species group Proposed subjective synonyms / Allied species

Nr Represented by the species*

1a S. albidoflavus s. albidoflavus S. canescens, S. coelicolor, S. felleus, S. gougerottii, S. intermedius, S. limosus, S. odorifer, S. rutgersensis, S. sampsonii 1b S. albidoflavus s. anulatus S. alboniger, S. albovinaceus, S. alboviridis, S. baarnensis, S. bacillaris, S. cavourensis, S. chrysomallus, S. citreofluorescens, S. cyaneofuscatus, S. fimicarius, S. fluorescens, S. globisporus, S. griseinus, S. griseobrunneus, S. griseus, S. lipmanii, S. parvus, S. pluricolorescens, S. setonii, S. spheroids, S. willmorei. Allied species: S. cremeus, S. niveus, S. sindensis 1c S. albidoflavus s. halstedii S. erythraeus, S. flavogriseus, S. flavovirens, S. griseolus, S. nigrifaciens, S. nitrosporeus, S. olivaceus, S. thermodiastaticus 5 S. exfoliatus S. filamentosus, S. hydrogenans, S. omiyaensis, S. roseolus, S. roseosporus, S. termitum, S. cinereoruber, S. gardneri, S. litocidini, S. narbonensis, S. nashvillensis, S. roseoviridis, S. umbrinus, S. violaceorectus, S. zaomyceticus 6 S. violaceus S. cellostaticus, S. violascens. Allied species : S. michiganensis, S. showdoensis, S. venezulae, S. spiroverticillatus, S. vinaceus 7 “S. roseus” 10 S. fulvissimus S. aureoveticillatus, S. longispororuber, S. spectabilis 12 S. rochei S. albogriseolus, S. althioticus, S. griseoaurantiacus, S. griseofuscus, S. parvulus, S. plicatus, S. pseudogriseolus, S. tendae, S. vinaceusdrappus, S. arabicus, S. calvus, S. griseorubens, S. matensis, S. mutabilis, S. rubiginosus, S. variabilis, S. werraensis, S. griseomycini 14 S. aureofaciens S. roseofulvus 15 S. chromofuscus S. galbus, S. argenteolus, S. flaviscleroticus, S. minutiscleroticus, S. viridosporus 16 S. albus S. almquistii, S. aminophilus, S. cacaoi, S. flocculus 17 S. griseoviridis S. chryseus, S. daghestonicus, S. murinus 18 S. cyaneus S. azureus, S. bellus, S. caelestis, S. chartreusis, S. coeruleofuscus, S. coeruleorubidus, S. coerulescens, S. curacoi, S. lanatus, S. arenae, S. echinatus, S. griseochromogenes, S. afghaniensis, S. janthinus, S. longisporus, S. purpurascens, S. roseoviolaceus, S. violarus, S. violatus, S. cinnabarinus, S. hawaiiensis, S. luteogriseus, S. neyagawaensis, S. resistomycificus, S. collinus, S. paradoxus, S. griseorubiginosus, S. pseudovenezulae, S. fumanus 19 S. diastaticus S. glomeroaurantiacus, S. humidus, S. olivochromogenes, S. flaveus, S. vastus, S. cinereus, S. lincolnensis, S. bottropensis, S. diastatochromogenes, S. rishiriensis, S. achromogenes, S. phaeoviridis, S. galilaeus, S. mirabilis 20 S. olivaceoviridis S. brasiliensis, S. canarius, S. carpinensis, S. corchorusii, S. regensis, S. spiralis 21 S. griseoruber S. coelescens, S. humiferus, S. tuirus, S. violaceolatus 22 S. poonensis S. roseiscleroticus 23 S. microflavus S. ambofaciens, S. eurythermus, S. griseosporeus, S. recifensis 27 S. viridochromogenes

57 continued 28 S. glaucescens 29 S. lydicus S. griseoplanus, S. libani, S. nigrescens, S. platensis, S. sioyaensis, S. albulus 31 S. antibioticus S. phaeofaciens, S. lucensis, S. misionensis, S. naganishii 32 S. violaceusniger S. endus, S. hygroscopicus, S. Melanosporofaciens, S. sparsogenes 33 “S. chromogenus” S. melanogenes, S. noboritoensis 34 S. nogalater 36 S. thermovulgaris S. thermonitrificans 37 S. griseoflavus S. cyanoalbus, S. hrsutus, S. pilosus, S. prasinopilosus, S. prasinus 39 S. longisporoflavus Allied species: S. bluensis 40 S. phaeochromogenes S. antibioticus, S. niger, S. purpurogeneiscleroticus, S. sclerotialus, S. violens 42 S. rimosus S. albofaciens, S. anandii, S. chrestomyceticus, S. kanamyceticus 55 S. griseocarneus S. ardum, S. luteoverticillatum, S, mashuense, S. olivoverticillatum 56 S. netropsis S. avidinii, S. eurocidicus S. flavopersicum, S. netropsis 58 S. blastmyceticus S. griseoveticillatus, S. mobaraensis, S. orinoci 59 S. roseoverticillatus 61 S. lavendulae S. colombiensis, S. flavotricini, S. goshikiensis, S. katrae, S. lavendulocolor, S. polychromogenes, S. racemochromogenes, S. subrutilus, S. toxytricini, S. virginiae, S. xanthophaeus 64 S. bikiniensis 65 S. purpureus 68 S. fradiae S. roseolilacinus 71 S. clavuligerus

Table 6: Proposed subjective synonyms and allied species as defined by Williams et al. (1983a).

58 2. TAXONOMY

Kämpfer et al. (1991) re-examined 821 strains of the genera Streptomyces and Streptoverticillium, including the strains used in Williams et al. (1983a), using a total of 329 physiological tests. A total number of 15 major clusters, 34 minor clusters and 40 single-membered clusters were defined at a 81.5 % similarity level. The phenetic data confirmed the major phena found in the study of Williams et al. (1983a). On the other hand, the taxonomic status of the minor and single-membered clusters were questioned. Hence, Kämpfer et al. concluded that the genus Streptomyces is overspeciated, as concluded already by Williams et al. (1983a). In addition, the genus Streptoverticillium should be reclassified within the genus Streptomyces.

Numerical classifications may be influenced by the selection of tests and strains, test error and genetic instability of the test strains (Goodfellow & O’Donnell, 1993; Schrempf et al., 1989). A further improvement of numerically defined classifications may be achieved with data derived by independent taxonomic methods i.e. chemotaxonomy and genotypy.

59 2.4 Chemotaxonomy

Technological advances in analytical methods such as electrophoresis, chromatography and spectroscopy led to the development and application of multiple chemotaxonomic tools for streptomycete classification, discriminating at different taxonomic levels. Application under highly standardized conditions is a prerequisite, as for phenotypical analysis. Data from these tools generally underpinned taxonomic insights obtained through phenotypy, DNA-DNA hybridizations or 16S rDNA sequencing (see Section 2.7). Impact of chemotaxonomy on Streptomyces systematics has been rather limited, so far, as only a few species and species groups of Williams et al. (1983a) have been analysed. Especially the S. albidoflavus species group (see Fig. 8, previous section) has been well characterized using an array of techniques.

►Cell wall structure analysis

Analysis of whole-cell hydrolysates shows that streptomycetes, including the former genera Chainia, Elytrosporangium, Microellobosporia and Streptoverticillium only contain the LL form of DAP occasionally with small amounts of meso-DAP (up to 16 %). The cell wall typically contains major amounts of hexa- and octahydrogenated menaquinones (Collins, 1985) with nine isoprene units and small amounts of di-, tetra- and decahydrogenated menaquinones (around 5 %). The share 9(H8) / 9(H6) may vary within the genus e.g. among the species S. niger and S. sclerotialus (Goodfellow et al., 1986b). Typical polar lipids (Minnikin et al., 1984) are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides (phospholipid type II). No characteristic sugars are found in the peptidoglycan layer. All these simple tests allow differentiation of Streptomyces isolates from other actinomycetes at genus level in a fast way (see Section 1).

►Fatty acid analysis (FAME) of whole-cells

Typical fatty acid profile of a Streptomyces sp. mainly comprises saturated straight chain and iso- and anteiso-branched chains (Table 7).

60 2. TAXONOMY

Fatty acids Relative amount

15:0 anteiso 13-18 % 16:0 iso 19-26 % 15:0 iso 7-22 % 17:0 anteiso 7-17 % 16:0 4-14 %

Table 7: Predominant cellular fatty acids from Streptomyces spp.

FAME data of Mergaert et al. (1995) confirmed the genomic homogeneity of the S. albidoflavus species group (Williams et al., 1983a), the heterogeneity of the S. cyaneus and S. anulatus groups and supported the reunification of the genus Streptoverticillium with Streptomyces (see Section 2.7). On the other hand, no correlation was found between the fatty acid groups of Saddler et al. (1987) and genomic species found in DNA-DNA hybridizations (Labeda & Lyons, 1991b). S. coeruleorubidus and S. purpurascens strains for instance were scattered amongst unrelated strains. This might be due to non-standardized cultivation of the strains. Although FAME analysis allows classification of large numbers of isolates in a fast way and inexpensive way, its taxonomic resolution is limited to intra-genus level.

► Polyacrylamide gel electrophoresis of whole-cell proteins (protein profiling)

In the study of Manchester et al. (1990), using SDS-PAGE of whole-cell proteins under denaturating conditions, the taxonomic consistency of the S. cyaneus, S. albidoflavus and S. anulatus species group (Williams et al., 1983a) was investigated. Most of the S. albidoflavus and S. anulatus strains were recovered in discrete clusters but the S. cyaneus and S. griseocarneus strains were scattered over several groups as revealed by fatty acid, numerical phenetic and DNA-DNA hybridisation data. The latter study concluded that protein profiling differentiates at species level and is hence useful for identification purposes.

61 ►Bidirectional polyacrylamide gel electrophoresis of ribosomal proteins

Analysis of representatives of nine Streptomyces clusters of the study of Williams et al. (1983a) in their ribosomal protein patterns revealed information around species level although S. lavendulae subsp. lavendulae and S. lavendulae subsp. grasserius showed a very similar pattern to that one of S. avidinii (Ochi, 1989, 1992a,b). This methodology necessitates a time- consuming sample preparation, suffers from difficulties to standardize and highly complex protein patterns for interpretation.

►Amino acid sequencing of the N-terminal sequences (20 amino acids) of ribosomal AT-L30 proteins

The study of Ochi & Hiranuma, 1994 showed that strains of the genera Kitasatospora and Streptoverticillium had identical or very similar terminal sequences to S. exfoliatus. In addition, a close relationship was observed with S. lavendulae. Thus, these data supported the reclassification of the genera Kitasatospora and Streptoverticillium in Streptomyces. In 1995, Ochi examined AT-L30 proteins of a higher number of species (81), representing the genus Streptomyces. Results showed a good congruence with corresponding numerical phenetic data of Williams et al. (1983a) and corresponding 16S rRNA sequence data.

►Phage typing

Streptomyces phages can be either polyvalent or species specific (Chater et al., 1986). Species-specific phages have been described for S. coelicolor Müller, S. griseus, S. griseinus, S. scabiei, S. venezulae, S. matensis, S. albus, S. azureus, S. caesius and S. violaceoruber (Anderson et al., 2001). Some cross- activity has also been detected with the genera Nocardia, Streptosporangium and mycobacteria. Data from Korn-Wendisch & Schneider (1992) supported the reclassification of the genus Streptoverticillium within Streptomyces. On basis of

62 2. TAXONOMY

these results, phage typing can be used for classification and identification at the genus and species level (Wellington & Williams, 1981).

►Serology

Serological tests were shown to be genus-specific and to some degree species- specific based on results obtained after the application of antisera raised against mycelia of streptomycetes (for overview: Anderson et al., 2001). Ridell et al. (1986) found a close relationship between S. lavendulae and formerly classified Streptoverticillium species.

►Curie-point pyrolysis mass spectrometry (PyMS)

In the study of Sanglier et al. (1992) PyMS was shown to be useful in the discrimination of S. violaceusniger strains from representative type strains assigned to the S. violaceusniger species group of Williams et al. (1983a). Ferguson et al. (1997) examined strains belonging to the S. albidoflavus species group and found a good correlation with numerical phenetic, fatty acid, DNA-DNA hybridization and protein profiling data showing that the species S. albidoflavus is a distinct and well defined species.

63 2.5 DNA-DNA hybridizations

DNA-DNA hybridization was the first molecular approach used routinely to measure the degree of genomic relatedness and the first one to be generally accepted for improving bacterial classification. It is referred in literature as the “gold standard” for species delineation (Wayne et al., 1987; Stackebrandt & Goebel, 1994). An average measure of nucleotide similarity of the entire genome is determined and is therefore more advantageous over those techniques that involve the comparison of individual genes only (see further). The DNA-DNA hybridization method uses single- stranded DNA (derived from native double stranded whole-cell DNA by denaturation) from two bacterial strains and compares their capacity to hybridise with each other to form double stranded DNA under different temperatures and ionic concentrations.

The optimal temperature for hybridization is calculated using the formula TOR = 0.51 x (G+C) + 47 °C (Gillis et al., 1970). Together with the ionic concentration this parameter determines the stringency of the reaction and hence influences the reproducibility of the results. The degree of relatedness between two strains is expressed as % homology. DNA-DNA hybridizations are taxonomically most suitable for resolving close relationships within a same genus. Its taxonomic power is more limited to measure relationships among strains at inter-genus level. It has been calculated that for reassociation under optimal hybridization conditions, the two DNA strands must exhibit at least 80 % sequence complementarity (Kim, 2004). Two strains sharing a homology value > 70 % (and ∆Tm1 < 5°C difference) belong to same species (Wayne et al., 1987). Values between 30 % and 70 % indicate a certain relationship most likely originating from gene clusters encoding similar metabolic pathways, etc. On the other hand, DNA-DNA homology values below 30 % indicate no relationship among two strains. Since the 1960s numerous DNA-DNA hybridisation techniques have been developed based either on immobilized DNA or free solution reassociation (Stackebrandt & Goebel, 1994): the agar gel method (McCarthy & Bolton, 1963), membrane filter method (Gillespie & Spiegelman, 1965), hydroxyapatite method (Brenner et al., 1969), the inititial renaturation method (De Ley et al., 1970) and the

1 difference in the melting temperature between the homologous and heterologous hybrids formed using standard denaturisation conditions

64 2. TAXONOMY

S1 endonuclease method (Crosa et al., 1973). Technological advances led in 1989 to the introduction of the fluorescent microplate method (Ezaki et al.,1989) requiring less amounts of pure DNA, easier to apply, and being less laborious and time- consuming than classic DNA-DNA hybridization protocols. This popular method, based on non-covalent binding reactions, was shown to correlate well with results obtained by the initial renaturation method (Goris et al., 1989) with similar reproducibility levels. Recently, a protocol was devised for covalently based reactions (Christensen et al., 2000). Despite the small sampling error (Sneath, 1970) and quite high reproducibility levels, DNA-DNA hybridization surveys only yield data on a pairwise reaction. Each similarity value has to be determined individually between every pair of organisms. The number of comparisons goes up exponentially with the number of strains to be compared. Inclusion of any new strain in the comparison requires the determination of its DNA similarity to every other strain in the set and any new potential new species should be hybridized with all species in the genus.

Within Streptomyces systematics, only few numerically phenetic defined major and minor species groups of Williams et al. (1983) have been taxonomically clarified using DNA-DNA hybridizations until now. • Early DNA-DNA homology studies date from 1969. In that year Monson et al. clarified the taxonomic status of the strains belonging to S. coelicolor and S. violaceoruber. It was shown that S. coelicolor A3(2) (a model organism used by streptomycete geneticists) is in fact a S. violaceoruber strain. • Mordarski et al. (1986) examined representatives of the S. albidoflavus species group consisting of subclusters S. albidoflavus, S. anulatus and S. halstedii, together with marker strains of S. albus, S. griseocarneus and S. lavendulae. They concluded that the S. albidoflavus strains formed a distinct genomic species. • The group of Labeda contributed considerably to Streptomyces systematics. In a first study, Labeda & Lyons, 1991(a) demonstrated that the S. violaceusniger cluster encompassed seven different genomic species. One of them i.e. S. hygroscopicus, S. endus was found to be a subjective synonym of S. hygroscopicus. In 1991(b), Labeda & Lyons found that the S. cyaneus species-group was genomically heterogeneous. Nine out of 18 representative strains from this cluster were assigned

65 to two DNA-DNA relatedness groups i.e. S. coeruleorubidus and S. purpurascens. They proposed respectively two and four synonymous species within these two groups. The strains of both groups could be distinguished by a few biochemical and morphological properties. Examination of red spored streptomycetes by Labeda (1998) supported the separation of the members of the S. fulvissimus and S. griseoviridis phenotypic clusters into six genomic species and the proposal of two synonymous species. A same heterogeneity was found among the S. lavendulae phenotypic cluster (Labeda, 1993). The species S. colombiensis was reduced to a synonym of S. lavendulae. Labeda (1996) also determined the levels of DNA-DNA relatedness among 35 species originally classified as Streptoverticillium. These strains represent eight of the 24 phenotypic clusters as defined by Locci & Schofield, 1989. On basis of DNA-DNA relatedness, 7 multi-membered and 13 single- membered clusters could be defined. Several species were found synonymous, but these taxa were not generally equivalent to the clusters suggested by phenetic numerical taxonomy data. Hatano et al. (2003) extended the latter study by the examination of more ‘Streptoverticillium’ species, including phenotypic re- examination. Data of Hatano et al. supported the earlier findings of Labeda (1996). It was shown that grouping based on aerial mass and substrate mycelium, melanin formation ability and sugar utilization pattern were somewhat different from the numerical classification of the verticilliate-forming ‘Streptoverticillium’ species based on 41 phenetic characters (Locci & Schofield, 1989). Finally, Labeda (1992) and Bouchek et al. (2000) showed that the potato pathogenic species S. scabiei and S. acidiscabies were unrelated to S. ipomoeae or to any other Streptomyces species producing blue spores. • Kim (2004) reported DNA-DNA homology values between 73 and 100 % among S. coelicolor Waksman 3443, S. coelicolor A3 (2), S. lividans ICSSB 1019, S. lividans 66, S. violaceolatus, S. coelescens, S. caesius, S. humiferus, S. lazureus to S. violaceoruber and hence should be considered as synonyms of latter species. The species S. rutgersensis subsp. rutgersensis and S. gougerotii showed a DNA-DNA relatedness of 91 %. Within the S. albidoflavus species group, the following species are synonyms of S. albidoflavus: S. limosus, S. sampsonii, S. odorifer, S. felleus, S. canescens and S. coelicolor Müller. Intermediate DNA-DNA homology values (<70 %) were shared among S. tendae, S. ambofaciens, S. tuirus, S. ciscaucasicus, S.

66 2. TAXONOMY

griseoruber, S. rutgersensis subsp. rutgersensis, S. cyaneogriseus, S. nogalater, S. eurythermus and S. intermedius with representatives of the S. violaceoruber and S. albidoflavus species group.

From all these studies it is clear that the phenetic approach of Williams et al., 1983a should be regarded as a method for grouping related species at the intra- genus to species level. Further improvement of the taxonomy of the genus Streptomyces is only possible upon analysis of all phenetically defined clusters in DNA-DNA hybridizations or equivalent genotypic tools.

67 2.6 Genotypic screening methods

With the advent of restriction enzymes and the PCR technology by Mullis in 1983, genotyping methods have become increasingly important in systematics. DNA- and PCR-based genomic fingerprinting methods are emerging as the most rapid, reliable and simple ways to characterize microbes

(Vaneechoutte, 1996; Louws et al., 1999). Probably the most discriminatory

PCR-based fingerprinting methods presently used are based on whole-genome analysis i.e. RAPD, PFGE, AFLP and rep-PCR fingerprinting (Fig. 9).

Fig. 9: Taxonomic discriminative power of genomic fingerprint techniques (Vandamme et al., 1996).

68 2. TAXONOMY

►Random Amplified Polymorphic DNA fingerprinting (RAPD)

RAPD involves PCR amplification of random fragments of genomic DNA using arbitrary primers. This technique has been mainly used with the aim of typing a few Streptomyces species i.e. S. ambofaciens, S. coelicolor, S. glaucescens, S. lividans, S. pristinaespiralis and S. rimosus (Martin et al., 2000) and it is thus difficult to determine its taxonomic resolution and value for the whole genus. The same study also showed that RAPD analysis is able to differentiate mutants belonging to a same clone.

►Pulsed Field Gel Electrophoresis (PFGE)

PFGE involves isolation of intact DNA from the cell followed by digesting using rare cutting restriction enzymes (hexa cutters) and separation of obtained large fragments on agarose gels. It is a typing tool, like RAPD, but with a significantly higher reproducibility. This has been demonstrated by the study of Beyazova & Lechevalier (1993) in which patterns of S. ipomoeae strains were recovered as two distinct subclusters at 75 % similarity (although this group of strains is known to form a coherent group). S. purpurascens and S. coeruleorubidus strains were not recovered in taxonomically coherent groups. Using PFGE, the instability of certain regions of the Streptomyces genome was proven (Leblond et al., 1991).

► Amplified Fragment Length Polymorphism (AFLP) fingerprinting

AFLP fingerprinting is a tool analysing the whole-genome and consists of (1) digestion of total genomic DNA using two rare cutting restriction enzymes (2) subsequent selective amplification of these fragments by PCR and (3) electrophoretic separation of fragments on high resolution polyacrylamide gels. Kim (2004) demonstrated that the S. violaceoruber species group formed a distinct cluster separate from the S. albidoflavus species group. The same conclusion could be drawn for the S. albidoflavus cluster and the other closely related species. It confirmed that S. felleus, S. canescens, S. odorifer, S.

69 coelicolor Müller, S. limosus and S. sampsonii group together, but S. rutgersensis subsp. rutgersensis, S. gougerotii and S. intermedius are separate from the other members of the S. albidoflavus species group. A good correlation was found with DNA-DNA homology data; strains exhibiting a DNA-DNA similarity higher than 72 % exhibited levels of AFLP similarity higher than 50 %. Kim (2004) suggested that AFLP fingerprinting could be used as a supplementary tool to DNA-DNA pairing for determining the taxonomic position and defining bacterial species within streptomycetes.

► Repetitive intergenic DNA sequences (rep)-PCR fingerprinting

Rep-PCR amplifies intervening sequences located between highly repetitive DNA motifs dispersed over the whole genome. Sadowsky et al. (1996) and Clark et al. (1998) investigated relationships among phytopathogenic and disease-suppressive Streptomyces strains. BOX primed PCR reactions were found stable, unique and reproducible (Sadowsky et al., 1996). In the study of Clark et al. (1998), a higher variation in patterns was found among S. ipomaeae strains using respectively the REP and ERIC primer while BOX-primed fingerprints showed higher similarities. Hence, rep-PCR fingerprinting is taxonomically discriminating at respectively strain level (REP and ERIC primer) and between strain and species (BOX primer).

► Ribotyping

This technique consists of four steps: (1) restriction of the bacterial chromosome with an endonuclease, (2) gel electrophoresis of the resulting fragments (3) transfer of the fragments to a membrane and (4) hybridisation of the gel with a lablelled probe complementary to the 16S and 23S rDNA (Grimont & Grimont, 1986). The first study applying this technique was used by Doering-Saad et al. (1992) who examined 40 potato scab-inducing and non- pathogenic streptomycete isolates. The resulting patterns showed a high diversity with little correlation with corresponding RFLP and phenetic data. Schumann et al. (2001) analysed 298 type strains differing in 20 or less

70 2. TAXONOMY

nucleotides in their 16S rDNA sequence. The majority of type strains differing in their 16S rDNA sequence are defined by individual ribogroups. In addition, type strains of different species may share the same ribogroup and identical 16S rDNA sequence. Comparison of molecular patterns with clustering of strains based on phenotypic data (Williams et al., 1983a & Kampfer et al., 1991) revealed a high, though not complete degree of correlation.

► Restriction Fragment Length Polymorphism (RFLP) of ribosomal RNA genes

In the study of Clarke et al. (1993) relationships were determined among 14 Streptomyces species. Thirteen of these species had been found previously to be losely related by phenetic numerical classification and DNA-DNA hybridization. Considerable variation was found in the RFLP patterns using a combined digest of 4 hexa-cutter restriction enzymes loaded on one gel. S. albidoflavus and S. anulatus were shown to be well defined species as found with other techniques (see above). In conclusion, RFLP analysis of ribosomal genes appeared to be an accurate and rapid strain identification tool for determining relationships between closely related Streptomyces species.

► RFLP analysis of PCR amplified gene

Steingrube et al. (1997) applied the RFLP methodology on a PCR amplified 439 bp fragment of the 65 kDa heat shock protein gene to examine 198 clinically important actinomycetes together with four strains of the species S. albus, S. griseus and S. somaliensis. MspI-derived RFLP patterns alone were sufficient for separating 74 % of the Streptomyces isolates from all other aerobic actinomycetes. HinfI produced very large fragments of 335 to 395 bp from 18 of 19 strains and further facilitated differentiation. These very large RFLP bands were unique to Streptomyces isolates and readily distinguished them from isolates of all other aerobic actinomycete species and taxa at genus level.

71 2.7 Phylogeny

2.7.1 16S rDNA sequence analysis

During the last two decades, bacterial taxonomy has been enriched with ribosomal chronometers i.e. the ribosomal rDNAs 5S, 16S and 23S for phylogeny, classification and for identification. Especially 16S rDNA sequence analysis played a major contribution in it. The 16S rDNA coding gene (about 1500 bp in length), required for the survival of the bacterial cell, is universally distributed among prokaryotes and consists of two evolutionary different evolving stretches DNA. The more conserved parts (e.g. α and β region in Streptomyces; Stackebrandt et al., 1991) allow to reveal deep-branching relationships such as the recognition of the domains Bacteria, Eucarya and Archaea (Woese et al., 1990; Winker & Woese, 1991) and phylogenetic allocation of streptomycetes among Bacteria (Embley & Stackebrandt, 1994; Section 1.3). The more variable parts of the 16S rRNA gene on the other hand, show a higher evolutionary rate of change and hence yield phylogenetic information at a lower hierarchic level i.e. between intra-genus and species. The taxonomic discriminative power of 16S rDNA sequencing is too limited to determine whether organisms belong to a same species (Fox et al., 1992; O’Donnell et al., 1994; Stackebrandt & Goebel, 1994), due to the high conservation of the 16S rRNA gene. Two strains that have less than 97 % 16S rDNA sequence similarity do not show a DNA-DNA homology value of more than 60 % but the opposite is not necessarily true. The advantages of 16S rDNA sequencing are multiple compared to DNA-DNA hybridization surveys: it is easy and relatively fast to determine through the PCR technology, semi-automated procedure, has a higher reproducibility, does not require specially trained scientists and generates absolute data. An international sequence depository bank was established, designated “the Ribosomal Database Project” to manage generated 16S rDNA sequences (and other sequencing data; Maidak et al., 1999; Cole et al., 2003; http://rdp.cme.msu.edu/html) and make it publicly available, through appropriate utilization software, as a basic tool for modern classification and identification purposes. Publicly available softwares e.g. BLAST (Altschul et al., 1990) and FASTA (Pearson & Lipman, 1988) allow to select all closest neigbours (> 97 % similarity) of a

72 2. TAXONOMY

strain provided that complete phylogenetic frameworks are available. Phylogenetic tree construction is subsequently performed using (i) an alignment algorithm either pairwise or multiple based e.g. CLUSTALX (Thompson et al., 1997) and followed by (ii) distance calculation among the entries e.g. Neighbour-Joining method (Saitou & Nei, 1987) using a phylogeny software package e.g. BioNumerics (Applied Maths, Belgium), PHYLIP (Felsenstein, 1993), Treecon (Van de Peer & De Wachter, 1994) or PAUP (Sworfford & Olsen, 1990). At the moment, RDP II and cooperating projects (EMBL, GenBank, DDBJ) comprise over 70000 16S rDNA sequences while almost 6000 prokaryotic species have been validly described (Euzéby, http://www.bacterio.cict.fr). Almost complete (> 1300 bases) and high quality 16S rDNA sequences (< 0.5 % ambiguities) are available in EMBL on 151 validly described Streptomyces species.

Within Streptomyces systematics, 16S rDNA sequencing studies have been used to determine the homogeneity of defined streptomycete species. Several major improvements of the taxonomy of Streptomyces have been proposed.

• Early 16S rDNA analyses, through 16S rRNA cataloguing, demonstrated a close relationship between representatives of the formerly classified genera Chainia, Elytrosporangium, Kitasatoa (= Kitasatospora), Microellobosporia, Streptoverticillium and Streptomyces (Fig. 10). • Streptomycetes causing scab formation on potato were shown to form a heterogeneous group based on numerical analysis of their almost complete 16S rDNA sequences thereby suggesting that strains causing this disease belong to several lineages (Takeuchi et al., 1996; Song et al., 2004). Subsequent DNA-DNA hybridisations confirmed this finding (see Section 2.5). • Identical, almost complete 16S rRNA sequences were shared among 4 strains of the species S. albidoflavus and the species S. canescens, S. coelicolor, S. felleus, S. limosus, S. odorifer and S. sampsonii. The latter group of species is closely related to the species S. gougerotii, S. intermedius and S. rutgersensis with respectively 15, 14 and 16 nucleotide differences (Fig. 10). A good correlation was found with numerical phenetic data of Williams et al. (1983a) suggesting that S. canescens, S. coelicolor, S. felleus, S. limosus, S. odorifer and S. sampsonii should be seen as subjective

73 synonyms of S. albidoflavus. Recent DNA-DNA hybridization data confirmed this, including the separation of S. gougerotii, S. intermedius and S. rutgersensis from the latter group (Kim, 2004). • Stackebrandt et al. (1991) found that 16S rRNA gene nucleotide 929 of S. ambofaciens (numbering system: Pernodet et al., 1989) was found to be unique to Streptomyces strains. A genus specific probe (5’ GCGTCGAATTAAGCCACA-3’) was generated incorporating this specific nucleotide position and its flanking region.

Several studies were conducted to determine the taxonomical significance of specific regions of the 16S rRNA gene.

• Witt & Stackebrandt (1990) examined 520 nucleotides of the 16S rDNAs of 40 strains belonging to 37 species. They represent 11 species groups of Williams et al. (1983a). Eleven strains have not been analysed by Williams et al. (1983a). A close relationship was shown between strains of the S. lavendulae cluster, S. purpureus cluster and Streptoverticillium strains (Fig. 10) as predicted by numerical phenetic and chemotaxonomic data. Hence, the genus Streptoverticillium was proposed to be reclassified within Streptomyces.

74 2. TAXONOMY

100

** # #

* 100

92 S. albidoflavus group

100 S. violaceoruber group

100

Fig. 10: Phylogenetic relationships between members of the genera Streptomyces, Kitasatospora and Streptacidiphilus based on almost complete 16S rRNA gene sequences. Tree constructed using the BioNumerics software (Neighbour joining, Jukes & Cantor correction). Outgroup is Intrasporangium calvum. Was formerly classified in *:Elytrosporangium; **: Chainia; #: Streptoverticillium. Important bootstrap values are given.

• Stackebrandt et al. (1991) reported the presence of conserved stretches DNA within the 16S rRNA gene i.e. α and β region (nucleotides 982 through 998 and 1102 through 1122; S. ambofaciens numbering). In a later study (Stackebrandt et al., 1992) both stretches were used to construct a phylogenetic tree corresponding well with a tree based on sequence analysis of a 1137 nucleotide stretch of the 16S rRNA gene. In addition, a good correlation was found with the numerical phenetic classification of Williams et al. (1983a). Analysis of the hypervariable part of the 16S rRNA gene alone i.e. the γ region (nucleotides 158 to 203) resulted in a phylogenetic tree showing less congruence with the tree based on analysis of the α and β region and the numerical phenetic data. Hence, phylogenetic relationships based on the analysis of the short γ region alone are questioned. This observation is in contradiction to a more recent study of Kataoka et al. (1997). In the latter study, a tree based on alignment of 120 nucleotides of the γ region (nucleotide positions 158 to 277; S. ambofaciens numbering [Pernodet et al., 1989]) of 89 streptomycetes, belonging to eight major clusters out of the study of Williams et al. (1983a), correlated well with a tree based on whole sequences. General topology was maintained only with longer evolutionary distances between the separate branches. The study of Kataoka et al. (1997) also supports the taxonomic status of the phenotypic groups as defined by Williams et al. (1983). Analysis of the large S. albidoflavus group, which had previously been shown to comprise three species groups and over 60 strains (Williams et al., 1983), was resolved into six groups using sequence comparisons of the γ region. Remarkably, the three phenotypic subgroups were maintained, although not clustering together. This is also the case when members of the 25 major cluster groups are analysed phylogenetically (Fig. 11). The only strains with identical γ regions were S. olivaceoviridis and S. griseoruber

76 2. TAXONOMY

representatives from clusters 20 and 21, respectively. Finally, Kataoka et al. (1997) concluded that the latter region can be used for the identification of unknown streptomycetes. The partial sequences of 485 representative streptomycetes are held in a database which is publicly available through EMBL.

Fig. 11: Genotypic analysis of the major streptomycete clusters, as defined by Williams et al. (1983a) through comparison of the 16S rDNA from nucleotides 158-276 including the γ region. Phylogenetic analysis was performed using the DNADIST and Neighbour programs of the PHYLIP software package. Bootstraps above 600 are given. The bar represents a distance of 100. Adapted from Anderson et al. (2001).

77 On the other hand, the complex history of the genus Kitasatospora (Omura et al., 1982) illustrates the importance to examine almost complete 16S rDNA sequences. Alignment of 520 nucleotide sequences with representative Streptomyces species showed a close relationship between these two genera as predicted through phenotypical analysis. Hence, Kitasatospora was proposed as a synonymous genus of Streptomyces by Wellington et al. (1992). Proposed reclassification was supported by N-terminal sequencing of ribosomal AT-L30 proteins (Ochi & Hiranuma, 1994). Using DNA-DNA hybridizations however, two distinct clusters were found among Kitasatospora species and original Streptomyces species questioning its reclassification. In addition, analysis of almost complete 16S rDNA sequences grouped Kitasatospora species separately from Streptomyces species in a separate monophyletic clade (Fig. 10). In combination with some discriminating phenotypic features (Table 1; Section 1) the genus Kitasatospora was again revived (Zhang et al., 1997).

2.7.2 Analysis of other stretches DNA

As an answer to the limited use of 16S rRNA sequences for the differentiation of closely related Streptomyces species, analysis of more variable DNA stretches has been introduced in systematics.

2.7.2.1 Sequencing of protein coding genes

Housekeeping genes, coding for essential metabolic proteins in the cell, are expected to show a higher evolution rate than the 16S rRNA gene. Their relevance in phylogeny in Streptomyces has been evaluated through two model sets of well characterized organisms proposed for the development of the definition of a post- genomic species concept for Streptomyces namely S. violaceoruber and S. albidoflavus species groups. In the study of Kim (2004), the gyrA (Kasai et al., 1998) and prcA (Tamura et al., 1995) genes displayed relatively higher levels of sequence variability (up to 7.4 % for prcA) than those of recA (Eisen, 1995), rnpB (Brown, 1999), rpoB (Kim et al., 1999, 2004; Ko et al., 2003) and 16S rDNA (Fig. 12). Sequencing of the most variable part of each of these genes (around 600 bp in

78 2. TAXONOMY

length) resulted in phylogenetic trees showing a similar topology to the 16S rDNA derived tree but with higher genetic distances (Kim et al., 2004; Kim, 2004) with exception of rpoB and rnpB. The latter two genes yielded trees with deep branches, reflecting evolutionary rates of change 5-9 times faster than 16S rDNA.

16S rRNA gyrA

prcA recA

rnpB rnpB rpoB

79 Fig. 12: Graphical representation of polymorphisms of 16S rDNA, gyrA, prcA, recA, rnpB and rpoB of representative strains of the S. violaceoruber and S. albidoflavus species groups and closely related species. Pi denotes the average number of nucleotide differences per site between two sequences, or nucleotide diversity. Adapted from Kim (2004).

Multi Locus Sequence Typing (MLST, Maiden et al., 1998; Cooper & Feil, 2004) is a recently developed tool combining sequence heterogeneity found in different genes. Preferably, these genes cover the whole genome. Given the fact that each sequence differing in one or two nucleotide sites is considered as a distinct allele, results in a unique allelic profile for each strain. In the study of Kim (2004), stretches of 5 housekeeping genes i.e. gyrA, prcA, recA, rnpB and rpoB were sequenced of 41 strains belonging to the S. violaceoruber and S. albidoflavus species group. Subsequent alignment of these sequences resulted in an allelic profile for each of these strains. Subsequent UPGMA derived tree showed a higher resolution to discriminate both species groups and their closest neighbours S. intermedius, S. gougerottii and S. rutgersensis than the corresponding 16S rDNA derived tree. Hence MLST is a promising tool for characterising and defining bacterial species groups. Its high discriminative power is mostly applied in other fields e.g. typing of closely related pathogenic strains (Helgason et al., 2004; Manning et al., 2003; Lemee et al., 2004), emergence and spreading of antibiotic resistant clones (Johnson et al., 2003; Gherardi et al., 2003, Wannet et al., 2004), studies of population genetics (Caugant, 2001) and epidemiological surveillances (Urwin & Maiden, 2003).

Despite the potential to provide phylogenetic information at a higher discriminatory level, up to now, a complete phylogenetic framework has not been created or made publicly available for the genus Streptomyces, using one or more of the above mentioned housekeeping genes neither with the lower discriminating 16S rRNA gene. This is mainly due to (i) the high number of validly described Streptomyces species (512 species, List with valid names version 07/2004, DSMZ) and (ii) relative high costs for sequencing.

80 2. TAXONOMY

2.7.2.2 16S rDNA-23S rDNA ITS analysis

Within the rrn operon, the 16S rRNA gene is separated from the 23S rRNA gene by a hypervariable spacer region (Gürtler & Stanisich, 1999). Within Streptomyces, this region consists of a mosaic of variable regions with intervening constant nucleotide blocks (Wenner et al., 2002). A high recombination frequency is thought to occur among these blocks resulting in differences in size (between 230 and 370 bp) and sequence composition. Heterogeneity in ITS sequences within a strain is further enhanced by the presence of several copies of the rrn operon and observed heterogeneity among these operons. Within the Streptomyces genome, 6-7 copies have been reported (Berger et al., 1996; La Farina et al., 1996; Kim et al., 1993; Suzuki et al., 1988) showing an average value of 2 % inter-operon heterogeneity (Ueda et al., 1999). The fact that every bacterial strain is supposed to have such a unique set of ITS spacer(s) and that their sequences evolve faster than the 16S rRNA gene, makes 16S-23S ITS analysis a powerful tool for the differentiation of recently diverged bacteria (Barry et al., 1991; Gürtler & Stanisich, 1996; Conrads et al., 2002; Goncalves & Rosato, 2002; Yoon et al., 1998; Zhang et al., 2001; Nour, 1998). As for 16S rDNA sequencing, a database facility has been established (RISSC; Garcia-Martinez et al., 2001; http://ulises.umh.es/RISSC) to manage 16S-23S ITS sequencing data generated. Within Streptomyces systematics, 16S-23S ITS analysis has been applied on a limited number of species. Hain et al. (1997) showed that both the heterogeneity in length and sequence composition of the 16S-23S spacer can be used to differentiate S. albidoflavus strains sharing identical 16S rDNA sequences. However, PCR amplification and subsequent direct sequencing yielded a mixture of fragments with sequence ambiguities necessitating the use of laborious cloning methods to be useful. Hence, analysis of the 16S-23S ITS alone is informative at intra-species level. Zhang et al. (1997) showed that Kitasatospora species, formerly classified as Streptomyces, have a nine nucleotide signature in their 16S-23S spacer. Alignment of 16S-23S spacer sequences showed that kitasatosporae and streptomycetes were consistently separated into two distinct clades supported by high bootstrap values and not affected by the different outgroups used. This observation was compliant

81 with 16S rDNA and phenotypic analysis and DNA-DNA hybridization homology studies to lift the genus Kitasatospora from the genus Streptomyces. Song et al. (2004) examined the 16S rRNA gene and 16S-23S ITS sequence of 34 Streptomyces strains including 11 scab-causing isolates. 16S-23S ITS regions of these isolates showed various lengths and highly variable sequence similarities (35- 100 %) within strains as well as intra- and interspecies. The species S. europascabiei and S. scabiei cluster together in a same 16S rDNA sequencing clade but could be separated on basis of their 16S-23S ITS sequences. On the other hand, for other species variability in 16S-23S ITS region was found too low for differentiation although separable in 16S rDNA sequencing (e.g. S. turgidiscabies and S. reticuliscabies; 98.6 % 16S rDNA similarity). Hence, 16S-23S ITS sequence analysis alone was found not useful for phylogenetic analysis in Streptomyces.

2.7.3 Whole genome sequencing and analysis

Questions are arising regarding the use of the 16S rRNA gene to study evolutionary processes on closely related genomes. This is due to the reported heterogeneity among multiple copies of the rRNA gene (Ueda et al., 1999) present in a single organism and the fact that lateral gene transfer is responsible for the presence of divergent rRNA genes in an organism (Wang & Zhang, 2000). This 16S rDNA sequence divergence may lead to changes in position in the phylogenetic tree. Besides the problem of paralogy, inherent imprecision in current alignment techniques may give conflicting phylogenetic trees. Thus, variations in choice of penalty for gaps and mismatches in alignment matrices is likely to change the topology of trees when additional species are added or when the order of addition is altered (Bansal & Meyer, 2002). Determination of sequences of the whole-genome and subsequent comparison with other genomes, using bioinformatics, is a new tool to examine recently diverged organisms. Parameters such as gene content, genomic organization and mean identity in orthologous genes reflect better subsequent evolutionary changes than those obtained with the 16S rRNA gene. In addition, information is obtained on pathways for regulation, secondary metabolism, level of pathogenicity, structural

82 2. TAXONOMY

prediction, factors involved in the emergence of new infectious diseases and drug development (Sanchez & Sali, 1999; Whittam & Bumbaugh, 2002; Yao, 2002). Currently over 200 whole-genome sequences of various genera have been determined. Among streptomycetes, two have been made publicly i.e. the model- organism S. coelicolor A(3)2 (Bentley et al., 2002) and the industrially important species S. avermitilis (Ikeda et al., 2003). Their genome size is respectively 8.7 and 9.03 Mb. A total number of 7825 genes were predicted in S. coelicolor, 7574 in S. avermitilis. This is almost twice the number of genes found in Escherichia coli K12 (4289 genes) or Bacillus subtilis 168 (4099 genes) and more than the eukaryote Saccharomyces cerevisiae (6294 genes) (Bernal et al., 2001). Comparison of both genomes leads to the identification of 5283 orthologous genes, and 2291 (S. coelicolor) and 2307 (S. avermitilis) genes that are unique to each strain. Besides regulatory functions, these unique genes code for proteins involved in secondary metabolism, polymer and xenobiotic degradation, and transposition (Ikeda et al., 2003). Of these, a total number of 965 S. coelicolor proteins, which comprise 12.3 % of total genes, were assigned regulatory functions (regulators and extracellular enzymes). This high number is correlated with the complex Streptomyces life-cycle (Weber et al., 2003).

84 3. AIMS & CONCEPTUAL FRAMEWORK

Aims & Conceptual framework

3.1 General aims

In contrast to most other bacterial taxa, classification and identification of species belonging to the genus Streptomyces is still dominated by the use of morphological and physiological traits. These approaches are time-consuming, the data interpretation requires a lot of expertise and the results often contradict with genotypic data. Only a few research groups in the world, mostly academic, are considered as experts in the classification and identification of these taxa. Both chemotaxonomic and genotypic information demonstrate the lack of discrimination of the currently used phenotypic classification as well as the instability of morphological traits. As a result, incorrect identifications may occur and synonymous names within a number of Streptomyces species have been elucidated. Taxonomy of the genus Streptomyces should evolve to a polyphasic alternative taking into account phenotypic and chemotaxonomic data. Ideally, this alternative should be reliable, fast and easily applicable to everyone, not only academics. However, due to the lack of minimal standards for defining Streptomyces species the polyphasic approach remains a laborious task. The over 500 validly described species in Streptomyces remain a major obstacle to set up large fundamental taxonomic studies, using the gold standard of systematics i.e. DNA-DNA hybridizations. Also as a result of this high number of species, new species descriptions still do not meet the criteria that are generally accepted in bacterial taxonomy. The phylogenetic position of a potentially novel species is determined upon comparison with an incomplete (public) 16S rRNA gene

85 sequence database because complete sequences are only available for 25 % of the validly described species. Considering the cut-off of 97 % sequence similarity as a level for possible species relatedness, within Streptomyces often too large numbers of related species are found supported by low bootstrap values. Hence, it is likely that for novel species descriptions not all nearest neighbours are included in subsequent DNA-DNA hybridizations.

3.2 Specific aims

This study aims to contribute to the establishment of a polyphasic taxonomy and to apply reliable alternatives to resolve at least three major bottlenecks in current Streptomyces systematics (Fig. 13). A first goal is to search for either a chemotaxonomic or genotypic screening tool with a taxonomic resolution at the species level. In addition, this method should be reliable, fast and inexpensive and show a high correlation with DNA-DNA hybridizations. It is our intention to analyze all publicly available Streptomyces species. Possible species relatedness will be further examined by DNA-DNA hybridizations. It is our aim to detect synonymous species in the genus Streptomyces and reduce the number of species. The database containing patterns of all Streptomyces species will be used for identification of new isolates. A second goal of the study focuses on the link between phenotypy and genotypy and to determine the taxonomic resolution of above mentioned screening method. The question raises whether the current phenotypic classification system of Streptomyces is confirmed at the genomic level or if genotypic taxonomy provides a more reliable classification system. A collection of S. virginiae strains including variants of virginiamycin producing strain S. virginiae 899 was analysed. A third goal aims to provide a fast method that may be a valuable alternative for 16S rRNA gene sequencing and allows to delineate nearest phylogenetic neighbours within the genus Streptomyces. Herefore, all type strains were screened using 16S- ITS RFLP fingerprinting, combining information of the 16S rRNA gene molecular clock and hypervariable 16S-23S ITS region. The taxonomic usefulness of obtained data is evaluated upon analysis of a subset of reference strains sequenced in their 16S rRNA gene.

86 3. AIMS & CONCEPTUAL FRAMEWORK

In a second part of this study the constructed reference framework is used to detect and describe novel species among a large collection of Chinese and Moroccan strains isolated from geographically diverse sources.

Identification of streptomycetes from Improving current Streptomyces taxonomy Chinese and Moroccan soil and description of new taxa

Collecting all Type strains

screening phylogeny classification Rep- Protein 16S-ITS PCR profiling RFLP Collection bioactive isolates selection

screening DNA-DNA hybridizations

application

Chapter 7. Identification of isolates Chapter 4. Detection and reclassification of synonymous names Description of new Streptomyces species Chapter 5. Intraspecies genotypic relatedness and correlation with phenotypic traits

Chapter 6. Phylogenetic grouping using 16S-ITS RFLP fingerprinting as a Fig. 13: Flowchart valuable alternative for 16S rRNA gene sequencing 88

PART 2

Experimental work

90 89 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Detection and reclassifcation of synonymous species

92 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

4.1 The search for synonyms among streptomycetes using SDS- PAGE of whole-cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violatus

B. Lanoot, M. Vancanneyt, I. Cleenwerck, L. Wang, W. Li, Z. Liu & J. Swings

Redrafted from Int J Syst Evol Microbiol 52, 823-829 (2002)

------SUMMARY A collection of 93 Streptomyces reference strains were investigated using SDS-PAGE of whole-cell proteins. Computer assisted numerical analysis revealed 24 clusters encompassing strains with very similar protein profiles. Five of them grouped several type strains with visually identical patterns. DNA-DNA hybridizations revealed homology values higher than 70 % among these type strains. Following the current species concept we propose that Streptomyces varsoviensis LMG 19360T is considered as a subjective synonym of Streptomyces violatus LMG 19397T; Streptomyces albosporeus subsp. albosporeus LMG 19403T as a subjective synonym of Streptomyces aurantiacus LMG 19358T, Streptomyces aminophilus LMG 19319T as a subjective synonym of Streptomyces cacaoi subsp. cacaoi LMG 19320T; Streptomyces niveus LMG 19395T and Streptomyces spheroides LMG 19392T as subjective synonyms of Streptomyces caeruleus LMG 19399T. ------

INTRODUCTION

At the introduction of the genus in 1943 (Waksman et al., 1943) classifications were based solely on morphology and pigmentation. The lack of standardized procedures for describing streptomycetes together with the unavailability of type strains led to species which were poorly described. Moreover antibiotics producing streptomycetes were described based on trivial differences in morphology for patent purposes. This unsound classification system has been improved by ‘The International Streptomyces Project’ (ISP) in 1966. Standard procedures for describing streptomycetes were developed and applied on 463 Streptomyces and (former) Streptoverticillium species. Type strains became publicly available when they were deposited in four collections. No attempts were undertaken by the ISP to determine synonymies among morphologically defined species. With the introduction of the principles of numerical taxonomy by Locci et al. (1981) and Williams et al. (1983), another view on the taxonomy of this complex genus emerged. Numerical taxonomy of phenotypic features without heavy emphasis on morphology and pigmentation allowed a more reliable classification than those previously defined. The taxonomic study of Williams et al. (1983) is therefore considered as a milestone in Streptomyces systematics. An extensive species reduction was proposed and probability matrices were created in order to identify isolates (Williams et al., 1989). In 1991 Kämpfer et al. updated the classification system of Williams et al. (1983) by extending the numbers of phenotypic traits and by including more taxa. Streptomyces systematics is gradually evolving from a phenotypically dominated taxonomy to a more polyphasic taxonomy comprising chemotaxonomic and genomic information. The large number of validly described Streptomyces species (more than 500) remains a major obstacle in order to set up large fundamental taxonomic studies, including DNA-DNA hybridizations and to establish the link between phenotypic and genomic information. Furthermore, due to the lack of minimal standards for defining Streptomyces species (Manfio et al., 1994), the polyphasic approach remains a difficult task. The aim of this study is to screen for synonymous taxa in a representative subset of the genus Streptomyces using SDS-PAGE of whole-cell proteins and to compare the data with these of Williams et al. (1989) and Kämpfer et al. (1991). It has been shown that under highly standardized cultivation conditions, SDS-PAGE of

94 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

whole-cell proteins is an excellent and generally applicable method for defining relationships at the species level among large numbers of strains (Vandamme et al., 1996; Descheemaeker et al., 1994). Manchester et al. (1990) and Vancanneyt et al. (1996) have already demonstrated the usefulness of SDS-PAGE of whole-cell proteins in streptomycete and pseudomonads systematics respectively.

MATERIALS AND METHODS

Bacterial strains and cultivation conditions

The strains used in this study can be found in Fig. 14. All strains were grown on Bennett’s agar slopes (using 0·01 M phosphate buffer pH 7·0) and incubated for 24 h at 28 °C unless indicated otherwise. Mycelial suspensions were prepared in 2 ml 0·1 M phosphate buffer (pH 7·0) and added to 250 ml flasks containing 80 ml of Bennett’s broth (0·01 M phosphate buffer, pH 7·0). The flasks were shaken for 24 h at 28 °C unless indicated otherwise. The same cultivation method was used for preparing protein or DNA extracts. In order to determine the influence of the incubation time on the resolution of protein profiles, protein extracts were prepared from Streptomyces griseus subsp. griseus LMG 5974 and Streptomyces rimosus LMG 5984T after 18, 36 and 51 hours. Furthermore the influence of the cultivation method (agar versus broth) was investigated.

Fig. 14: Dendrogram derived from unweighted pair group average linkage of r values (expressed as percentage) of protein profiles. Clusters are delineated at a similarity level of 90 %.

96 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

SDS-PAGE of whole-cell proteins

Protein extracts were prepared according to the protocol of Manchester et al. (1990) with some modifications indicated above. SDS-PAGE of the whole-cell proteins, scanning and normalization of electrophoretic patterns, was performed as described by Pot et al. (1994) (Fig. 15). The similarity between all traces was calculated using the Pearson product moment correlation coefficient with the software package GelCompar version 4·2 (Applied Maths). Out of the obtained similarity matrix a dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) algorithm. ------

0 250 500 750 1000

OD0

B I O D B M

0 100 200 300 0400

Fig. 15: Principle of SDS-PAGE of whole-cell proteins Adapted from Pot B. (UGent) ------

DNA-DNA hybridizations

DNA was prepared of 250-500 mg cells using the method of Pitcher et al. (1989) supplemented with a lysozyme (Serva) step (10 mg ml-1 in 1x TE buffer). DNA was finally dissolved in 0·1 SSC. Only high molecular DNA was retained for further applications. Microplate hybridizations (Fig. 16) were performed using the microplate method described by Ezaki et al. (1989) using a model FL-2575 microplate reader

(Towa Scientific) for the fluorescence measurements and black Maxisorp FluoroNunc microplates (Nunc A/S). Biotinylated probe DNA was sheared by ultrasonication (30 s, duty cycle 50, output control 1) using a Branson Sonifier Model 250 equipped with microcup horn and then hybridized with single-stranded unlabeled DNA, non- covalently bound to microplate wells. The optimal temperature for hybridization was

52 °C which was calculated using the formula TOR = [0.51 x (G+C) + 47 °C] –36 °C, where 36 °C is the correction for the presence of 50 % formamide. Salmon sperm DNA was used as the negative control in all experiments.

98 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

------Principle of the microplate DNA-DNA hybridization method

Immobilization of reference DNA:

DNA suspended in PBS-Mg Wash once with PBS Incubate for 4 h at 30°C Dry the plate at 45°C

Labeling of probe DNA:

Extraction of free photobiotin Add photobiotin with butanol

Illuminate with 400 W lamp for 30 min

Actual hybridization:

Probe DNA is added Wash with SSC

Hybridization for 3 h (T dependent of %G+C)

Enzymatic development:

Streptavidin-beta-D-galactosidase is added Wash with SSC

Incubate for 10 min at 37°C Add 4-methylumbelliferyl- beta-D-galactoside

Fig. 16: Fluorescent microplate method of Ezaki et al. (1989) Figure adapted from Goris J. (UGent) ------

Determination of mol% G + C

The method of Tamaoka & Komagata (1984) was used. DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase (Sigma) followed by separation using reversed-phase High-Performance Liquid Chromatography (Waters).

RESULTS AND DISCUSSION

SDS-PAGE of whole-cell proteins

Before routine application of SDS-PAGE of whole-cell proteins the method was optimized using S. griseus subsp. griseus LMG 5974 and S. rimosus LMG 5984T. It was investigated to what extent factors like cultivation time and medium (agar vs broth), are influencing the resolution of protein profiles. Our results indicated that high-resolution and reproducible protein patterns were obtained only from cultures grown in liquid medium, whereas cultures grown on solid media yielded a high background (due to proteinase activity) and consequently gave a lower resolution. It was also demonstrated that the growth phase of cells in liquid medium should be preferably within the log phase what means approximately 36 hours for fast growing streptomycetes. Protein patterns of strains harvested at the early stationary phase (51 hours), analogous to the conditions used in the study of Manchester et al. (1990), had a clearly lower resolution. Duplicate protein extracts were prepared in order to verify the reproducibility of the growth conditions and preparation of extracts. A correlation value above 92 % was observed between duplicate protein patterns loaded on separate gels. Using the above-mentioned growth conditions, protein patterns were generated from 93 Streptomyces strains (Fig. 14) belonging to different phenotypically defined clusters. After processing of the data a cluster analysis was performed (Fig. 14). A 90 % similarity level was used as guide line to delineate 24 clusters (Fig. 14) encompassing strains with very similar protein profiles. A correlation level (r) of 0·90 or higher in protein patterns of bacterial strains has often been used as an indication for possible relatedness at the species level (Kersters, 1985; Manchester et al., 1990).

DNA-DNA hybridization experiments

In the present study, delineated clusters do not contain strains of a single species, but several type strains of different validly described Streptomyces species. Five clusters (1, 5, 8, 12 and 19; Fig. 17) contain type strains with identical profiles

100 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

(only small differences in intensity of bands). Other type strains grouping in the respective clusters and all members of the other clusters show highly similar, but not identical patterns (minor differences in presence or absence of bands). DNA-DNA hybridization experiments were performed in order to investigate whether synonymous names may be found among the species in these clusters. ------

LMG 19358T LMG 19403T LMG 19320T LMG 19319T LMG 19393T LMG 19335T LMG 19360T LMG 19397T LMG 19399T LMG 19395T LMG 19392T LMG 19301T LMG 19327T LMG 19302T

Fig. 17: All strains found in this study possessing visually identical protein profiles, except for cluster 22, encompassing strains with similar protein profiles. A correlation is made with the numerical phenotypic studies of Williams et al. (1989) and Kämpfer et al. (1991). MWM (Molecular Weight Marker): from left to right: beta-galactosidase (116000), bovine albumin (66000), egg albumin (45000), glyceraldehyde-3-phosphate dehydrogenase (36000), carbonic anhydrase (29000), trypsinogen (24000), trypsin inhibitor (21100) and lysozyme (14200) ------

The DNA-DNA hybridization results among strains of the five selected clusters, sharing identical patterns, is presented in Table 8. Within cluster 1, Streptomyces albosporeus subsp. albosporeus LMG 19403T and Streptomyces aurantiacus LMG 19358T share a DNA homology of 74 %. Within cluster 5, Streptomyces cacaoi subsp. cacaoi LMG 19320T and Streptomyces aminophilus LMG 19319T are 100 %

101

related. Within cluster 8, Streptomyces endus LMG 19393T shows a relatedness of 92 % (data from Labeda et al., 1991) with Streptomyces hygroscopicus subsp. hygroscopicus LMG 19335T. Within cluster 12, Streptomyces varsoviensis LMG 19360T and Streptomyces violatus LMG 19397T show a DNA relatedness of 90 %. Within cluster 19, Streptomyces spheroides LMG 19392T shares a DNA relatedness of 77 % and 84 %, respectively, with Streptomyces niveus LMG 19395T and Streptomyces caeruleus LMG 19399T. S. caeruleus LMG 19399T is highly related to S. niveus LMG 19395T as they share a DNA homology of 100 %. These data confirm that streptomycetes possessing visually identical protein patterns are genomically more than 70 % related and represent the same genomic species. In a second step it was investigated what level of DNA homology is found among the strains showing minor differences in protein profiles (r > 0·90, but differentiated by presence or absence of a few bands). Three strains belonging to cluster 22 (r ≥ 0·93), i.e. Streptomyces anulatus LMG 19301T, Streptomyces griseus subsp. griseus LMG 19302T and Streptomyces microflavus LMG 19327T, were studied. As presented in Table 8, S. anulatus LMG 19301T is sharing a genomic relatedness of respectively 61 % and 45 % to S. griseus subsp. griseus LMG 19302T and S. microflavus LMG 19327T. S. griseus subsp. griseus LMG 19302T and S. microflavus LMG 19327T show a genomic relatedness of 43 %. These intermediate DNA homology values confirm that strains possessing highly similar protein patterns but showing minor qualitative differences, may share DNA homologies below 70 % and may be considered as distinct taxa. Numerical analysis of SDS-PAGE patterns of soluble proteins seems to be a perfect method for screening for highly related strains. Visual identity is indispensable if relationship at the species level is needed.

102 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

DNA relatedness (%) to: SDS-PAGE Name as cluster received / Strain 1 2 3 4 5 6 7 8 9 10 11 12

1 1 100 ______

2 74(7) 100 ______5 3 _ _ 100 ______4 _ _ 100(6) 100 ______12 5 _ _ _ _ 100 ______6 _ _ _ _ 90(9) 100 ______19 7 ______100 _ _ _ _ _ 8 ______77(4) 100 _ _ _ _ 9 ______85(2) 100(5) 100 _ _ _ 22 10 ______100 _ _ 11 ______43(2) 100 _ 12 ______61(6) 45(12) 100

Table 8: DNA hybridization between several Streptomyces taxa. 1, S. albosporeus LMG 19403T; 2, S. aurantiacus LMG 19358T ; 3, S. cacaoi cacaoi LMG 19320T ; 4, S. aminophilus LMG 19319T ; 5, S. violaceus LMG 19360T ; 6. S. violatus LMG 19397T ; 7, S. spheroides LMG 193952T ; 8, S. niveus LMG 19395T ; 9, S. caeruleus LMG 19399T ; 10, S. griseus griseus LMG 19302T ; 11, S. microflavus LMG 19327T; 12, S. anulatus LMG 19301T. Values are means ± SD; -, not determined.

103

A good correlation exists between SDS-PAGE of whole-cell proteins data of the present study and published DNA hybridization data (e.g. Labeda et al., 1991; Kim et al., 1999). In the study of Labeda et al. (1991) it has been proposed to consider S. endus as a subjective synonym of S. hygroscopicus. Both type strains have identical protein profiles (cluster 8; Fig. 17) confirming their conclusions. In the same study it was observed that the phenotypically similar species Streptomyces violaceusniger and S. hygroscopicus show an intermediate DNA homology level of 42 %. In our study both strains are sharing highly similar (r = 0·89) but not identical protein profiles. Kim et al. (1999) recently clarified the taxonomic status of thermophilic streptomycetes. It was for instance shown that Streptomyces thermoviolaceus and Streptomyces thermovulgaris are distinct taxa as they share a low DNA homology value of 10 %. This low value is confirmed by different protein patterns (r = 0·83).

Comparison with phenotypic data

A low correlation is observed between the protein profiles and the numerical phenotypic data provided by Williams et al. (1989) and Kämpfer et al. (1991). We expected that taxa sharing high DNA-DNA homology values would group within the same phenotypic cluster. Only for the type strains of S. aminophilus LMG 19319T and S. cacaoi subsp. cacaoi LMG 19320T the observed high relatedness (100 %, Table 8) is in correlation with their phenotypic cluster assignment (Fig. 17) as both strains are classified in speciesgroup Cat. I, sp. 9 according to Williams et al. (1989) and in cluster 31 according to Kämpfer et al. (1991). A partial correlation with former studies was observed in the following case: S. hygroscopicus subsp. hygroscopicus LMG 19335T and S. endus LMG 19393T (DNA homology of 92 %) are both grouped in speciesgroup Cat. I, sp. 16 by Williams et al. (1989), but are belonging to cluster 56 and 41, respectively, according to Kämpfer et al. (1991). S. niveus LMG 19395T and S. spheroides LMG 19392T (DNA homology of 77 %) are belonging to the speciesgroup Cat. I, sp. 2 according to Williams et al. (1989) but are classified in cluster 43 and 40, respectively, according to Kämpfer et al. (1991). S. caeruleus LMG 19399T, which is belonging to the same DNA homology group (85 % and 100 % DNA homology respectively to S. spheroides LMG 19392T and S. niveus LMG 19395T), is grouped in Cat. IV, sp. 7 by Williams et al. (1989) and cluster 58 by Kämpfer et al.

104 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

(1991). In two other cases, no correlation existed between our data and those of other authors i.e. S. varsoviensis LMG 19360T and S. violatus LMG 19397T (sharing 90 % DNA homology) are classified in Cat. III, sp. 3 and Cat. I, sp. 11 according to Williams et al. (1989) and cluster 37 and 50 according to Kämpfer et al. (1991). S. aurantiacus LMG 19358T and S. albosporeus subsp. albosporeus LMG 19403T (sharing a DNA homology of 74 %), are classified in Cat. II, sp. 13 and Cat. IV, sp. 1 according to Williams et al. (1989) and in cluster 12 and 63 by Kämpfer et al. (1991). These results clearly confirm that the phenotypic grouping provided by Williams et al. (1989) and Kämpfer et al. (1991) do not always reflect the genomic relationships. In the genus Streptomyces, (conventional) phenotypic data still dominate the classification system. In other genera containing less species, this phenotypic classification system (i.e. genus Helicobacter, Dewhirst et al., 2000) has already gradually evolved into a polyphasic approach (in which genomic data are most often used as a basis for delineation of taxa). It is logical that we should pursue an analogous polyphasic taxonomy in the genus Streptomyces, and that it is no longer acceptable to use different criteria for speciation in different groups of bacteria. Genotypy should preferably precede phenotypy. Only when genome based groups are defined the search for phenotypic traits can start and may be successful or not. From our study, it is clear that SDS-PAGE of whole-cell proteins, applied under highly standardized conditions, is a valuable phenotypic tool for screening for synonyms within the genus Streptomyces provided that DNA-DNA hybridization experiments are performed to confirm genomic relatedness at the species level. Based on the data from this paper we propose the following synonyms in the genus Streptomyces: S. varsoviensis LMG 19360T is a subjective synonym of S. violatus LMG 19397T; S. albosporeus subsp. albosporeus LMG 19403T is a subjective synonym of S. aurantiacus LMG 19358T, S. aminophilus LMG 19319T is a subjective synonym of S. cacaoi subsp. cacaoi LMG 19320T; S. niveus LMG 19395T and S. spheroides LMG 19392T are subjective synonyms of S. caeruleus LMG 19399T. Emended descriptions are given below and are based on the species descriptions as given by Williams et al. (1989) and the original descriptions.

105

Emended description of Streptomyces cacaoi subsp. cacaoi. Waksman in Bunting 1932, 515-517; Waksman and Henrici 1948, 951AL.

Spore chains are Spirales; the spore surface is smooth. The aerial mass is in the white-yellow color series. The reverse side of a colony has no distinctive pigments. Melanin pigment is not formed and soluble pigments are not produced. The following carbohydrates are utilized for growth: D-glucose, L-arabinose, D-xylose, D-mannitol and D-fructose. No growth or only trace of growth on i-inositol and rhamnose. The G + C content of the DNA is 73 mol%. The type strain is IMRU 3082 (LMG 19320). Subjective synonym: Streptomyces aminophilus IJSB 30:371AL ATCC 14961 (LMG 19319)

Emended description of Streptomyces caeruleus (based on the original descriptions of S. caeruleus and S. spheroides) (Baldacci 1944) Pridham, Hesseltine and Benedict 1958, 60AL (Actinomyces caeruleus Baldacci 1944, 180)

Spore chains are Rectiflexibiles or Spirales. The spore surface is smooth. The aerial mass is yellow or gray. The reverse side of a colony is dark blue-violet to black (for S. caeruleus) and is pale yellow to light yellowish brown (for S. spheroides). Melanoid pigments are not formed. Bluish-black to greenish-black diffusible pigments are produced on some media (for S. caeruleus). Carbon utilization: for S. caeruleus: only on D-glucose. Very poor growth is observed on Pridham and Gottlieb carbon-utilization medium using other carbon sources. For S. spheroides: utilization of D-glucose, L-arabinose, sucrose, D-xylose, D-mannitol, D-fructose and rhamnose. Growth is possible on i-inositol and raffinose. The G + C content of the DNA is 71 mol%. The type strain is IPV 930 (ISP 5103, LMG 19399). Subjective synonyms: Streptomyces niveus Smith, Dietz, Sokolski and Savage 1956, IJSB.30:394AL NRRL 2466 (LMG 19395) and Streptomyces spheroides Wallick et al. 1956, 30:401 AL DSM 40292 (LMG 19392)

106 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Emended description of Streptomyces aurantiacus (Rossi-Doria 1891), Waksman in Waksman and Lechevalier 1953, 53AL Streptothrix aurantiaca Rossi- Doria 1891, 417. Spore chains are Spirales, the spore surface is smooth. Aerial mycelium (if developed) is belonging to the red color series. The reverse side of a colony is yellow, yellow-brown, red, red-brown. Melanoid pigments are not formed and soluble pigments are not produced. The following carbohydrates are utilized for growth: D- glucose, L-arabinose, rhamnose, D-xylose, raffinose, sucrose (only for S. aurantiacus), i-inositol (only for S. aurantiacus), D-galactose (only for S. albosporeus subsp. albosporeus), meso-inositol (only for S. albosporeus subsp. albosporeus). The G + C content of the DNA is 70 mol%. The type strain is INMI 1373 (LMG 19358). Subjective synonym: Streptomyces albosporeus subsp. albosporeus IJSB 30:370AL DSM 40795 (LMG 19403)

Emended description of Streptomyces violatus. Actinomyces violatus Artamonova and Krasil’nikov (1960); Streptomyces violatus (Artamonova and Krasil’nikov) Pridham 1970, 30(147).

Spore chains are Spirales, Retinaculiaperti or Rectiflexibiles; the spore surface is spiny (for S. violatus) or smooth (for S. varsoviensis). The aerial mass is in the white, yellow or red color series. The reverse side of a colony is yellow, yellow-brown to reddish-violet. Melanoid pigments are formed (sometimes slowly) and red to violet pigments are produced (last feature only seen for S. violatus). The following carbohydrates are utilized for growth: D-glucose and D-mannitol. The following carbohydrates are only used by S. violatus for growth: D-fructose, L-arabinose, D- xylose, rhamnose, sucrose, raffinose, i-inositol and D-mannitol. The G + C content of the DNA is 71 mol%. The type strain is INMI 1205 (LMG 19397). Subjective synonym: Streptomyces varsoviensis IJSB 30:403AL ATCC 14631c (LMG 19360).

107

Acknowledgements. This study was performed in the frame of the Belgian-Chinese program: ‘Identification and classification of actinomycetes, specifically bioactive Streptomyces strains isolated from Chinese soil’, supported by OSTC Belgium (BL/02/C10) and MOST/NSFC P.R. China. We thank Dirk Dewettinck (UGent) for excellent technical assistance in performing SDS-PAGE of whole-cell proteins. The authors are also indebted to Prof. R. M. Kroppenstedt (DSMZ, Germany) and Dr. T. Kudo (JCM, Japan) for providing cultures.

108 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

B. Lanoot, M Vancanneyt, P. Dawyndt, M. Cnockaert, J. Zhang, Y. Huang, Z. Liu & J. Swings

Redrafted from System Appl Microbiol 27, 84-92 (2004)

------SUMMARY The type strains of 451 validly described Streptomyces species were screened using the BOX-PCR fingerprint technique. Of the different primers tested, the BOX primer yielded the most robust and reproducible band patterns. The majority of the species (350) had a unique pattern each. A total number of 30 clusters were delineated comprising species with nearly identical patterns. Four of these clusters grouped synonymous Streptomyces species known from previous studies (Labeda, 1998; Labeda & Lyons, 1991b; Lanoot et al., 2002). A high correlation exists between BOX-PCR fingerprinting and DNA-DNA homology data. In the present study, the latter was confirmed for 7 additional BOX clusters and led to the following emended descriptions: S. cochleatus is a subjective synonym of S. cinereorectus, S. roseoflavus of S. fradiae, S. roseodiastaticus of S. tricolor, S. roseosporus of S. filamentosus, S. distallicus of S. colombiensis, S. arabicus of S. vinaceus and S. phaeoviridis of S. phaeopurpureus. Although not yet confirmed by DNA-DNA pairing studies, rep-PCR data suggested the presence of at least 33 additional junior synonyms. ------

109

INTRODUCTION

Since the introduction of the genus Streptomyces by HENRICI and WAKSMAN in 1943, morphological and phenotypic properties have been dominating classification systems (Anderson & Wellington, 2001). Because phenotypic properties often provide insufficient taxonomic resolution at species level, synonymies among species names exist (Labeda & Lyons, 1991a-b; Labeda, 1998; Lanoot et al., 2002). Classification of streptomycetes has become more transparent since the application of genotypic approaches but practically the high number of validly described species in the genus (over 500) remains the main obstacle in Streptomyces taxonomy. It is also striking that new species descriptions (Li et al., 2002; Petrosyan et al., 2003) still do not meet the criteria that are generally accepted in bacterial taxonomy cfr. Stackebrandt et al. (1994) and Wayne et al. (1987). Moreover, the phylogenetic position is determined upon comparison with an incomplete 16S rDNA sequence database as complete sequences are available only for 25 % of the validly described species. Considering the cut-off of 97 % sequence similarity as a level for possible species relatedness, within Streptomyces often too large numbers of related species are found for subsequent DNA-DNA pairing studies. As a result of this, not all nearest phylogenetic neighbours are included for further analyses. For the present study we have selected the fast and inexpensive BOX-PCR technique (Versalovic et al., 1991) (Fig. 17), to construct a genotypic database comprising fingerprints of 473 Streptomyces’ type strains. BOX-PCR is based on the observation that outwardly facing oligonucleotide primers, complementary to interspersed repeated sequences, enable the amplification of differently sized DNA fragments, consisting of sequences lying in between these elements. Rep-PCR fingerprinting has been applied in numerous taxonomic studies on plant-associated, environmental, medical and food-associated bacteria (De Bruijn, 1992; Georghiou et al., 1994; Judd et al., 1993; Louws et al., 1994; Rademaker et al., 1998). An overview on its optimisation, advantages and usefulness for speciation of streptomycetes is presented. We investigated how similar Streptomyces’ genomes are and if highly related species are still present. DNA-DNA pairing studies were performed to validate the BOX-PCR findings for Streptomyces. In other studies a high correlation has

110 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

already been found between DNA-DNA hybridisation and rep-PCR data (Nick et al., 1999; Rademaker et al., 2000; Sawabe et al., 2002).

------

PCR-reaction: Gel electroforesis: MW ABMW

Microbe A

PCR-reaction: Microbe B

Fig. 18: Principle of the rep-PCR fingerprint technique (Versalovic et al., 1991) Adapted from Heyrman J. (UGent) ------

MATERIALS AND METHODS

Bacterial strains and cultivation conditions

The strains used in this study are listed in Table 9 and 10. All strains were grown on Bennett’s agar (0.1 % yeast extract, 0.1 % beef extract, 0.2 % casein hydrolysate, 1 % glucose, 2 % agar; final pH 7.3) and incubated at their appropriate temperature as found in literature. Mycelial suspensions were prepared in 2 ml 0·1 M phosphate buffer (0.22 % Na2HPO4.12H2O, 0.062 % NaH2PO4.1H2O; final pH 7·0) and added to 250 ml flasks containing 80 ml of Bennett’s broth for small scale DNA preparation. For large-scale DNA preparations 160 ml of medium was added to 500 ml flasks. The flasks were shaken for 24 h at the appropriate temperature.

111

BOX-PCR fingerprinting

For DNA extraction the protocol of Gevers et al., 2001, using a combination of glass beads and enzymes, was applied with the following modifications (1) 100 – 200 mg of cells were taken for extraction of DNA (2) cells were frozen for 24 h instead of 1 h (3) incubation time for the cell lysis was depending on the strain and was between 60 and 90 min. (4) extracted DNA was finally resuspended in 200 µl 1x TE buffer (1 mM EDTA, 10 mM Tris/HCl). Only DNA’s with OD 260/280 ≥ 1.70 were used for further analysis. For optimisation several primers were evaluated using different PCR protocols i.e. BOXA1R (Rademaker et al., 2000; Sadowsky et al., 1996; Versalovic et al., 1991), (GTG)5 (Sadowsky et al., 1996), ERIC1R & ERIC2 (Versalovic et al., 1991) and REP1R-I & REP2-I (Versalovic et al., 1991). Only BOXA1R primer was retained for routine analysis using the protocol of Rademaker et al., 2000 with modifications. One PCR reaction (reaction volume 25 µl) contained 5 µl 5x Gitschier buffer (16.6 ml 1 M (NH4)2SO4, 67 ml 1 M Tris-HCl pH 8.8, 6.7 ml 1 M MgCl2, 1.3 ml of a 1/100 dilution of 0.5 M EDTA pH 8.8, 2.08 ml 14.4 M β-mercaptoethanol, add double distilled water till 200 ml), 0.4 µl BSA (10 mg ml-1, BioLabs), 1.25 µl dNTP (2 mM each; Pharmacia), 2.5 µl DMSO 100 % (Sigma), 1 µl primer (BOXA1R, 0.3 µg µl- 1, 5’- CTACGGCAAGGCGACGCTGACG-3’, Pharmacia), 2 U Red Goldstar Taq polymerase (EuroGentec) and 50 ng purified DNA. PCR program consisted of the following steps run on a Perkin Elmer 9600 thermocycler: 95 °C 7 min, 30 cycles of 90 °C 30 s – 53° C 1 min. – 65 °C 8 min, 65 °C 16 min. After PCR 5 µl of loading dye (6 x) was added to the samples and 9 µl of this mixture was loaded on a 2 % agarose gel (LSI agar, 15 x 15 cm). A 5.5 µl aliquot of a molecular size marker (100 bp and 500 bp, Bio-Rad Laboratories) was loaded for every 5 samples. PCR products were separated on 15 x 15 cm gels using 130 V (cst), 400 mA for 220 min. in freshly prepared 1x TBE (1.08 % Tris, 0.55 % boric acid, 4 ml 0.5 M EDTA pH 8.0) using a Bio-Rad PowerPac 300 power supply. The gels were stained for 20 min. in a 1x TBE bath supplemented with EtBr (1 mg l-1). Destaining was done for 1 min. in Milli-Q water. A photograph of the gel was stored on disk as TIFF file through a CCD coupled camera by using the ColorVision software. Gels were imported into the software package BioNumerics version 2.5 (Applied Maths, Belgium). Similarity matrices of densitometric curves of the gel tracks were calculated using the Pearson

112 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Product Moment Correlation Coefficient followed by tree construction using UPGMA algorithm. Settings: optimization 0.49 %, the first 5.3 % and last 16.1 % of data points of each lane were excluded from the analysis.

DNA-DNA pairing studies

Extraction of DNA was performed using the same protocol as given above for BOX-PCR analysis but slightly modified for application on large scale (x 10). Vortexing with beads of the SDS (Sodium dodecyl sulphate, 20 %) treated cells was done for 30 s. After incubation at 37 °C and 65 °C respectively TEK 1 x (1 mM EDTA, 10 mM Tris/HCl pH 8.0) was added to a final volume of 20 ml followed by the addition of 6 ml 5 M NaCl and gentle shaking. Subsequent chloroform/isoamylalcohol (24:1 v/v) aided extraction and precipitation of the DNA was performed as described by Pitcher et al., 1989. Resuspended DNA was deproteinized upon treatment with proteinase K (150 µl of a 10 mg ml-1 solution on 3 ml DNA solution, 55 °C for 20 min.) followed by RNAse treatment. Purified DNA was finally resuspended in 0.3 à 0.5 ml

0.1 SSC (0.0876 % NaCl, 0.03152 % C6H8O7.6H2O, 0.018 % NaOH in dd water; final pH 7.0) stored at 4 °C. Determination of % G + C content was determined using the protocol of Tamaoka et al., 1984. DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase (Sigma) followed by separation using reversed-phase high-performance liquid chromatography (Waters). DNA-DNA hybridizations were performed using the fluorometric method (Ezaki et al., 1989) using a model FL-2575 microplate reader (Towa Scientific) for the fluorescence measurements and white Maxisorp FluoroNunc microplates (Nunc A/S). Biotinylated probe DNA was sheared by ultrasonication (30 s, duty cycle 50, output control 1) using a Branson Sonifier Model 250 equipped with microcup horn and then hybridized with single-stranded unlabeled DNA, non-covalently bound to microplate wells. Hybridizations were performed at 52 °C for 3 h in a hybridization solution containing 50 % formamide. Salmon sperm DNA was used as the negative control in all experiments. DNA-DNA homology data generated in this study are presented as mean of reciprocal values.

113

RESULTS AND DISCUSSION

Selection of primers and reproducibility

Tested on a set of randomly chosen strains, (GTG)5, REP and ERIC primer pairs yielded low resolution patterns with a lot of smear, only containing a few distinct bands in the range 0.1-0.7 kb. No further attempts were undertaken to improve the

REP-, ERIC- or (GTG)5-PCR fingerprinting. The BOXA1R primer resulted in high resolution fingerprints with the fragment sizes ranging from less than 0.2 kb to approximately 2.0 kb. Less visible fragments above 2.0 kb were occasionally found. For strain typing of Streptomyces ipomoeae isolates Clark et al., 1998 generated high-resolution BOX, REP and ERIC primed fingerprints. They concluded that BOX primed fingerprints were taxonomically the least discriminating of the three primers tested. According to these authors the BOX primer is thought to target conserved regions in the genome. We have decided to apply the BOX primer for screening of 473 Streptomyces’ type strain genomes. For BOX-PCR fingerprints intergel reproducibility ranged between 87 and 94 % due to differences in background intensities caused by the ethidium bromide staining.

Numerical analysis

High resolution fingerprints were obtained for all strains mentioned in Table 9 and 10. The complete dendrogram is given in Annex IV. Numerical analysis of the data revealed three groups of fingerprints: (1) unique patterns among 350 strains (Table 9, profiles not shown), (2) a total number of 30 clusters, representing 75 strains, possessing nearly identical BOX patterns (clusters 1 to 30, Table 10, profiles shown in Fig. 19) and (3) a number of strains (48, footnote Table 10) linked to and showing a significant similarity to one of the above-mentioned clusters (cluster 4, 9, 18, 19, 21 and 22) but remaining unclustered because of the differences in one or more bands (profiles not shown) and their uncertain affiliation.

114 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

S. abikoensis LMG 20386T, S. aburaviensis LMG 19305T, S. achromogenes s. achromogenes LMG 20387T, S. achromogenes s. rubradiris LMG 20388T, S.acidiscabies LMG 19856T, S. acrimycini LMG 21798T, S. aculeolatus LMG 19906T, S. afghaniensis LMG 20390T, S. alanosinicus LMG 20391T, S. albaduncus LMG 20392T, S. albireticuli LMG 20393T, S. albofaciens LMG 20394T, S. alboflavus LMG 21038T, S. albogriseolus LMG 20395T, S. albolongus LMG 20396, S. alboniger LMG 20397T, S. albospinus LMG 20398T, S. albosporeus s. albosporeus LMG 19403T, S. albosporeus s. labilomyceticus LMG 20400T, S. alboverticillatus LMG 20401T, S. alboviridis LMG 20403T, S. albulus LMG 20404T, S. albus s. pathocidicus LMG 20406T, S. althioticus LMG 20408T, S. amakusaensis LMG 19350T, S. ambofaciens LMG 20409T, S. anandii LMG 8600T, S. antibioticus LMG 20412T, S. ardus LMG 20415T, S. arenae LMG 20416T, S. argenteolus LMG 5967T, S. armeniacus LMG 20418T, S. atratus LMG 20420T, S. atroaurantiacus LMG 20421T, S. atrovirens LMG 20422T, S. auranticus LMG 19358T, S. aurantiogriseus LMG 19298T, S. aureocirculatus LMG 21794T, S. aureoversilis LMG 20425T, S. aureoverticillatus LMG 20426T, S. avidinii LMG 20428T, S. azaticus LMG 20429T, S. azureus LMG 20430T, S. bacillaris LMG 8585T, S. bikiniensis LMG 19367T, S. biverticillatus LMG 20433T, S. blastmyceticus LMG 20434T, S. bluensis LMG 5969T, S. bobili LMG 20436T, S. bottropensis LMG 20437T, S. brasiliensis LMG 20438T, S. bungoensis LMG 20439T, S. cacaoi s. asoensis LMG 20440T, S. caelestis LMG 20441T, S. californicus LMG 19309T, S. canarius LMG 20443T, S. canus LMG 19329T, S. caniferus LMG 20446T, S. capillispiralis LMG 19909T*, S. carpaticus LMG 20448T,S. carpinensis LMG 19913T, S. catenulae LMG 20449T, S. cavourensis s. cavourensis LMG 20450T, S. cavourensis s.washingtonensis LMG 20451T, S. cellostaticus LMG 20452T, S. celluloflavus LMG 21796T, S. cellulosae LMG 19315T, S. chartreusis LMG 20455T, S. chattanoogensis LMG 19339T, S. chrestomyceticus LMG 20457T, S. chromofuscus LMG 19317T, S. chryseus LMG 20458T, S. chrysomallus s. fumigatus LMG 21793T, S. cinereoruber s. fructofermentans LMG 20463T, S. cinereospinus LMG 20464T, S. cinereus LMG 21310T, S. cinerochromogenes LMG 20466T, S. cinnabarinus LMG 20467T, S. cinnamonensis LMG 20468T, S. cirratus LMG 20473T, S. clavuligerus LMG 20477T, S. coelicoflavus LMG 20480T, S. coelicolor LMG 8571T, S. coeruleofuscus LMG 20482T, S. coeruleoprunus LMG 20483T, S. coerulescens LMG 8590T, S. collinus LMG 20486T, S. cremeus LMG 20489T, S. crystallinus LMG 20490T, S. curacoi 20491T, S. cuspidosporus LMG 20492T, S. cyaneofuscatus LMG 20493T, S. cyaneus LMG 20494T, S. cyanoalbus LMG 19343T, S. cystargineus LMG 20495T, S. diastaticus s. ardesiacus LMG 20497T,S. diastatochromogenes LMG 20498T, S. djakartensis LMG 21795T, S. durhamensis LMG 20501T, S. echinatus LMG 5972T, S. ederensis LMG 20504T, S. ehimensis LMG 20505T, S. erumpens LMG 20507T, S. erythraeus LMG 20508T, S. eurocidicus LMG 20509T, S. eurythermus LMG 20510T, S. exfoliatus LMG 19307T, S. fervens s.melrosporus LMG 19897T, S. filipinensis LMG 19333T, S. fimbriatus LMG 20513T, S. finlayi LMG 19373T, S. flaveolus LMG 19328T, S. flavidofuscus LMG 20515T, S. flavidovirens LMG 19387T, S. flavofungini LMG 21799T, S. flavotricini LMG 19880T*, S. flavovariabilis LMG 19905T, S. flocculus LMG 19889T, S. floridae LMG 19899T, S. fragilis LMG 19874T, S. fulvissimus LMG 19310T, S. fulvorobeus LMG 19901T, S. fumanus LMG 19882T, S. fumigatiscleroticus LMG 19911T, S. galbus LMG 19879T, S. galilaeus LMG 21790T, S. gancidicus LMG 19898T, S. gardneri LMG 19876T, S. gelaticus LMG 19376T, S. geysiriensis LMG 19893T, S. glaucescens LMG 19330T, S. glaucosporus LMG 19907T, S. glaucus LMG 19902T, S. globisporus s.globisporus LMG 8578T, S. globosus LMG 19896T*, S. glomeratus LMG 19903T, ,S. gobitricini LMG 19910T, S. graminofaciens LMG 19892T, S. griseinus LMG 19875T, S. griseoaurantiacus LMG 21045T, S. griseobrunneus LMG 19877T, S. griseocarneus LMG 19383T, S. griseochromogenes LMG 19891T, S. griseoflavus LMG 19344T, S. griseofuscus LMG 19885T, S. griseoloalbus LMG 21308T, S. griseolosporeus LMG 19962T*, S. griseolus LMG 19878T, S. griseoplanus LMG 19923T, S. griseoruber LMG 19325T, S. griseorubens LMG 19931T, S. griseorubiginosus LMG 19941T, S. griseosporeus LMG 19947T, S. griseoverticillatus LMG 19944T, S. griseus s. solvifaciens LMG 19952T, S. hachijoensis LMG 19928T, S. hawaiiensis LMG 5975T, S. heliomycini LMG 19960T, S. helvaticus LMG 19940T, S. herbaricolor LMG 19929T, S. hiroshimensis LMG 19924T, S. hirsutus LMG 19927T, S. humidus LMG 19936T, S. hydrogenans LMG 19948T, S. hygroscopicus s.angustmyceticus LMG 19958T, S. hygroscopicus s.decoyicus LMG 19954T, S. hygroscopicus s.glebosus LMG 19950T, S. hygroscopicus s.ossamyceticus LMG 19951T, S. iakyrus LMG 19942T, S. indiaensis LMG 19961T, S. indigoferus LMG 19930T, S. intermedius LMG 19304T, S. inusitatus LMG 19955T, S. ipomoeae LMG 20520T, S. kanamyceticus LMG 19351T, S. kashmirensis LMG 19937T, S. kasugaensis LMG 19949T, S. katrae LMG 19945T, S. kifunensis LMG 19957T, S. kunmingensis LMG 20521T, S. kurssanovii LMG 19933T, S. lanatus LMG 19380T, S. lateritius LMG 19372T, S. laurentii LMG 19959T, S. lavendofoliae LMG 19935T, S. lavendulae i.e. subspecies lavendulae LMG 19925T* and subspecies grasserius LMG 19938T*, S. lavenduligriseus LMG 19943T, S. lavendulocolor LMG 19934T, S. levis LMG 20090T, S. libani s. rufus LMG 20087T, S. lienomycini LMG 20091T, S. lilacinus LMG 20059T, S. lincolnensis LMG 20068T, S. lomondensis LMG 20088T, S. longisporoflavus LMG 19347T, S. longispororuber LMG 20082T, S. longisporus LMG 20053T, S. longwoodensis LMG 20096T, S. lucensis LMG 20065, S. luridus LMG 19365T, S. lusitanus LMG 20078T, S. luteofluorescens LMG 20522T, S. luteogriseus LMG 20073T, S. luteosporeus LMG 20085T, S. luteoverticillatus LMG 20045T, S. lydicus LMG 19331T, S. macrosporus LMG 20089T, S. malachitofuscus LMG 20067T, S. malaysiensis LMG 20099T, S. massasporeus LMG 19362T, S. matensis LMG 20055T, S. mauvecolor LMG 20100T, S. megasporus LMG 20092T, S. melanogenes LMG 20056T, S. melanosporofaciens LMG 20066T, S. michiganensis LMG 20042T, S. mirabilis LMG 20076T, S. misakiensis LMG 19369T, S. mobaraensis LMG 20086T, S. morookaensis LMG 20074T, S. murinus LMG 10475T, S. mutabilis LMG 20054T, S. mutomycini LMG 20098T, S. naganishii LMG 21042T, S. narbonensis LMG 20043T, S. nashvillensis LMG 20064T, S. netropsis LMG 5979T, S. neyagawaensis LMG 20080T, S. niger LMG 20101T, S. nitrosporeus LMG 20044T, S. niveoruber LMG 19379T, S. noboritoensis LMG 19337T, S. nodosus LMG 19430T, S. nogalater LMG 19338T, S. nojiriensis LMG 20094T, S. noursei LMG 5982T, S. novaecaesareae LMG 20069T, S. ochraceiscleroticus LMG 19349T, S. olivaceiscleroticus LMG 20081T, S. olivaceoviridis LMG 19324T, S. olivaceus LMG 19394T, S. olivochromogenes LMG 20071T, S. olivoreticuli s. cellulophilus LMG 20097T, S. olivoreticuli s. olivoreticuli LMG 20050T, S. olivoverticillatus LMG 20058T, S. omiyaensis LMG 20075T, S. orinoci LMG 20079T, S. pactum LMG 19357T, S. pallidus LMG 19396T, S. paracochleatus LMG 20095T, S. paradoxus LMG 20523T, S. parvisporogenes LMG 20072T, S. parvulus LMG 21789T, S. parvus LMG 20524T, S. peucetius LMG 20084T, S. phaeochromogenes LMG 19348T, S. phaeofaciens LMG 20070T, S. phosalacineus LMG 20102T, S. platensis LMG 20046T, S. plicatus LMG 20288T, S. polychromogenes LMG 20287T, S. poonensis LMG 19326T, S. prasinopilosus LMG 19345T, S. prasinosporus LMG 19346T, S. prunicolor LMG 19311T, S. pseudoechinosporeus LMG 21052T, S. pseudogriseolus LMG 20252T*, S. pseudovenezuelae LMG 20276T, S. pulveraceus LMG 20322T, S. puniceus LMG 20258T, S. purpeofuscus LMG 20283T, S. purpurascens LMG 20526T, S. purpureus LMG 19368T, S. purpurogeneiscleroticus LMG 20331T, S. racemochromogenes LMG 20273T, S. ramulosus LMG 19354T, S. rectiverticillatus LMG 20292T, S. rectiviolaceus LMG 20310T, S. regensis LMG 20300T, S. resistomycificus LMG 21797T, S. rimosus s. paromomycinus LMG 20308T, S. rimosus s. rimosus LMG 19352T, S. rishiriensis LMG 20297T, S. rochei LMG 19313T, S. roseofulvus LMG 20263T, S. roseolilacinus LMG 20264T, S. roseolus LMG 20265T, S. roseoverticillatus LMG 20255T, S. roseoviridis LMG 20266T, S. rubiginosus LMG 20268T, S. rubrogriseus LMG 20318T, S. rutgersensis s. castelarensis LMG 20304T, S. salmonis LMG 20306T, S. sapporonensis LMG 20324T, S. scabiei LMG 20323T, S. sclerotialus LMG 20528T, S. septatus LMG 8604T, S. setae LMG 20529T, S. showdoensis LMG 20298T, S. somaliensis LMG 21049T*, S. spectabilis LMG 5986Tt2, S. spheroides LMG 19392T, S. spinoverrucosus LMG 20321T, S. spiralis LMG 20332T, S. spiroverticillatus LMG 20254T, S. sporocinereus LMG 20311T, S. spororaveus LMG 20313T, S. subrutilus LMG 20294T, S. sulfonofaciens LMG 20325T, S. syringium LMG 20320T, S. tanashiensis LMG 20274T, S. tauricus LMG 20301T, S. tendae LMG 19314T, S. termitum LMG 20289T, S. thermoalcalitolerans LMG 19858T, S. thermocarboxydovorans LMG 19860T, S. thermocoprophilus LMG 19857T, S. thermodiastaticus LMG 20302T, S. thermolineatus LMG 20309T, S. thioluteus LMG 21037T, S. torulosus LMG 20305T, S. toxytricini LMG 20269T, S. tubercidicus LMG 19361T, S. tuirus LMG 20299T, S. variegatus LMG 20315T, S. varsoviensis LMG 20083T, S. vastus LMG 21043T, S. venezulae LMG 19308T, S. vinaceusdrappus LMG 20296T, S. violaceochromogenes LMG 20271T, S. violaceorubidus LMG 20319T, S. violaceusniger LMG 19336T, S. violascens LMG 20272T, S. violens LMG 20303T, S. virginiae LMG 20534T, S. viridiviolaceus LMG 20282T, S. viridobrunneus LMG 20317T, S. viridochromogenes LMG 20260T, S. viridodiastaticus LMG 20279T, S. viridoflavus LMG 20277T, S. vitaminophilus LMG 21051T, S. wedmorensis LMG 21050T, S. werraensis LMG 21047T, S. xanthochromogenes LMG 19366T, S. xanthocidicus LMG 19370T, S. xantholiticus LMG 19402T, S. xanthophaeus LMG 21039T, S. yerevanensis LMG 21053T, S. yokosukanensis LMG 21040T, S. zaomyceticus LMG 19853T.

Table 9: Strains possessing unique patterns

*: Weak patterns obtained

115

CLUSTERS DELINEATED REFERENCE NOMENCLATURE: SHOWING HIGH DNA HOMOLOGY PROPOSED / NAME

1 S. roseiscleroticus LMG 20284T, S. ruber LMG 20285T - S. ruber † 2 S. phaeopurpureus LMG 20051T, S. phaeoviridis LMG 20061T present study S. phaeopurpureus 3 S. flaviscleroticus LMG 19886T, S. minutiscleroticus LMG 20062T - S. flaviscleroticus † 4 S. anthocyanicus LMG 20411T, S. sannanensis LMG 20329T - S. anthocyanicus † 5 S. ciscaucasicus LMG 20474T, S. clavifer LMG 20476T - S. clavifer † 6 S. enissocaesilis LMG 20506T, S. sioyaensis LMG 20531T - S. sioyaensis † 7 S. thermogriseus LMG 20532T, S. thermovulgaris LMG 19342T - S. thermovulgaris † 8 S. aureorectus LMG 19908T, S. asterosporus LMG 20419T, S. calvus LMG 20442T, S. virens LMG 20316T - S. calvus † 9 S. flavovirens LMG 20516T, S. nigrifaciens LMG 20048T - S. flavovirens † 10 S. graminearus LMG 19904T, S. griseomycini LMG 19883T, S. griseostramineus LMG 19932T - S. griseomycini † 11 S. daghestanicus LMG 20496T, S. griseoviridis LMG 19321T Labeda, 1998 S. griseoviridis 12 S. griseus s. alpha LMG 19953 T, S. griseus s. cretosus LMG 19946T#, S. lipmanii LMG 20047T, S. microflavus LMG 19327T, S. willmorei LMG 21046T# - S. microflavus † 13 S. colombiensis LMG 20487T, S. distallicus LMG 20499T present study S. colombiensis 14 S. caereuleus LMG 19399T, S. niveus LMG 19395T Lanoot et al., 2002 S. caeruleus 15 S. umbrinus LMG 20280T, S. misionensis LMG 20063T - S. umbrinus † 16 S. albovinaceus LMG 20402T, S. mediolani LMG 20093T present study S. albovinaceus 17 S. aminophilus LMG 19319T, S. cacaoi s. cacaoi LMG 19320T Lanoot et al., 2002 S. cacaoi s. cacaoi 18 S. citreofluorescens LMG 20475T, S. chrysomallus s. chrysomallus LMG 20459T, S. fluorescens LMG 8579T, S. anulatus LMG 19301T - S. anulatus † 19 S. flavofuscus LMG 19900T, S. setonii LMG 20291T - S. setonii † 20 S. albus s. albus LMG 20405T, S. almquistii LMG 21307T, S. gibsonii LMG 19912T, S. rangoonensis LMG 20295T - S. albus s. albus † 21 S. roseodiastaticus LMG 20327T, S. tricolor LMG 20328T present study S. tricolor 22 S. canescens LMG 20445T, S. felleus LMG 20511T, S. limosus LMG 8570T, S. sampsonii LMG 8574T - S. sampsonii † 23 S. arabicus LMG 20414T, S. vinaceus LMG 20533T present study S. vinaceus 24 S. filamentosus LMG 20512T, S. roseosporus LMG 20262T present study S. filamentosus 25 S. erythrogriseus LMG 19406T, S. griseoincarnatus LMG 19316T, S. labedae LMG 19956T, S. variabilis LMG 20270T - S. variabilis † 26 S. chibaensis LMG 20456T, S. corchorusii LMG 20488T - S. chibaensis † 27 S. fradiae LMG 19371T, S. roseoflavus LMG 20535T present study S. fradiae 28 S. aureofaciens LMG 5968T, S. avellaneus LMG 20427T, S. psammoticus LMG 20525T - S. aureofaciens † 29 S. endus 19393T, S. hygroscopicus s. hygroscopicus LMG 19335T Labeda et al., 1999 S. hygroscopicus 30 S. cinereorectus LMG 20461T, S. cochleatus LMG 20478T present study S. cinereorectus

Table 10: Numerical analysis of BOX-PCR patterns of 473 Streptomyces type strains with delineation of 30 clusters containing strains with nearly identical patterns§.

§: strains with uncertain affiliation which show a significant similarity to one of the 30 delineated clusters (see above) or to each other:

S. coelescens LMG 20479T, S. humiferus LMG 20519T, S. litmocidini LMG 20052T, S. violaceolatus LMG 20293T and S. violaceoruber LMG 20256T (linked to cluster 4) ; S. flavogriseus LMG 19887T (linked to cluster 9) ; S. praecox LMG 20290T (linked to cluster 18) ; S. baarnensis LMG 20431T, S. fimicarius LMG 21044T (linked to cluster 19) ; S. rameus LMG 20326T (linked to cluster 21) ; S. albidoflavus LMG 21791T, S. champavatii LMG 20454T, S. globisporus s. caucasicus LMG 19895T and S. odorifer LMG 8572T (linked to cluster 22) ; grouping together: S. bellus LMG 19401T and S. coeruleorubidus LMG 20484T ; S. griseoluteus LMG 19356T and S. recifensis LMG 20261T ; S. flavoviridis LMG 19881T and S. pilosus LMG 20049T ; S. goshikiensis LMG 19884T and S. sporoverrucosus LMG 20314T ; S. cinereoruber s. cinereoruber LMG 20462T and S. violaceorectus LMG 20281T ; S. badius LMG 19353T and S. sindenensis LMG 21041T ; S. antimycoticus LMG 20413T and S. sporoclivatus LMG 20312T ; S. kishiwadensis LMG 19939T and S. mashuensis LMG 8603T ; S. bambergiensis LMG 19299T and S. prasinus LMG 20259T ; S. ghanaensis LMG 19894T and S. viridosporus LMG 20278T ; S. atroolivaceus LMG 19306T and S. olivoviridis LMG 20057T ; S. pluricolorescens LMG 8576T and S. rubiginosohelvolus LMG 20267T ; S. libani s. libani LMG 20077T and S. nigrescens LMG 19332T ; S. diastaticus s. diastaticus LMG 19322T, S. gougerotii LMG 19888Tand S. rutgersensis s. rutgersensis LMG 8568T ; S. janthinus LMG 8591T, S. roseoviolaceus LMG 8594T, S. violaceus LMG 20257T, S. violarus LMG 20275T and S. violatus LMG 19397T.

#: not included in DNA-DNA hybridisations †: suggested name

116 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Taxonomic discriminative power

The majority i.e. 350 of the 473 type strains analysed had a unique BOX pattern (Table 9) confirming the high discriminative power of the BOX-PCR fingerprint technique and the reported high diversity within the genus Streptomyces. When considering the group 2 clusters with nearly identical BOX patterns (clusters 1 to 30, Table 10, Fig. 19), for four of them (cluster 11, 14, 17, 29; Table 2) previous taxonomic studies revealed DNA homology values above 70 %. Emended descriptions have been proposed for S. cacaoi subsp. cacaoi (Lanoot et al., 2002), S. caeruleus (Lanoot et al., 2002), S. griseoviridis (Labeda, 1998) and S. hygroscopicus (Labeda & Lyons, 1991b). In the present work, seven clusters were investigated by DNA-DNA pairing here (clusters 2, 13, 21, 23, 24, 27, 30; Table 10, Fig. 19). It was found that S. phaeoviridis has a DNA homology of 93 % with S. phaeopurpureus (cluster 2), S. distallicus has a DNA homology of 100 % with S. colombiensis (cluster 13), S. roseodiastaticus has a DNA homology of 100 % with S. tricolor (cluster 21), S. arabicus has a DNA homology of 98 % with S. vinaceus (cluster 23), S. roseosporus has a DNA homology of 93 % with S. filamentosus (cluster 24), S. roseoflavus has a DNA homology value of 85 % with S. fradiae (cluster 27), S. cochleatus is sharing a DNA homology value of 100 % with S. cinereorectus (cluster 30). In cluster 12 we found that S. microflavus, S. lipmanii and S. griseus subsp. alpha share DNA homology values in the range of 95-100 %. As a result of these experiments emended descriptions of the following seven species are proposed below: S. cinereorectus, S. colombiensis, S. filamentosus, S. fradiae, S. phaeopurpureus, S. tricolor, S. vinaceus. For 19 remaining clusters (i.e. cluster 1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 19, 20, 22, 25, 26 and 28) (Table 10, Fig. 19), grouping in totally 52 species with nearly identical BOX patterns, no DNA-DNA homology studies have been performed yet. Based on previous results which showed in all cases a high correlation between nearly identical BOX patterns and high DNA-DNA homology values we presume synonymous species names are present within the mentioned clusters (information present in Table 2). BOX-PCR genomic fingerprinting is found a fast and worthful tool to detect highly related genomes.

117

P earso n cor relation (Opt: 0.49 %) [5.1% -8 0.6% ] rep-PCR re p-PC R

% correlation DNA-DNA

0 homology value 20 40 60 80 10 .S. roseiscleroticus LMG 20284 T 1 .S. ruber LMG 20285 T .S. phaeoviridis LMG 20061 T 80 % # 2 .S. phaeopurpureus LMG 20051 T .S. minutiscleroticus LMG 20062 T 3 .S. flaviscleroticus LMG 19886 T .S. sannanensis LMG 20329 T 4 .S. anthocyanicus LMG 20411 T .S. clavifer LMG 20476 T 5 .S. ciscaucasicus LMG 20474 T .S. sioyaensis LMG 20531 T 6 .S. enissocaesilis LMG 20506 T .S. thermogriseus LMG 20532 T 7 .S. thermovulgaris LMG 19342 T .S. virens LMG 20316 T .S. calvus LMG 20442 T .S. asterosporus LMG 20419 T 8 .S. aureorectus LMG 19908 T .S. nigrifaciens LMG 20048 T 9 .S. flavovirens LMG 20516 T .S. graminearus LMG 19904 T .S. griseomycini LMG 19883 T 10 .S. griseostramineus LMG 19932 T .S. daghestanicus LMG 20496 T 95 % £ 11 .S. griseoviridis LMG 19321 T .S. willmorei LMG 21046 T .S. griseus s. cretosus LMG 19946 T .S. lipmanii LMG 20047 T .S. microflavus LMG 19327 T 100 % 99 % # 95 % 12 .S. griseus s. alpha LMG 19953 T .S. colombiensis LMG 20487 T 100 % # 13 .S. distallicus LMG 20499 T .S. caeruleus LMG 19399 T 100 % * 14 .S. niveus LMG 19395 T .S. umbrinus LMG 20280 T 15 .S. misionensis LMG 20063 T .S. mediolani LMG 20093 T .S. albovinaceus LMG 20402 T 16 .S. cacaoi s. cacaoi LMG 19320 T 100 % * 17 .S. aminophilus LMG 19319 T .S. anulatus LMG 19301 T .S. citreofluorescens LMG 20475 T .S. fluorescens LMG 8579 T 18 .S. chrysomallus s. chrysomallus LMG 20459 T .S. setonii LMG 20291 T 19 .S. flavofuscus LMG 19900 T .S. albus s. albus LMG 20405 T .S. gibsonii LMG 19912 T .S. almquistii LMG 21307 T 20 .S. rangoonensis LMG 20295 T .S. tricolor LMG 20328 T 100 % # 21 .S. roseodiastaticus LMG 20327 T .S. canescens LMG 20445 T .S. felleus LMG 20511 T 22 .S. limosus LMG 8570 T .S. sampsonii LMG 8574 T .S. arabicus LMG 20414 T 98 % # 23 .S. vinaceus LMG 20533 T .S. roseosporus LMG 20262 T 93 % # 24 .S. filamentosus LMG 20512 T .S. erythrogriseus LMG 19406 T .S. griseoincarnatus LMG 19316 T .S. variabilis LMG 20270 T 25 .S. labedae LMG 19956 T .S. chibaensis LMG 20456 T 26 .S. corchorusii LMG 20488 T .S. fradiae LMG 19371 T 85 % # 27 .S. roseoflavus LMG 20535 T .S. psammoticus LMG 20525 T .S. aureofaciens LMG 5968 T 28 .S. avellaneus LMG 20427 T .S. hygroscopicus s. hygroscopicus LMG 19335 T 92 % $ 29 S. . endus LMG 19393 T S. . cinereorectus LMG 20461 T 100 % # 30 S. . cochleatus LMG 20478 T Fig. 1 Fig. 19: Dendrogram showing 30 clusters containing strains with nearly identical rep patterns. Available DNA-DNA homology values from literature and the present study are indicated. #: present study *: Lanoot et al., 2002; £: Labeda, 1998; $: Labeda & Lyons, 1991b.

118 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

The affiliation of group 3 streptomycetes (48 strains) mentioned in the footnote of Table 10, was uncertain. They showed a significant similarity to one of the delineated clusters (clusters 4, 9, 18, 19, 21 and 22, Table 10) or to each other. Our study shows that DNA-DNA homology values in the range 50 to 80 % are difficult to correlate with a level of similarity in BOX patterns and is species dependent. Labeda & Lyons (1991a) found a DNA homology of 76 % between S. coeruleorubidus and S. bellus (Fig. 19). S. janthinus, S. roseoviolaceus, S. violatus and S. violarus share a DNA-DNA homology value in the range 48-80 % (Labeda & Lyons, 1991a; Fig. 20). Although exceptional, also among the strains with unique patterns (Table 9) DNA- DNA homology values above 70 % are found S. curacoi shares a DNA-DNA homology value between 72 and 75 % with S. coeruleorubidus and S. bellus. S. afghaniensis and S. purpurascens share a DNA-DNA homology value of 72 % (Labeda & Lyons, 1991a) ; S. roseoverticillatus and S. biverticillatus share a DNA- DNA homology value of 77 % (Labeda, 1996); S. spheroides shares a DNA-DNA homology value between 77 and 85 % with S. niveus and S. caeruleus (cluster 14, Table 10; Lanoot et al., 2002; S. griseus and S. alboviridis share a DNA-DNA homology value of 97 % (Witt & Stackebrandt, 1990).

------

DNA-DNA homology .S. bellus LMG 19401 T 76 % .S. coeruleorubidus LMG 20484 T (LABEDA et al., 1991) .S. janthinus LMG 8591 T .S. roseoviolaceus LMG 8594 T 48-80 % .S. violarus LMG 20275 T (LABEDA, 1996) .S. violatus LMG 19397 T

Fig. 20: Selection of BOX patterns of strains with uncertain affiliation and corresponding DNA-DNA homology values. ------

In conclusion among the 451 Streptomyces species studied, BOX-PCR suggested the delineation of at least 30 clusters of synonymous species names. For 11 of them (7 in the present study) emended descriptions have already been

119

proposed. Considering the remaining 19 clusters (grouping 52 species), DNA-DNA homology studies may confirm another 33 junior synonyms. The present study shows that the rep-PCR fingerprint technique using the BOX primer is a fast, reproducible tool to reduce the number of synonymous species names present within the genus Streptomyces. The BOX-PCR technique, in combination with an extensive database, may be a valuable identification tool for Streptomyces.

EMENDED DESCRIPTIONS

Streptomyces phaeopurpureus (Shinobu 1957, 63AL)

Spore chains are Rectiflexibiles, the spore surface is smooth. The aerial mass is in the gray or red color series, white color might be observed on glycerol-asparagine agar. The reverse side of a colony is yellow to yellow brown modified by red or orange. Substrate mycelium pigment is not a pH indicator. Melanoid pigments are formed. Orange, yellow or reddish pigment is formed in the medium. D-glucose, L- arabinose, D-xylose, sucrose, rhamnose, i-inositol, raffinose, D-mannitol and D- fructose are utilized for growth. The G + C content of the DNA is 72,3 mol%. The type strain LMG 20051 shows a DNA-DNA homology value of 80 % with Streptomyces phaeoviridis LMG 20061 which is a later synonym.

Streptomyces cinereorectus (Terekhova and Preobrazhenskaya 1986)

Spore chains can have spirals, hooks or can be straight. The spore surface is smooth. Substrate mycelium is colourless over grey-yellowish to grayish brown. The aerial mass is in the gray series or can be white. Melanoid pigments can be formed. A brown soluble pigment can be formed in the medium. A good growth is observed on D-glucose. No or poor growth on: i-inositol, raffinose. The utilisation of L- arabinose, D-fructose and D-mannitol can vary. The G + C content of the DNA is 72,8 mol%. The type strain LMG 20461 shows a DNA-DNA homology value of 100 % with Streptomyces cochleatus LMG 20478 which is a later synonym.

120 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Streptomyces colombiensis (Pridham, Hesseltine and Benedict 1958, 76AL

Emended description based on the description of S. colombiensis). Spore chains are Spirales; RA morphology may be observed. The spore surface is smooth. The aerial mass is in the red color series. The reverse side of a colony is moderate reddish to strong brown, grayish yellow, orange yellow or yellowish brown. Melanoid pigments may be formed. No pigments are found in the medium. D-glucose and D- fructose are utilized. No growth or trace of growth with L-arabinose, i-inositol, D- mannitol, rhamnose, sucrose, raffinose or D-xylose. The G + C content of the DNA is 71,7 mol%. The type strain LMG 20487 shows a DNA-DNA homology value of 100 % with Streptomyces distallicus LMG 20499 which is a later synonym.

Streptomyces tricolor (Wollenweber 1920) Waksman 1961, 158AL (Actinomyces tricolor Wollenweber 1920, 13.)

Spore chains are Spirales; the spore surface is smooth. The aerial mass is in the grey color series. The reverse side of a colony is red to blue (based on description of S. tricolor). Melanoid pigments may be formed (for S. tricolor). Blue diffisible pigments are produced in the medium. No reports available on the utilisation of carbon sources for S. tricolor. S. roseodiastaticus utilises D-glucose, L-arabinose, L-rhamnose and D-xylose. The G + C content of the DNA is 72,6 mol%. The type strain LMG 20328 has a DNA-DNA homology value of 100 % with Streptomyces roseodiastaticus LMG 20327 which is a later synonym.

Streptomyces fradiae (Waksman and Curtis 1916) Waksman and Henrici 1948b, 954AL (Actinomyces fradii (sic) Waksman and Curtis 1916, 125).

Spore chains are Retinaculiaperti or Spirales; the spore surface is smooth. The aerial mass is in red or yellow color series. The reverse side of a colony is moderate to deep orange over grayish yellow to yellowish brown. Melanoid pigments are not formed. No pigments in the medium are found. D-glucose, L-arabinose are utilized for growth. No growth or trace of growth on i-inositol, rhamnose, sucrose, raffinose and D-mannitol. The utilisation of D-xylose and D-fructose is variable. The G + C content

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of the DNA is 74,0 mol%. The type strain LMG 19371 has a DNA-DNA homology value of 85 % with Streptomyces roseoflavus LMG 20535 which is a later synonym.

Streptomyces vinaceus (Jones 1952, 47AL)

Spore chains can be Spirales or Retinaculiaperti; Rectiflexibiles morphology may appear. The spore surface is smooth, warty or spiny. The aerial mass is in the red or gray color series. White aerial mycelium may also be present on glycerol- asparagine agar. The reverse side of a colony is strong brown over orange yellow, grayish yellow to yellowish pink. Melanoid pigment might be formed. No pigment or a trace of yellow or pink may be found in the medium. D-glucose, D-mannitol and D- fructose are utilized for growth. Utilization of L-arabinose, rhamnose, D-xylose, sucrose and i–inositol can vary. No growth or only trace of growth on raffinose. The G + C content of the DNA is 73,1 mol%. The type strain LMG 20533 has a DNA-DNA homology value of 98 % with Streptomyces arabicus LMG 20414 which is a later synonym.

Streptomyces filamentosus (Okami and Umezawa in Okami, Okuda, Takeuchi, Nitta and Umezawa 1953, 153AL)

Spore chains are Rectiflexibiles, the spore surface is smooth. The aerial mass is in the red color series. White or yellow color possible on glycerol-asparagine agar The reverse side of a colony has no distinctive pigments. Melanoid pigment and pigments in the medium are not formed. D-glucose, L-arabinose, D-xylose and rhamnose carbohydrates are utilized for growth. No growth or trace of growth on i- inositol, D-mannitol and raffinose. Utilization of D-fructose is doubtful. Variable reports on utilization of sucrose. The G + C content of the DNA is 73,3 mol%. The type strain LMG 20512 has a DNA-DNA homology value of 93 % with Streptomyces roseosporus LMG 20262, which is a later synonym.

122 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Acknowledgements. We are grateful to A. Balcaen and E. De Brandt for determining mol % G + C content. V. Chepurnov (UGent) is acknowledged for translating the original description of S. cinereorectus from Russian into English language. This study was performed in the frame of the Belgian-Chinese program: ‘Identification and classification of actinomycetes, specifically bioactive Streptomyces strains isolated from Chinese soil’, supported by OSTC Belgium (BL/02/C10) and MOST/NSFC P.R. China.

123

124 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

B. Lanoot, M. Vancanneyt, A. Van Schoor, Z. Liu & J. Swings

Redrafted from Int J Syst Evol Microbiol, in press (2005a)

------SUMMARY A DNA-DNA hybridisation survey was performed on 13 Streptomyces species and two subspecies, dispersed over 5 genotypically defined clusters as delineated by Lanoot et al. (2004). Within each of the latter clusters, strains shared DNA-DNA homology values above 70 %. On the basis of the recommendations of Wayne et al. (1987) and Stackebrandt et al. (1994) the following 8 validly described Streptomyces species are considered as later synonyms: S. nigrifaciens as a synonym of S. flavovirens; S. citreofluorescens, S. chrysomallus subsp. chrysomallus and S. fluorescens as synonyms of S. anulatus; S. chibaensis as a synonym of S. corchorusii; S. flaviscleroticus as a synonym of S. minutiscleroticus; S. lipmanii, S. griseus subsp. alpha, S. griseus subsp. cretosus and S. willmorei as synonyms of S. microflavus. Emended descriptions are proposed. ------

125

INTRODUCTION

Since the introduction of the genus Streptomyces by Henrici and Waksman in 1943 classification systems basically relied on the use of morphological and phenotypic criteria. As shown in literature these classification schemes often lack taxonomic discriminative power at species level. Genotypic approaches revealed synonymies among several species exist as was shown for e.g. the S. violaceusniger, S. cyaneus and S. lavendulae species groups (Labeda et al., 1991a- b; Labeda, 1993). The high number of validly described species however remains a major practical obstacle in an overall genotypic reclassification of Streptomycetes. During a recent study, 451 validly described Streptomyces species have been screened using BOX-PCR. Numerical analysis of patterns resulted in the delineation of 30 clusters, each comprising multiple type strains with nearly identical patterns. For ten of them (clusters 2, 11, 14, 17, 21, 23, 24, 27, 29, 30 in Lanoot et al., 2004) taxonomic reclassifications have been proposed and resulted in the emended descriptions of 7 species (Lanoot et al., 2004). In the present study, the taxonomic relationship between the type strains within clusters 3, 9, 12, 18, 26 (clusters delineated in Lanoot et al., 2004) was investigated by DNA-DNA hybridisations.

MATERIALS AND METHODS

Strains investigated

The strains investigated were S. minutiscleroticus LMG 20062T and S. flaviscleroticus LMG 19886T (cluster 3), S. nigrifaciens LMG 20048T and S. flavovirens LMG 20516T (cluster 9), S. lipmanii LMG 20047T, S. microflavus LMG 19327T, S. willmorei LMG 21046T, S. griseus s. alpha LMG 19953T, S. griseus subsp. cretosus LMG 19946T (cluster 12), S. citreofluorescens LMG 20475T, S. chrysomallus LMG 20459T, S. fluorescens LMG 8579T and S. anulatus LMG 19301T (cluster 18), S. chibaensis LMG 20456T and S. corchorusii LMG 20488T (cluster 26). All strains were grown on Bennett’s agar medium (0.1 % yeast extract, 0.1 % beef

126 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

extract, 0.2 % casein hydrolysate, 1 % glucose, 2 % agar; final pH 7.3) and incubated at 28 °C for 24 h.

DNA-DNA hybridisations

DNA was extracted using a glass beads-mutanolysine based lysis method as described previously (Lanoot et al., 2004). Determination of mol % G + C content was determined via HPLC using the protocol of Tamaoka et al. (1984) and were for all strains in the range from 70 to 73 %. Genomic relatedness between the strains of each cluster was determined by DNA-DNA hybridisations using the fluorescence based microplate method of Ezaki et al. (1989). The hybridisation temperature was 52 °C.

RESULTS AND DISCUSSION

The mean DNA-DNA reassociation value between S. minutiscleroticus LMG 20062T and S. flaviscleroticus LMG 19886T (cluster 3) was 96 %, between S. nigrifaciens LMG 20048T and S. flavovirens LMG 20516T (cluster 9) was 95 %, between S. corchorusii LMG 20456T and S. chibaensis LMG 20488T (cluster 26) was 94 %. Species belonging to cluster 18 (LMG 20475T, LMG 20459T, LMG 8579T and LMG 19301T) shared DNA-DNA homology values in the range 90 to 97 % (Table 11). Members of cluster 12 (LMG 20047T, LMG 19327T, LMG 21046T, LMG 19953T, LMG 19946T) shared DNA-DNA homology values in the range 78 to 99 % (Table 11).

Our study also showed that the species S. griseus with its four subspecies is genotypically heterogeneous and was clustering in BOX-PCR group 12 (S. griseus subsp. cretosus LMG 19946T, S. griseus subsp. alpha LMG 19953T; Lanoot et al., 2004) or possessing a unique pattern (S. griseus subsp. griseus LMG 19302T, S. griseus subsp. solvifaciens LMG 19952T). In contradiction to their phenotypical homogeneity, low DNA-DNA homology values in the range 26 to 53 % (Table 11) were shared among the type strain of the species S. griseus subsp. griseus LMG 19302T and three other subspecies i.e. S. griseus subsp. alpha LMG 19953T, S.

127

griseus subsp. cretosus LMG 19946T and S. griseus subsp. solvifaciens LMG 19952T. Two subspecies i.e. S. griseus subsp. cretosus, S. griseus subsp. alpha shared a DNA-DNA homology value of 98 % (Table 11). In the present study, latter two subspecies are reclassified as S. microflavus.

128 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

BOX- Name PCR Number % G+C % DNA-DNA homology (SD) cluster * content

LMG 19327T LMG 20047T LMG 21046T LMG 19953T LMG 19946T LMG 19302T LMG 19952T LMG 19301T LMG 8579T LMG 20475T LMG 20459T

S. microflavus LMG 19327T 71 100

S. lipmanii LMG 20047T 71 92(8) 100

12 S. willmorei LMG 21046T 71 91(3) 85(12) 100

S. griseus subsp. alpha LMG 19953T 70 100 99(1) 78(8) 100

S. griseus subsp. cretosus LMG 19946T 70 100 90(10) 90(10) 98(3) 100

S. griseus subsp. griseus SEP LMG 19302T 72 _ _ _ 43(5) 53(5) 100

S. griseus subsp. solvifaciens SEP LMG 19952T 73 _ _ _ 30(3) _ 26(12) 100

S. anulatus LMG 19301T 71 ______100

S. fluorescens LMG 8579T 71 ______90(5) 100 18 S. citreofluorescens LMG 20475T 72 ______97(3) 92(6) 100

S. chrysomallus subsp. chrysomallus LMG 20459T 71 ______94(6) 94(6) 93(7) 100

Table 11: DNA-DNA reassociation values among Streptomyces species of 5 delineated BOX-PCR clusters. * as defined by Lanoot et al. (2004) bold: reclassified species

129

Emended descriptions

On basis of DNA-DNA homology relatedness the 13 validly described Streptomyces species and two subspecies actually consist of the following 5 species with later synonym(s). Phenotypic characteristics are given in Table 12.

S. flavovirens (Waksman 1923) Waksman and Henrici 1948; 30:382 (AL) LMG 20516T (=DSM 40062=NRRL B1329) and S. nigrifaciens Waksman 1961; 30:394 (AL) LMG 20048T (=DSM 40071=NRRL B-2094) are synonyms of each other. As S. flavovirens was described first, its name is given priority on S. nigrifaciens for renaming both synonyms.

S. anulatus (Beijerinck 1912) Waksman 1957; 30:372 (AL) LMG 19301T (=AS4. 1421=DSM 40361) Later synonyms: S. chrysomallus subsp. chrysomallus Lindenbein 1952; 30:376 (AL) LMG 20459T (=AS4. 1676=DSM 40128), S. citreofluorescens Korenyako et al. (1960); 30:377 (AL) LMG 20475T (=AS4. 1652=DSM 40265) and S. fluorescens Korenyako et al. (1960); 30:382 (AL) LMG 8579T (=DSM 40203=NRRL B-2873).

S. corchorusii Ahmad and Bhuiyan, (1958); 30:378 (AL) LMG 20488T (=AS4. 1592=DSM 40340) Later synonym: S. chibaensis Suzuki et al. (1958) ; 30:376 (AL) LMG 20456T (=AS4. 1654=DSM 40220)

S. minutiscleroticus Thirumalachar et al. (1965) Goodfellow et al. (1986) LMG 20062T (= DSM 40301=NRRL B-12202) Later synonym: S. flaviscleroticus Thirumalachar and Sukapure 1964 Goodfellow et al. (1986) LMG 19886T (=DSM 40270=NRRL B-12173)

S. microflavus Krainsky 1914 Waksman and Henrici 1948; 30:392 (AL) LMG 19327T (=AS4. 1428=DSM 40331) Later synonyms: S. griseus subsp. alpha Ciferri 1927 Pridham 1970; 30:387 (AL) LMG 19953T (=DSM 40937=NRRL B-2249), S. griseus subsp. cretosus Pridham

130 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

1970; 30:387 (AL) LMG 19946T (=DSM 40561=NRRL B-2252), S. lipmanii (Waksman and Curtis 1916) 30:391 (AL) Waksman and Henrici, 1948; LMG 20047T (=DSM 40070=NRRL B-1229) and S. willmorei Erikson 1935 Waksman and Henrici, 1948; 30:405 (AL) LMG 21046T (=DSM 40459=NRRL B-1332).

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Carbon utilization

Name Number Spore chain Spore Aerial mass Reverse-side Melanoid Glc Ara Fru Ino Man Raf Rha Suc Xyl morphology surface color color pigment S. microflavus LMG 19327T RF smooth GR, Y ND - + V + +/- + +/- + V + S. lipmanii LMG 20047T RF smooth Y ND - + +/- + +/- + +/- + V + S. willmorei LMG 21046T RF smooth GR, Y, W ND - + +/- + +/- + +/- + +/- + S. griseus subsp. griseus LMG 19302T RF smooth Y ND - + +/- + +/- + +/- +/- +/- + S. anulatus LMG 19301T RF, SP or RA smooth Y, W ND - + + + - + - + - + S. fluorescens LMG 8579T RF smooth Y, W ND - + + + +/- + +/- + +/- + S. citreofluorescens LMG 20475T RF smooth Y ND - + + + +/- + +/- + +/- + S. chrysomallus LMG 20459T RF smooth Y ND - + + + +/- + +/- + +/- + S. flaviscleroticus LMG 19886T SP smooth lacks Y-BR - + + + +/- + +/- + +/- + S. minutiscleroticus LMG 20062T SP smooth GR, Y Y-BR - + + + + + +/- + +/- + S. flavovirens LMG 20516T RF smooth GR C, GY-Y - + + +/- - + - + - + S. nigrifaciens LMG 20048T RF smooth GR ND - + + V +/- + +/- + +/- + S. chibaensis LMG 20488T SP or RF smooth GR, Y ND - + + + + + + + + + S. corchorusii LMG 20456T SP or RF smooth GR ND - + + + + + + + + +

Table 12: Phenotypic characteristics of the reclassified species S. anulatus, S. corchorusii, S. minutiscleroticus, S. flavovirens and S. microflavus and later synonym(s). Data taken from the original descriptions.

Abbreviations: Spore chain morphology: RF, Rectiflexibiles; SP, Spirales; RA, Retinaculiaperti; Color of aerial mycelium / Reverse side: BR, Brown; Y, Yellow; W, White; C, Colorless; ND, not distinctive; GR, Gray; Carbon utilization: Glc, D-glucose; Ara, L-arabinose; Fru, D-fructose; Ino, i-inositol; Man, D-mannitol; Raf, raffinose; Rha, L-rhamnose; Suc, sucrose; Xyl, D-xylose; -, negative; +, positive; +/-, no growth or trace of growth; V, variable reports on utilization.

132 4. RECLASSIFICATION OF SYNONYMOUS SPECIES

Acknowledgements. BL is grateful to L. Lebbe and E. De Brandt for determining mol % G + C content. This study was performed in the frame of the Belgian-Chinese program: ‘Identification and classification of actinomycetes, specifically bioactive Streptomyces strains isolated from Chinese soil’, supported by the Federal Public Planning Service Science Policy of Belgium (BL/02/C10) and MOST / NFSC P.R. China.

133

134 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

Intra-species genotypic relatedness of S. virginiae strains and correlation with phenotypic traits

135

136 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

Phenotypic and genotypic characterisation of variants

of the virginiamycin producing strain 899 and

its relatedness to the type strain of Streptomyces virginiae

B. Lanoot, M. Vancanneyt, B. Hoste, M. C. Cnockaert, M. Piecq, F. Gosselé

& J. Swings

Redrafted from Syst Appl Microbiol, in press (2005b)

------

SUMMARY. A representative set of 19 variants, with a known genealogy, of the virginiamycin producing strain Streptomyces virginiae 899 was investigated phenotypically and genotypically. Colour of the aerial and substrate mycelium were very variable both among spontaneous variants and those obtained after induced mutagenesis. At genotypic level, all variants showed nearly identical BOX patterns, not reflecting the phenotypic heterogeneity observed. More than 40 years of forced mutational pressure did not cause huge chromosomal distortions but was most likely limited to base substitutions. The species S. virginiae, including besides producers of virginiamycin the type strain and non-type strains producing other bioactive compounds, is genomically heterogeneous on the basis of BOX-PCR fingerprinting and DNA-DNA hybridizations. The virginiamycin producing strain 899 does not belong to the species S. virginiae despite its phenotypic similarity to the latter. ------

137

INTRODUCTION

Actinomycetes, more particularly streptomycetes, are well known producers of a variety of economically interesting secondary metabolites such as antimicrobial compounds, antitumour agents, herbicides and other bioactive molecules. Different groups of antibiotics produced by Streptomyces were described, e.g. macrolides, aminoglycosides, polyethers, glycopeptides, and streptogramins (Vandamme, 1984). The latter family comprises structurally related macrocyclic lactone peptolides such as mikamycin (Arai et al., 1958), pristinamycin (Preud’homme et al., 1965) and virginiamycin (Biot, 1984; Cocito, 1979), produced by “Streptomyces mitakaensis”, “S. pristiniaespiralis” and S. virginiae, respectively. Streptomyces virginiae strain 899 (=ATCC 13161) was isolated and described by De Somer and Van Dijck (1955) and has been used in an extensive industrial strain selection program run by Phibro Animal Health. Besides morphologically differentiated variants, other economically interesting overproducers but also mutants, blocked in the biosynthesis of one or both synergistic factors M and S of the virginiamycin complex, were isolated (Biot, (1984); Piecq and Gosselé, unpublished results). Studies on Streptomyces rimosus highlighted the instability of morphological traits and showed the resulting taxonomic implications i.e. incorrect identifications based on a phenotypical classification approach (Al-Jawadi et al., 1985; Gravius et al., 1993). The same risk exists for Streptomyces virginiae: a single strain can show different morphological phenotypes, which can result in misidentification. In order to avoid this, Streptomyces taxonomy is evolving from a phenotypic classification (Locci, 1989) towards a genotypic alternative (Anderson & Wellington, 2001). The question raises whether the phenotypic instability of Streptomyces is reflected at the genomic level and if genotypic taxonomy can resolve the classification problems. The aim of the present study is to determine the genotypic relationship among a representative set of variants of strain S. virginiae 899, and check the correlation, if any, to their phenotype i.e. colour, sporulation and virginiamycin production. Compared to previous studies (Gravius et al., 1993; Rezanka et al., 1992), our study used strains originating from a long term mutational programme. The origin of all variants is clearly described in a genealogical tree.

138 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

Two molecular fingerprint tools were applied, taxonomically discriminating at different levels i.e. (i) the robust BOX-PCR fingerprint technique (Lanoot et al., 2004; Rademaker et al., 2000; Versalovic et al., 1994), applied on all variants and (ii) AFLP fingerprinting on selected strains. The latter method proved to be highly reproducible for tracing subtle genomic insertions or deletions (Keim et al., 1997) while BOX-PCR fingerprinting yields information at the intra-species level. In a second part of this study, the classification of strain 899 into the species S. virginiae was verified using the genotypic tool BOX-PCR fingerprinting and DNA- DNA hybridizations. In addition, genotypic relationships were determined between the type strain of S. virginiae, strains belonging to the same species but producing other bioactive compounds, and two functionally related species (i.e. “S. pristinaespiralis” and “S. mitakaensis”) using the same methodology.

MATERIALS AND METHODS

Strains investigated and growth conditions

In total, 27 Streptomyces strains divided over two groups were investigated (Table 13). All strains were grown aerobically on Bennett’s agar medium during one week at 28 °C or in Bennett’s liquid medium for 72 h at 27 °C on a shaking platform (150 rpm). A first group consists of 19 variants of S. virginiae 899, the original virginiamycin producer (ATCC 13161). The variants were chosen among several hundred thousands of strains isolated over the last 40 years in an industrial strain selection program run by Phibro Animal Health. They were chosen based on their virginiamycin production (non-producers, overproducers, producers of virginiamycin factors M or S), their sporulation ability and the colour of substrate and aerial mycelium. Moreover, the selected variants cover as much as possible the entire genealogical tree. Majority of them were obtained by successive rounds of induced mutagenesis. U.V., MNNG (NTG) and gamma radiation were the most applied mutagenic treatments. Some other treatments or selection criteria were also used such as resistance to virginiamycin, curing from the lysogenic phages, resistance to amino acid analogues and auxotroph reversion, as described by Biot (1984). Three deletion mutants obtained by gene disruption in the so-called vgnIV region,

139 implicated in virginiamycin production (Dehottay and Piecq, unpublished results), were also included in the study (PD 282, PD 170 and PD 321). Finally, variants V899-1 to V899-6 were recovered in 1996 through natural selection (repeated sub- culturing and single colony isolation) of a lyophilized culture of original strain 899. The genealogy of the strains investigated is presented in Fig. 21. A second group of strains included culture collection strains: the type strain of S. virginiae, LMG 20534T, four other S. virginiae strains (LMG 21028, LMG 21048, LMG 21537, MAFF 116014) and two streptogramin producing strains belonging to the species “S. pristinaespiralis” (ATCC 25486T) and “S. mitakaensis” (ATCC 15297).

------

year of isolation 1954 899 (Phibro) 1955 1961 1962 844 1963 1973 1974 R4237 1975 1985 1986 1987 1988 1989 1990 PD170 1991 1992 PD282 PD321 1993 180059 1994 1995 236379 1996 1997 277277 V899-1 V899-2 V899-3 V899-4 V899-5 V899-6 1998 1999 286164 285184 2000 286763 287524 V1403 2001 2002 2003

Fig. 21: Genealogical tree of S. virginiae strain 899 and its variants. Dotted line: spontaneous variants Continuous line: induced mutagenesis ------

140 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

Genotyping

Extraction of DNA

DNA was extracted from liquid cultures using a glass beads-mutanolysine procedure as described by Lanoot et al. (2004).

Rep-PCR fingerprinting

PCR protocol using BOXA1R primer and electrophoresis of subsequent PCR products were performed as indicated by Lanoot et al. (2004). Digitized gel images were loaded into the software package BioNumerics version 2.5 (Applied Maths, Belgium). Similarity matrices of densitometric curves of the gel tracks were calculated using the Pearson Product Moment Correlation Coefficient followed by tree construction using UPGMA algorithm. Settings: optimization 0.49 %, the first 5.1 % and last 29.6 % of data points of each lane were excluded from the analysis. Reproducibility was at least 85 %.

AFLP analysis

The procedures followed were based essentially on the description of Janssen et al. (1996). An overview of the technique is given in Fig. 22. One µg of DNA was digested with ApaI and TaqI (Amersham Biosciences) and subsequently ligated using T4 ligase (Amersham Biosciences) to equimolar amounts of oligonucleotide sequence complements 5’-TCGTAGACTGCGTACAGGCC-3’ and 5’- TGTACGCAGTCTAC-3’ complementary to the ApaI restriction halfsite and 5’- GACGATGAGTCCTGAC-3’ and 5’-CGGTCAGGACTCAT-3’ complementary to the TaqI restriction halfsite. Templates thus obtained were precipitated with 1.25 M ammonium acetate and 50 % (v/v) isopropanol, washed with 70 % (v/v) ethanol and resuspended in 100 µl buffer containing 10 mM Tris, 0.1 mM EDTA pH 8.0. Templates were selectively amplified by PCR using six different combinations of the primer with core sequence 5’-GACTGCGTACAGGCCC-3’ (+ additional selective base A, G or T at 3’ end) and primer 5’-CGATGAGTCCTGACCGA-3’ (+ additional selective base C at 3’ end) or 5’-GATGAGTCCTGACCGA-3’ (+ additional selective base A or G at 3’ end). The primer complementary to the TaqI restriction site and

141 adaptor was radioactively labelled at the 5’ end using [γ-32P] ATP (Amersham Biosciences) using T4 polynucleotide kinase (Amersham Biosciences). Amplified fragments were separated by electrophoresis on 5 % denaturing polyacrylamide gels with 8.3 M urea and TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA; pH 8.4) as running buffer. After drying, the AFLP fingerprints were visualized autoradiographically by exposing the dried gels to Hyperfilm-MP (Amersham Biosciences), scanned and stored as TIFF files.

------

Double digest

Genomic DNA with two restriction enzymes Ligation of adaptors

Selective

PCR amplification

Selective bases

Fig. 22: Simplified overview of the AFLP fingerprint technique. Adapted from Hauben, L. (UGent). Sequences and restriction enzymes shown are examples. *: marked (radioactive)

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142 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

% G+C determination and DNA-DNA hybridization experiments

The mol % G+C content of DNA was determined using the protocol of Tamaoka and Komagata (1984) in which DNA is hydrolysed into nucleosides with nuclease P1 (Sigma) and bacterial alkaline phosphatase (Sigma) followed by separation with reversed-phase high-performance liquid chromatography. DNA-DNA hybridizations were performed using the fluorometric method (Ezaki et al., 1989) using a model FL-2575 microplate reader (Towa Scientific) for the fluorescence measurements and white Maxisorp FluoroNunc microplates (Nunc A/S). Biotinylated probe DNA was sheared by ultrasonication (30 s, duty cycle 50, output control 1) using a Branson Sonifier Model 250 equipped with microcup horn and then hybridized with single-stranded unlabeled DNA, non-covalently bound to microplate wells. Hybridizations were performed at 55 °C for 2 hours in a hybridization solution containing 50 % formamide. Salmon sperm DNA (Sigma) was used as the negative control. Experiments were repeated twice; data presented are the mean values.

RESULTS AND DISCUSSION

Relationships among derivates of strain S. virginiae 899

Three phenotypic traits were clearly distinguishable amongst the 19 variants of Streptomyces virginiae 899 investigated in this study i.e. (i) colour of substrate or aerial mycelium, (ii) sporulation efficiency and (iii) virginiamycin production (Table 13).

143

Species Strain Phenotype Remarks Bioactive component (a) Slope aspect (b) S. virginiae 899 and its derivates Nature Production Sporulation Mycelium color Aerial Substrate 899 (=ATCC 13161) virg. (M+S+) 1 ++ bw-gr bg original virginiamycin producer (De Somer & Van Dijck, 1955) V899-1 virg. (M+S+) 10 - rd-br rd-br V899-2 virg. (M+S+) 10 + bw incol V899-3 virg. (M+S+) 1 + gr br V899-4 virg. (M+S+) 1 ++ gr rd V899-5 virg. (M+S+) 1 + gr bg V899-6 virg.* (M- S-) 0 + gr br-bg 844 virg. (M+S+) 10 - br rd-br cured from its phage (=R81 in Biot, 1984) R4237 virg. (M+S+) 100 - incol incol revertant from tryptophan auxotroph (=SV422 in Biot, 1984) V1403 virg. (M+S+) 1000 + bw-bg bg 285184 virg. (M+S+) 1000 + bw bg 286164 virg. (M+S+) 1000 ++ bw bg 286763 virg. (M+S+) 1000 - incol incol 287524 virg. (M+S+) 1000 - incol incol 180059 virg.* (M+S-) 1000 ++ bw bg 277277 virg.* (M- S+) 1000 + bw br-bg 236379 virg.* (M- S+) 1000 + bw-bg bg PD170 virg.* (M- S-) 0 - incol incol deleted vgnIV ≈40kb (c) PD282 virg.* (M- S-) 0 + gr gr-br deleted vgnIV ≈20kb (c) PD321 virg.* (M- S-) 0 + gr incol deleted vgnIV ≈26kb (c)

S. virginiae LMG 20534T (=DSM 40094) actithiazic acid + cycloserine N.A. + wh bg-br Type strain; soil (USA) S. virginiae LMG 21028 (=DSM 916) lipoxamycin N.A. + bw bg-br soil (Sebek, 1971) S. virginiae LMG 21048 (=DSM 40669) - N.A. ++ wh-gr bg-br S. virginiae LMG 21537 (=IFO 3392) actithiazic acid + cycloserine N.A. + wh-gr bg-br soil (Anzai et al., 1994) S. virginiae MAFF 116014 virginiamycin 1 + bw-gr bg “S. pristiniaespiralis“ATCC 25486T pristinamycin N.A. N.A. N.A. N.A. Type strain “S. mitakaensis” ATCC 15297 mikamycin N.A. N.A. N.A. N.A. soil (Arai et al., 1958)

144 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

Table 13: Strains examined

(a) virg.: virginiamycin producing organism. virg.*: particular non producing variants originating from virginiamycin M and S producing strains. Virginiamycin production levels are given in arbitrary units, relative to the amount originally produced by the strain 899. M and S are synergistic factors of virginiamycin.

(b): determined on Bennett’s medium after 3 weeks of incubation at 28 °C. sporulation: “-“ = no spores on the slant surface; “+” = thin or irregular lawn of spores; “++” = thick and uniform layer of spores aerial = colour of agar slope upper surface / substrate = colour of agar slope reverse side br=brown; rd=red; bg=beige; bw=broken white; gr=(light) grey; incol=without distinctive colour, wh=white

(c): as shown by Southern blotting with the corresponding DNA region (Dehottay and Piecq, unpublished results).

N.A.: not available ------

Colour of the substrate mycelia on Bennett’s agar medium varied from colourless (R4237) to red-brown (844), differing from the greyish-beige of their ancestor (899). Sporulation efficiencies on Bennett’s medium varied from non sporulation (286763) to highly sporulated (286164). Aerial mycelium, when present, showed less variation, namely between broken white (285184) and grey (PD282) and exceptionally red brown (V899-1). This phenotypic variability was found, regardless of the way the strain 899 derivates were obtained: e.g. the variants of V899-1 to 6, obtained by natural selection were as different from strain 899 as mutants obtained after gene disruption, such as PD 282 (Fig. 23).

145

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A B C D E

Fig. 23: Differences in colour of substrate mycelium of the type strain of S. virginiae LMG 20534T and S. virginiae 899 derived variants. A= LMG 20534T, B= V899-1, C= V899-2, D= V899-4 and E= PD 282 ------

Among the collection of spontaneous variants of ancestor strain 899, some traits of the colony morphology were found indicative for those variants producing higher concentrations of virginiamycin. Higher producers V899-1 and V899-2 have a crater- like colony morphology while poor or non producers are uniform, round shaped colonies (V899-3, V899-4), circular, concentric waved colonies (V899-5) or scrambled shaped colonies (V899-6)(data not shown). In a study on S. rimosus mutants, crater morphology was found to be generally linked to high oxytetracyclin production (Gravius et al, 1993). However, a unique colony phenotype was not found that separates unequivocally virginiamycin producers (M+S+) from non producers (M+S-, M-S+, M-S-) on the genealogical tree of S. virginiae 899. Such instability of phenotypic traits is an intrinsic feature of streptomycetes. The observed phenotypic heterogeneity among the 19 strains derived from ancestor strain 899, was investigated genotypically. The fast and easily applicable

146 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

BOX-PCR fingerprinting technology (Versalovic et al., 1994) was selected as overall screening tool primarily as it is expected to yield information on the whole genome. Indeed, in silico matching of the BOX primer against the genome of S. coelicolor A3(2) revealed a theoretical total number of 46 hits, dispersed over the entire genome, using stringent matching conditions (Fig. 24). Distances between two following hits were varying between 5.7 kb and 1037 kb. Hence major chromosomal alterations, when present, should be detectable.

548 kb

1 2

: Origin of replication

2 1

Fig. 24: In silico matching of the BOXA1R primer to the whole-genome sequence of S. coelicolor A3(2).

Sequence adopted from the Sanger Institute, accession nr AL645882 Matching conditions: 100 % similarity required for the last 3 nucleotides

147

More than 40 years of mutagenic pressure on S. virginiae strain 899, either induced or spontaneous, yielded no major differences among the 20 BOX-PCR patterns generated. All variants derived from strain 899 showed BOX patterns identical to the ancestor strain 899 (Fig. 25, Cl. 1). Only minor quantitative differences in bands were found, which are not significant. In contrast to the morphological heterogeneity observed within S. virginiae, BOX-PCR fingerprints predicted a high relatedness (Lanoot et al., 2004).

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Pe arson corre lati on (Op t:0.49 %) [5 .7 %-70 .4 %] rep-PCR rep-PCR Fingerprint type % correlation 1500 bp 1000 bp 500 bp 300 bp

0 40 60 80 10 .899 (ATCC 13161)899 (ATCC 13161) .V899-1 V899 -1 .V899-3 V899 -3 .V899-5 V899 -5 .V899-4 V899 -4 .PD282 PD 282 .286763 286763 .286164 286164 .844 844 .PD170 PD 170 .V899-6 V899 -6 FPT 1 .V1403 V140 3 .236379 236379 .285184 285184G .180059 180059 .287524 287524 cl.1 .R4237 R4237 .PD321 PD 321 .V899-2 V899 -2 .MAFF 116014 MAFF 116014 .277277 277277 .LMG 21028 LMG 21028FPT 2 .ATCC 15297 ATCC 15297 cl.2 .LMG 20534T LMG 20534T FPT 3 .LMG 21048 LMG 21048 .LMG 21537 LMG 21537FPT 4 .ATCC 25486 ATCC 25486 ig. 4: Dendrogram showing BOX-PCR fingerprints of strain 899, 19 mutants derived fro Fig4 Fig. 25: Dendrogram showing BOX-PCR fingerprints of strain 899, 19 variants

derived from 899, 5 S. virginiae strains and 2 functionally related species.

Underlined: WT strains from culture collections

AFLP fingerprinting, a highly reproducible typing tool (Janssen et al., 1996) with a higher taxonomic resolution (strain level) than BOX-PCR fingerprinting (between strain and species), yielded few additional information, compared with the BOX-PCR

148 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

data, concerning the eventual genetic variability of the strain 899 derivatives. Among six primer combinations applied, the following combinations give two different bands among the patterns of strain 899 and the deleted mutant PD170 (Fig. 26): combination 1: 5’-GACTGCGTACAGGCCC(A)-3’ – 5’-CGATGAGTCCTGACCGA(C)- 3’; combination 2: 5’-GACTGCGTACAGGCCC(A)-3’ – 5’ GATGAGTCCTGACCGA(G)-3’(additional selective base between brackets). ------X Y X Y

primer primer comb comb 1 2

Fig. 26: Discriminative AFLP fingerprints of strain 899 and its deleted mutant PD 170 obtained with following primer combinations-selective base: Primer combination 1: 5’-GACTGCGTACAGGCCC(A)-3’ – 5’-CGATGAGTCCTGACCGA(C)-3’

149

Primer combination 2: 5’-GACTGCGTACAGGCCC(A)-3’-5’-GATGAGTCCTGACCGA(G)-3’ X= strain PD 170, Y=strain 899; Arrows indicate clear differences in band patterns.

------

We can conclude that the reported phenotypic instability is poorly reflected at the genotypic level by the techniques used.

Classification of strain 899 in the species S. virginiae

The species S. virginiae was described by Grundy et al. (1952), possessing red or grey coloured aerial mycelium, greyish yellow to yellow-brown substrate mycelium on yeast-malt extract, oatmeal or glycerol-asparagine agar medium, retinaculiaperti spore chains, a smooth spore surface and produces melanoid pigments (Shirling & Gottlieb, 1968). Phenotypically, strain 899 looks similar to the type strain of S. virginiae LMG 20534T: same spore morphology, similar mycelial colour on Bennett’s agar medium (De Somer & Van Dijck, 1955) and few differences in carbohydrate utilization and enzymatic activity (Anzai et al., 1994). For instance, strain 899 and the type strain of S. virginiae (LMG 20534T) only differ in 6 out of 52 traits investigated: ß-D-galactosidase activity, antibiotic susceptibility to lincomycin, growth at 5 °C, nitrate reduction and milk peptonization at 27 °C (Anzai et al., 1994). On the other hand, at genotypic level both strains showed different BOX-PCR patterns (Fig 25, Cl. 1 and Cl. 2), predictive for a low relationship. A low DNA-DNA similarity of 39 % was found for both strains. This is in accordance with the 47% DNA similarity reported in a previous study (Anzai et al., 1994). As a result, the virginiamycin producing strain 899 does not belong to S. virginiae and should be reclassified. The lack of a database comprising whole 16S rDNA sequences on all validly described Streptomyces species however hampers to determine all its closest phylogenetic neighbours, preventing a correct reclassification.

Relationship of S. virginiae LMG 20534T to other S. virginiae strains producing other bioactive compounds and functionally related species

S. virginiae LMG 20534T also looks phenotypically similar to the strains LMG 21028, LMG 21537, LMG 21048 and MAFF 116014 (Anzai et al., 1994). The

150 5. INTRA-SPECIES GENOTYPIC RELATEDNESS & PHENOTYPIC TRAITS

phenotypic homogeneity of all these S. virginiae strains was however not confirmed at genotypic level. Indeed, numerical analysis of the BOX-PCR patterns of the five S. virginiae strains LMG 20534T, LMG 21028, LMG 21048, LMG 21537 and MAFF 116014 revealed a high heterogeneity (Fig. 25). Profiles were dispersed over two clusters (Cl. 1 and 2) and two separate positions, representing four diverse fingerprint types (FPT 1 to 4). Cluster 1 contained besides the nearly identical BOX patterns of strain 899 and its 19 derived mutants also the pattern of the virginiamycin producing strain MAFF 116014 (FPT 1). The type strain of S. virginiae LMG 20534T and LMG 21048 (Cl. 2) only showed a few bands of difference in the high molecular weight zone (FPT 3). Strains LMG 21028 (FPT 2) and LMG 21537 (FPT 4) each showed unique patterns. DNA-DNA similarity studies underpinned the observations found in BOX-PCR. Strain LMG 21048 (FPT 3) shares a DNA-DNA relatedness of 93 % with strain LMG 20534T supporting its classification within S. virginiae. On the contrary, intermediate DNA-DNA similarity values between 40 and 43 % were shared among LMG 20534T and the strains LMG 21028 (FPT 2), LMG 21537 (FPT 4) and MAFF 116014 (FPT 1). These similarity values confirm the results published earlier by Anzai et al. (1994) for the strains LMG 20534T- LMG 21028 (48 %) and LMG 20534T- LMG 21537 (49 %). A DNA-DNA similarity value on LMG 20534T - MAFF 116014 was not determined in the latter study. It is clear that the named S. virginiae strains do not form a single species in spite of their morphological and biochemical homogeneity. Strains LMG 21028 and LMG 21537 do not belong to S. virginiae and should be reclassified. The functionally related streptogramin producing species “S. pristinaespiralis” and “S. mitakaensis” had different BOX-PCR patterns from each other and to all S. virginiae reference strains investigated (Fig. 25), confirming that production of related bioactive compounds can not be used as a taxonomic criterion for classification.

In conclusion, the taxonomy of the S. virginiae strains studied, based on morphology and other phenotypic traits would be different from a classification based on an analysis at the genetic level. Indeed, based on phenotypic traits, the 19 variants arising from the ancestor strain 899 would have been described as different species whereas the wild type strains from culture collections would form a homogenous group. However, genetic analysis reveals a higher divergence between

151 the collection strains than between the 19 variants from 899. Using the BOX-PCR method, 4 fingerprint types (FPTs) were obtained for the S. virginiae collection strains, expected to correspond to several species as verified by DNA/DNA hybridisation, compared to a single FPT for all the 19 variants of strain 899, including strain MAFF 116014. Only a typing methodology such as AFLP fingerprinting was able to distinguish between the wild type strain 899 and a deleted mutant PD 170. Thus, BOX-PCR fingerprinting proved to be a convenient tool for species delimitation in Streptomyces.

Acknowledgments. BL is grateful to Dirk Gevers (Plant System Biology, UGent) for generating the in-silico distribution of the BOX primer within the genome of S. coelicolor A3(2). OSTC Belgium grant BL/02/C10 supported this work. We thank Dr. Miyazaki (MAFF, Japan) for providing strain MAFF 116014.

152 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

Phylogenetic grouping using 16S-ITS RFLP fingerprinting as a valuable alternative for 16S rRNA gene sequencing

153 154 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

6.1 Preliminary study

16S rRNA gene RFLP fingerprinting (also called ARDRA) is an alternative to sequencing to obtain phylogenetic information (Heyndrickx et al., 1996) (Fig. 27). Therefore one or a combination of several tetra-cutters are used to generate fingerprints consisting of ideally 10-15 bands. In order to obtain as much information as possible from the 16S rRNA marker gene, fingerprints were generated in-silico, using the software package GeneCompar Version 2.0. Thus a set of 34 representative almost complete streptomycete 16S rRNA gene sequences were downloaded from EMBL and analysed using a battery of restriction enzymes. Based on these results, four restriction enzymes were selected i.e. BstUI, RsaI, TaqI and NIaIII and applied on a representative subset of 78 Streptomyces reference strains. Highly reproducible and informative fingerprints were generated with a sufficient number of well-spreaded bands, especially those obtained with BstUI and NIaIII (Fig. 28).

155 DNA PREPARATION AMPLIFICATION OF 16S rDNA RESTRICTION DIGEST GENE Bacterial cells Amplification in ThermoCycler Polymerase Chain Reaction PCR product (PCR)

Taq polymerase Restriction Primer pA Enzymes 5’ 3’ DNA (BstUI, RsaI, 16S rDNA TaqI, NIaIII) 3’ 5’ ds DNA Primer pH

Restriction fragments

SCANNING

NORMALISATION & COMBINING OF THE FOUR RESTRICTION PATTERNS

ELECTROPHORESIS NUMERICAL ANALYSIS

Fig. 26: Overview of the ARDRA fingerprint technique

15615 6 Fig. 27: Principle of the 16S rRNA gene RFLP technique. 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

Fig. 28: Dendrogram showing a selection of combined 16S RFLP fingerprints of 14 Streptomyces reference strains obtained with restriction enzymes BstUI, RsaI, TaqI and NIaIII.

Numerical analysis of the combined patterns, using the GelCompar software, resulted in the delineation of 17 clusters containing strains with identical RFLP profiles (Table 14). Upon comparison with a 16S tree, based on publicly available whole 16S rRNA gene sequences (not shown), 16S RFLP was shown to differentiate at a low taxonomic level i.e. intra-genus level.

157 Cluster Species

1 S. exfoliates, S. bikiniensis 2 S. finlayi, S. californicus, S. griseus, S. microflavus, S. atroolivaceus, S. anulatus, S. kanamyceticus, S. badius, S. parvus, S. xanthochromogenes, S. albus s. albus LMG 19318, S. olivaceus LMG 19374 3 S. aburaviensis, S. xanthocidicus 4 S. xantholiticus, “S. cyaneogriseus”, S. niveoruber 5 S. aurantiacus, S. albosporeus s. albosporeus 6 S. noboritoensis, S. longisporoflavus, S. phaeochromogenes, S. halstedii 7 S. venezulae, S. lateritius 8 S. endus, S. hygroscopicus s. hygroscopicus, S. ochraceiscleroticus, S. aminophilus, S. cacaoi, “S. hygroscopicus s. ascomyceticus” 9 S. canus, S. filipinensis, S. spheroides, S. niveus, S. caeruleus, S. lanatus, S. fradiae, S. violaceusniger 10 S. thermovulgaris, S. prasinosporus, S. chromofuscus 11 S. thermoviolaceus s. thermoviolaceus, S. nodosus, S. albus s. albus LMG 19389 12 S. chattanoogensis, S. nigrescens, S. lydicus, S. tubercidicus, S. ramulosus 13 S. rimosus subsp. rimosus (LMG 19390), S. rimosus LMG 19352T 14 S. amakusaensis, S. fulvissimus, S. prasinopilosus, S. bambergiensis, S. poonensis, S. cyanoalbus, S. intermedius, S. nogalater, S. aurantiogriseus, “S. caelicus”, S. violatus, S. prasinus 15 S. griseoruber, S. antibioticus LMG 19334 16 “S. malachiticus”, S. olivaceus LMG 19394T, S. tendae 17 S. bellus, S. flaveolus, S. rochei, S. griseoincarnatus, S. massasporeus, S. erythrogriseus, S. cellulosae

Table 14: Delineation of 17 groups of closely related Streptomyces species based on 16S RFLP fingerprinting using 4 restriction enzymes.

Digestion with a fifth restriction enzyme would certainly have improved the taxonomic discriminative power of 16S RFLP but would become to laborious for application on all validly described Streptomyces species. In order to increase its differentiation capacities significantly, the hypervariable 16S-23S ITS region was included in 16S RFLP as discussed more in detail in Section 6.2.

158 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

6.2 Phylogenetic grouping of streptomycetes using 16S-ITS RFLP fingerprinting

B. Lanoot, M. Vancanneyt, B. Hoste, K. Vandemeulebroecke, M. C. Cnockaert, P. Dawyndt, Z. Liu & J. Swings

Submitted to Research in Microbiology.

------SUMMARY A total number of 463 Streptomyces and Kitasatospora type strains were screened using 16S-ITS RFLP fingerprinting (combined restriction digest using enzymes BstUI and HaeIII). In total 59 clusters could be delineated each comprising multiple strains with nearly identical patterns. A good correlation was found with the phylogeny as revealed by 16S rDNA sequencing. The majority of strains assigned to a particular 16S-ITS RFLP cluster classify in the corresponding 16S sequencing cluster, whether a 16S similarity cut-off value of 97 % or 98 % was used. We conclude that the taxonomic resolution of 16S-ITS RFLP fingerprinting is higher compared to 16S rDNA sequencing and provides a tool to reduce the number of laborious DNA-DNA hybridisations necessary to discover potentially new species within Streptomyces. ------

159 INTRODUCTION

16S rDNA sequences are highly conserved in evolution (Woese, 1987) and provide an excellent means to infer relationships at the intergeneric and –species level (Stackebrandt & Goebel, 1994). In Streptomyces systematics, however, sequences of only about 180 species are available and deposited in public databases. Furthermore, bootstrap values are often too low to delineate stable phylogenetic clusters in the 16S rDNA tree. Hence, it is likely that for novel species descriptions not all nearest neighbours are included in subsequent DNA-DNA hybridisations what may result in the description of junior synonymous species (Hatano et al., 2003; Kim et al., 1999; Labeda & Lyons, 1991a-b, Lanoot et al., 2002, 2004). Awaiting the availability of a complete 16S rDNA framework for the genus Streptomyces, we provide an alternative means to delineate reliable species groups. In total 463 Streptomyces and Kitasatospora type strains were screened using RFLP fingerprinting of the 16S rRNA gene and its adjacent variable 16S-23S ISR region (Hain et al., 1997; Wenner et al., 2002). Its taxonomic usefulness for grouping closely related species was determined upon comparison with a subset of 158 almost complete16S rDNA sequences, part of them determined in this study.

MATERIALS AND METHODS

Strains and growth conditions

A total number of 463 type strains were analysed, consisting of 432 Streptomyces species, 24 subspecies and 7 Kitasatospora species (Annex II). All strains were cultivated on Bennett’s agar and incubated at their appropriate temperature as recommended by the DSMZ and LMG catalogues.

16S-ITS RFLP fingerprinting

An overview of the technique is given in Fig. 29. Genomic DNA was extracted from 100-200 mg cells (wet weight) using a standardized extraction protocol (Lanoot

160 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

et al., 2004). It mainly consists of an EDTA containing washing step followed by a weakening of the cell wall using a combination of the enzymes lysozyme and mutanolysin. Cells were finally lysed using SDS and vortexing with glass beads. Removal of proteins, DNA precipitation and RNAse treatment were performed as described by Pitcher et al. (1989). Amplification of the 16S rRNA gene and adjacent 16S-23S ITS region was performed using the following PCR mix: one PCR reaction (reaction volume 25 µl) contained 12.5 µl Premix solution (Qiagen), 0.61 µl primer pA (50 ng µl-1, details given further below), 0.61 µl primer BL 235R (50 ng µl-1, 5’- GCG CCC TTA AAA ACT TGG -3’, position 3-20 on 23S rDNA, E.coli numbering, Pharmacia), and 100 ng purified DNA. PCR program consisted of the following steps run on a GeneAmp 9600 PCR System (Applied Biosystems): Hot Start 95 °C 5 min, 30 cycles of 94 °C 1 min – 53° C 1’20’’ – 72 °C 1 min, 72 °C 10 min. Amplified products were digested using restriction enzymes BstUI and HaeIII, according to the manufacturers’ recommendations (Biolabs). Digests were loaded on a 8% polyacrylamide gel (1 mm, 1x TAE, 80 W, 300 V, 400 mA, 19 °C) and run for exactly 2 hours. The gels were stained for 20 min. in a 1x TBE bath supplemented with ethidium bromide (1 mg l-1). Destaining was done for 1 min in Milli-Q water. A photograph of the gel was stored on disk as TIFF file through a CCD coupled camera using the ColorVision software. Gels were normalised through the use of molecular weight marker 8 (Boehringer-Mannheim) and subsequent analysis with the software package BioNumerics version 3.0. Of each fingerprint, the first 5.3 % and last of the 14.6 % data points were excluded from the numerical analysis. All bands within a profile were assigned except for those with a peak height of less than 1/4 of the average peak height found within the profile. The similarity among digitized profiles was calculated using the Dice coefficient (band tolerance position 0.86 %). Out of the resultant similarity matrix, a UPGMA dendrogram was derived. An average reproducibility level of 96 % similarity was found among duplicate profiles obtained after new a DNA preparation, PCR, digestion and loading on different gels.

161 DNA PREPARATION AMPLIFICATION OF 16S rDNA RESTRICTION DIGEST – ITS FRAGMENT Bacterial cells ThermoCycler

PCR PCR product

Forward Primer Restriction Enzymes 5’ 3’ 23S rDNA (BstUI, HaeIII) 16S rDNA 16S rDNA 16S rDNA ITS 3’ 5’ ds DNA Reversed Primer Restriction fragments

SCANNING

NORMALISATION & COMBINING OF THE FOUR RESTRICTION PATTERNS

NUMERICAL ANALYSIS PAGE ELECTROPHORESIS

162 Fig. 29: Principle of 16S rDNA-ITS RFLP fingerprinting. 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

16S rDNA sequencing

Genomic DNA was prepared according to the protocol of Lanoot et al. (2004). 16S rDNA was amplified using the combination of a conserved forward primer pA (5’- AGAGTTTGATCCTGGCTCAG-3’), MH1 (5’-AGTTTGATCCTGGCTCAG-3’) or ARI C/T (5’-CTGGCTCAGGAC/TGAACGCTG-3’) and reverse primer MH2 (5’- TACCTTGTTACGACTTCACCCCA-3’) or pH (5’-AAGGAGGTGATCCAGCCGCA-3’), hybridising respectively at positions 8-27, 10-27, 19-38, 1507-1485 and 1541-1522, according to the Escherichia coli numbering system. PCR products were purified using NucleoFast 96 PCR Plates (Macherey-Nagel) according to the manufacturer's instructions. Sequencing reactions were performed using the BigDye Terminator

Cycle Sequencing Kit (Applied Biosystems) and purified using the Montage SEQ96 Sequencing Reaction Cleanup Kit (Millipore), based on the protocols of the manufacturer. Sequencing was achieved using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Primers used for sequencing are listed in Coenye et al. (1999). Sequence assembly was performed using the program AutoAssembler (Applied Biosystems). Almost complete (around 1500 bp) 16S rRNA gene sequences determined in this study and retrieved from the EMBL/GenBank databases were aligned, their editing, alignment and reformatting was performed with the BIOEDIT software version 6.0.7 (Hall, 1999) and ForCon (Raes & Van de Peer, 1999). Evolutionary distances were calculated using the Jukes-Cantor algorithm. A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) with the TREECON program (Van de Peer & De Wachter, 1994). Tree topology was evaluated by bootstrap analysis based on 500 resamplings. The root position of the tree was estimated by using Intrasporangium calvum DSM 43043T as outgroup organism. The complete 16S rDNA sequences of 67 type strains determined in this study have been deposited in EMBL / GenBank under accession numbers AJ781320 to AJ781383 and AJ781753 to AJ781755.

163 RESULTS AND DISCUSSION

Choice of restriction enzymes

Among a collection of commercially available tetra-cutter restriction enzymes three were selected, i.e. BstUI, HaeIII and TaqI being cheap and possessing a GC rich recognition sequence. A combination digest with two restriction enzymes was applied, to obtain more restriction fragments. Three combinations i.e. BstUI-HaeIII, BstUI-TaqI and HaeIII-TaqI were evaluated in silico (GeneCompar, Applied Maths) on combined 16S rDNA and ITS sequence (one sequence) of 12 Streptomyces species downloaded from EMBL / GenBank: S. albidoflavus, S. coelicolor, S. acidiscabies, S. aurantiacus, S. griseus s. griseus, S. griseochromogenes, S. bottropensis, S. diastatochromogenes, S. ipomaeae, S. tendae, S. setonii and S. mauvecolor. Theoretical fingerprints consisted of 10 to 15 bands for all combinations. Restriction enzymes BstUI and HaeIII were finally selected for overall screening because of the highest number of unique bands among the patterns of non related species (S. diastatochromogenes, S. coelicolor, S. griseus s. griseus, S. griseochromogenes and S. mauvecolor (data not shown)) and possessing the best distribution of these bands along the profile.

Evaluation of taxonomic resolution

In a first step 16S-ITS RFLP data were compared for those 158 type strains for which a complete 16S sequence was available. Among these 158 strains analysed, a total number of 34 clusters were visually delineated (R1 to R34, Fig. 29, Table 15) corresponding with a correlation level between 78 and 85 %. In order to correlate our 16S-ITS RFLP and 16S sequencing data, cluster delineation in the 16S rDNA tree was performed on basis of 2 criteria: (i) strains sharing a 16S rDNA similarity above 97%, resulting in 4 major clusters (SI to SIV, Fig. 1), and (ii) strains sharing a 16S rDNA similarity above 98 %, resulting in a total number of 25 subclusters (designated S1 to S25, Fig. 30).

164 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

16S sequencing 16S-ITS RFLP clusters S. rubrogriseus AJ781373 Subclusters** Cluster* 5% 5162 S. violaceolatus AJ781365 41 86 S. humiferus AF503491 S. violaceoruber AF503492 R 22 26 S. lienomycini AJ781353 S 1 89 S. tendae D63873 S. violaceorubidus AJ781374 R 6 46 78 S. ambofaciens M27245 S. paradoxus AJ276570 42 100 S. griseoincarnatus AJ781321 R 1 30 S. erythrogriseus AJ781328 S 2 5 S. griseoflavus AJ781322 4139 S. heliomycini AJ781343 R 5 S. malachitofuscus AJ781347 3 91 S. graminearus AJ781333 R 4 S. glaucus AJ 781332 S 3

39 S. fumanus AJ399463 R 12 19 S. afghaniensis AJ399483 48 S. azureus AJ399470 S 4 S. tuirus AF503493 83 S. hawaiiensis AJ399466 39 S. massasporeus AJ781323 S. luteogriseus AJ399490 7 S. purpurascens AB045888 S I R 1 27 100 S. violarus AJ399477 S. arenae AJ399485 19 S. janthinus AJ 399478 2 97 S. violaceus AJ781755 64 S. violatus AJ399480 S. roseoviolaceus AJ399484 S. odorifer Z76682 100 S. limosus Z76679 S. felleus Z76681 S. canescens Z76684 R 8 S. coelicolor Z76678 S 5 2 S. sampsonii D63871 10 S. albidoflavus Z76676 69 S. eurythermus D63870 S. nogalater AB045886 48 R 4 88 S. gougerotii Z76687 5 13 99 S. rutgersensis subsp. rutgersensis Z76688 R 9 S. intermedius Z76686 R 8 S. coeruleofuscus AJ399473 R 1 S. spinoverrucosus AJ781376 35 62 S. iakyrus AJ399489 13 S. bellus AJ399476 R 1 S. lomondensis AJ781352 45 S. indiaensis AJ781344 R 4 S. variegatus AJ781371 11 45 S. thermoviolaceus subsp. thermoviolaceus Z68095 R 3 53 S. thermoviolaceus subsp. apingens Z68096 S 6 27 S. thermodiastaticus Z68101 7 S. thermocarboxydovorans U94489 R 4 S. bluensis X79324 5 R 7 57 S. somaliensis AJ007403 R 33 15 S. thermocoprophilus AJ007402 S. glomeratus AJ781754 44 R 9 4 S. tricolor AJ781380 100 S 7 R 2 S. rameus AJ781379 S. longisporus AJ399475 R 22 59 S. lanatus AJ399469 S. echinatus AJ 399465 89 S. torulosus AJ781367 60 S. ipomoea AY207593 R 26 39 S. neyagawaensis AJ399493 S. bottropensis D63868 18 63 36 S 8 S. scabiei D63862 23 S. diastatochromogenes D63867 R 25 90 S. olivochromogenes AY094370 32 S. mirabilis AF112180 S 9 R 20 S. tauricus AB045879 S II 100 100 S. albosporeus AJ781327 S 10 R 24 19 3 S. aurantiacus AJ781383 19 S. griseochromogenes AJ310923 R 22 23 S. griseorubiginosus AJ781339 S. ciscaucasicus AY508512 43 R 19 30 S. pseudovenezulae AJ399481 57 S 11 S. galilaeus AB045878 51 R 22 27 S. chartreusis AJ399468 S. resistomycificus AJ310926 22 S. cinnabarinus AJ399487 50 S. capoamus AB045877 97 S. galbus X79852 S 12 90 R 22 S. longwoodensis AJ781356 S. flavovariabilis AJ781334 3

165 97 90 X79852 BLlongwo 16S sequencing 16S-ITS RFLP clusters BLflavov Subclusters** Clusters* 3 28 S. clavuligerus AB045869 S. inusitatus AJ781340 R 13 S. globosus AJ781330 R 31 S. nojiriensis AJ781355 36 77 82 26 50 S. spororaveus AJ781370 100 S. subrutilus X80825 S 13 S. virginiae D85123 99 S. venezulae AB045890 15 39 S. lateritius AJ781326 R 22 S. viridobrunneus AJ781372 41 S 14 37 37 S. tanashiensis AJ781362 S. bikiniensis X79851 R 23 S. laurentii AJ781342 14 R 22 100 S. gobitricini AJ781335 S. lavendofoliae AJ781336 S 15 R 14 18 100 S. graminofaciens AJ781329 S 16 S II S. peuceticus AB045887 R 21 33 S. mauvecolor AJ781358 R 34 53 S. pulveraceus AJ781377 69 S. mediolani AJ781354 91 100 S. atroolivaceus AJ781320 S. mutomycini AJ781357 36 R 23 S. setonii D63872 S 17 34 53 S. fulvorobeus AJ781331 S. argenteolus AB045872 100 S. mashuensis X79323 S. kishiwadensis AJ781338 S 18 R 11 98 S. lydicus Y15507 47 S. chattanoogensis AJ621611 38 S. tubercidicus AJ621612 S. libani s. rufus AJ781351 R 23 50 100 S. platensis AB045882 S. catenulae AJ621613 S 19 53 99 S. chrestomyceticus AJ621609 9 S. albofaciens AB045880 37 R 10 31 84 S. rimosus AB045883 59 S. erumpens AJ621603 R 23 100 27 S. olivaceiscleroticus AJ781350 S. albulus AB024440 S. gibsonii AJ781753 100 R 18 S. rangoonensis AJ781366 S 20 100 S. sporoclivatus AJ781369 S III S. melanosporofaciens AJ271887 6 52 98 S. hygroscopicus AJ391820 R 16 98 S. sporocinereus AJ781368 S 21 63 96 S. endus AJ391821 S. violaceusniger AJ391822 S. malaysiensis AF117304 R 15 2 S. morookaensis AJ781349 S. griseocarneus X99943 R 17 16 58 S. sapporonensis AJ781378 40 S. kashmirensis AJ781337 R 12 72 S. roseoverticillatus AJ781361 R 13 710 S. lilacinus AJ781346 22 S. thioluteus AJ781360 R 12 S. parvisporogenes AJ781348 S 22 S. olivoreticuli AJ781345 74 2 S. biverticillatus AJ781381 44 48 S. syringium AJ781375 R 13 S. viridoflavus AJ781363 100 K. paracochleata U93328 R 28 26 81 K. cochleata U93316 S 23 62 K. cystargineus U93318 51 K. setae AB022868 S. phosalacineus AJ781359 S 24 R 27 100 S. purpureus AJ781324 83 S IV 46 S. aureofaciens Y15504 K. kifunense AB022874 40 R 29 S. misakiensis AJ781325 S 25 93 87 S. purpeofuscus AJ781364 K. azatica U93312 R 30 S. thermovulgaris Z68094 R 32 Intrasporangium calvum AJ566282

166 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

Fig. 30: Neighbour Joining tree based on almost complete 16S rDNA sequences of 158 Streptomyces and Kitasatospora type strains in correlation with corresponding 16S- ITS RFLP fingerprinting data.

*: clusters containing strains with an overall similarity above 97% **: clusters containing strains with an overall similarity above 98% ------

When considering the 4 clusters SI to SIV at the 97 % similarity level, a good correlation was found with 16S-ITS RFLP data. Strains grouping in a particular 16S- ITS RFLP cluster generally group in the same 16S sequencing cluster and often represent a separate subcluster. For instance among cluster SI, 12 16S-ITS RFLP clusters (R1 to R9, R12, R22, R33) could be distinguished as shown in Fig. 30. It is clear from the latter figure that each of these 16S-ITS RFLP clusters consist of a homogeneous set of nearly identical patterns (e.g. R1 and R8). Analogous conclusions are valid for the other three major 16S rDNA clusters (SII to SIV). These data indicate that 16S-ITS RFLP fingerprinting allows, among species yielding 16S sequence similarities above 97 %, to define phylogenetically stable groups at a higher taxonomic level. Within the complex Streptomyces systematics, a 16S sequence similarity cut-off value of 98 % is often applied for species delineation. Below this value two strains most likely share a DNA-DNA homology value less than 70 % (Goodfellow et al., 1997). However, still the number of species for subsequent hybridisations is often too high and a selection of nearest neighbours is made based on the topology of the tree. Researchers often ignore low bootstrap values at the branch points of the tree (Fig. 30). Being aware of the instability of the constructed tree in the present study, a significant correlation was found between our 16S-ITS RFLP data and phylogeny on basis of 16S rDNA sequencing. All strains of 20 16S-ITS RFLP clusters (R2, R3, R5, R6, R10, R11, R14, R15, R16, R18 to R21, R24 to R30) were grouping in a single S subcluster (Fig. 30). For 7 16S-ITS RFLP clusters, a perfect match was found e.g. S. kishiwadensis and S. mashuensis (R11, S18), S. gobitricini and S. lavendofoliae (R14, S15). In other cases, multiple 16S-ITS RFLP clusters were found in a sequencing cluster showing that 16S-ITS RFLP fingerprinting has a higher taxonomic

167 resolution than 16S rDNA sequencing even at a similarity cut-off value of 98 %. Cluster S21 for instance consists of the 16S-ITS RFLP clusters R15 and R16, each grouping strains with nearly identical patterns (Fig. 29). Among the Kitasatospora species group (S23 to S25), formerly belonging to the genus Streptomyces (Zhang et al., 1997), a close relationship was found with the Streptomyces species S. purpeofuscus, S. misakiensis, S. aureofaciens, S. purpureus and S. phosalacineus. Exceptionally, although few variation is found among patterns of strains belonging to a certain 16S-ITS RFLP cluster, corresponding strains are scattered over multiple 16S sequencing clusters e.g. strains belonging to cluster R22 found in sequencing clusters S1, S11, S12, S13 and S14. Within the 16S rDNA tree, 12 strains constituted a separate lineage. In 16S-ITS RFLP fingerprinting all of them had a unique pattern, allowing differentiation from each other and from other clusters delineated. Despite its high reproducibility and easy applicability, 16S-ITS RFLP analysis generates fingerprints and no absolute data (e.g. 16S rDNA sequencing), implicating that generated data are only comparable under highly standardized conditions. On basis of the latter results, we conclude that the cluster delineation is phylogenetically valid and is supported by sequencing information. These observations allow us to extrapolate the conclusions to the whole set of 463 profiles (Annex V). In total 59 clusters are delineated comprising between 2 and 68 strains (Table 15).

In conclusion, 16S-ITS RFLP fingerprinting allowed to group species at a phylogenetic level with a higher taxonomic resolution than 16S sequence analysis. The methodology is therefore useful in species discrimination with a well-considered minimal set of species for DNA-DNA hybridisations studies and further polyphasic analysis.

Acknowledgements. BL is grateful to Nancy Ghysels for excellent technical assistance. This study was financed by the Federal PPS Science Policy of Belgium (grant BL/02/C10) and MOST/NFSC P.R. China (grant 30370002).

168 6. PHYLOGENETIC GROUPING USING 16S-ITS RFLP

Cluster Strains as received

R 1 S. afghaniensis LMG 20390T, S. arenae LMG 20416T, S. azureus LMG 20430T, S. bellus LMG 19401T, S. coeruleofuscus LMG 20482T, S. coeruleorubidus LMG 20484T, S. coerulescens LMG 8590T, S. erythrogriseus LMG 19406T, S. griseoflavus LMG 19344T, S. griseoincarnatus LMG 19316T, S. griseorubens LMG 19931T, S. hawaiiensis LMG 5975T, S. iakyrus LMG 19942T, S. janthinus LMG 8591T, S. labedae LMG 19956T, S. levis LMG 20090T, S. longispororuber LMG 20082T, S. luteogriseus LMG 20073T, S. massasporeus LMG 19362T, S. matensis LMG 20055T, S. paradoxus LMG 20523T, S. purpurascens LMG 20526T, S. roseoviolaceus LMG 8594T, S. tuirus LMG 20299T, S. variabilis LMG 20270T, S. violaceus LMG 20257T, S. violarus LMG 20275T, S. violatus LMG 19397T , S. violaceochromogenes LMG 20271T R 2 S. tricolor LMG 20328T, S. rameus LMG 20326T, S. roseodiastaticus LMG 20327T, S. sclerotialus LMG 20528T R 3 S. thermoviolaceus s. apingens LMG 20307T, S. thermoviolaceus s. thermoviolaceus LMG 19359T R 4 S. cellulosae LMG 19315T, S. djakartensis LMG 21795T, S. gancidicus LMG 19898T, S. graminearus LMG 19904T, S. griseomycini LMG 19883T S. griseostramineus LMG 19932T, S. indiaensis LMG 19961T, S. lomondensis LMG 20088T, S. malachitofuscus LMG 20067T, S. nodosus LMG 19340T, S. nogalater LMG 19338T, S. pseudogriseolus LMG 20252T, S. rubiginosus LMG 20268T, S. thermocarboxydovorans LMG 19860T, S. thermodiastaticus LMG 20302T, S. werraensis LMG 21047T R 5 S. acrimycini LMG 21798T, S. albaduncus LMG 20392T, S. heliomycini LMG 19960T R 6 S. griseofuscus LMG 19885T, S. parvulus LMG 21789T, S. tendae LMG 19314T R 7 S. albus subsp. pathocidicus LMG 20406T, S. bluensis LMG 5969T R 8 S. albidoflavus LMG 21791T, S. anandii LMG 20410T, S. arabicus LMG 20414T, S. canescens LMG 8567T, S. champvatii LMG 20454T, S. coelicolor LMG 8571T, S. eurythermus LMG 20510T, S. felleus LMG 20511T, S. globisporus subsp. caucasicus LMG 19895T, S. griseus subsp. solvifaciens LMG 19952T, S. intermedius LMG 19304T, S. limosus LMG 8570T, S. odorifer LMG 8572T, S. sampsonii LMG 8574T, S. vinaceus LMG 20533T, S. violaceorubidus LMG 20319T R 9 S. chromofuscus LMG 19317T, S. diastaticus subsp. diastaticus LMG 19322T, S. glomeratus LMG 19903T, S. gougerotii LMG 19888T, S. rutgersensis s. rutgersensis LMG 8568T R 10 S. albofaciens LMG 20394T, S. rimosus LMG 19352T R 11 S. kishiwadensis LMG 19939T, S. mashuensis LMG 8603T R 12 S. abikoensis LMG 20386T, S. alanosinicus LMG 20391T, S. ehimensis LMG 20505T, S. fumanus LMG 19882T, S. hiroshimensis LMG 19924T, S. kashmirensis LMG 19937T, S. lavendulocolor LMG 19934T, S. lilacinus LMG 20059T, S. luteoverticillatus LMG 20045T, S. olivoreticuli subsp. olivoreticuli LMG 20050T, S. parvisporogenes LMG 20072T, S. roseolilacinus LMG 20264T, S. thioluteus LMG 21037T R 13 S. albireticuli LMG 20393T, S. antibioticus LMG 20412T, S. biverticillatus LMG 20433T, S. distallicus LMG 20499T, S. eurocidicus LMG 20509T S. hachijoensis LMG 19928T, S. inusitatus LMG 19955T, S. lincolnensis LMG 20068T, S. melanogenes LMG 20056T, S. netropsis LMG 5979T, S. olivaceus LMG 19394T, S. olivoreticuli subsp. cellulophilus LMG 20097T, S. olivoverticillatus LMG 20058T, S. roseoverticillatus LMG 20255T, S. salmonis LMG 20306T, S. syringium LMG 20320T, S. varsoviensis LMG 20083T, S. viridoflavus LMG 20277T R 14 S. gobitricini LMG 19910T, S. lavendofoliae LMG 19935T, S. michiganensis LMG 20042T R 15 S. malaysiensis LMG 20099T, S. orinoci LMG 20079T, S. violaceusniger LMG 19336T R 16 S. antimycoticus LMG 20413T, S. endus LMG 19393T, S. hygroscopicus s. hygroscopicus LMG 19335T, S. melanosporofaciens LMG 20066T, S. rutgersensis subsp. castelarensis LMG 20304T, S. sporocinereus LMG 20311T, S. sporoclivatus LMG 20312T R 17 S. alboverticillatus LMG 20401T, S. ardus LMG 20415T, S. blastmyceticus LMG 20434T S. cinnamoneus subsp. albosporus LMG 20469T, S. cinnamoneus subsp. cinnamoneus LMG 8602T, S. cinnamoneus subsp. lanosus LMG 20471T, S. cinnamoneus subsp. sparsus LMG 20472T, S. griseocarneus LMG 19383T , S. griseoverticillatus LMG 19944T, S. sapporonensis LMG 20324T R 18 S. albus subsp. albus LMG 20405T, S. almquistii LMG 21307T, S. flocculus LMG 19889T S. gibsonii LMG 19912T, S. rangoonensis LMG 20295T R 19 S. ciscaucasicus LMG 20474T, S. clavifer LMG 20476T S. ederensis LMG 20504T, S. longisporoflavus LMG 19347T, S. griseorubiginosus LMG 19941T, S. pseudovenezuelae LMG 20276T R 20 S. cinereoruber subsp. fructofermentans LMG 20463T, S. mirabilis LMG 20076T, S. olivochromogenes LMG 20071T R 21 S. kurssanovii LMG 19933T, S. peucetius LMG 20084T, S. xantholiticus LMG 19402T R 22 S. achromogenes subsp. achromogenes LMG 20387T, S. anthocyanicus LMG 20411T, S. aureocirculatus LMG 21794T, S. avidinii LMG 20428T, S. bobili LMG 20436T, S. bungoensis LMG 20439T, S. canarius LMG 20443T, S. cavourensis subsp. cavourenesis LMG 20450T, S. cinereospinus LMG 20464T, S. cinnamonensis LMG 20468T, S. cirratus LMG 20473T, S. coelescens LMG 20479T S. cremeus LMG 20489T, S. durhamensis LMG 20501T S. flavotricini LMG 19880T, S. galbus LMG 19879T, S. galilaeus LMG 21790T, S. goshikiensis LMG 19884T, S. griseochromogenes LMG 19891T, S. griseoloalbus LMG 21308T, S. griseolosporeus LMG 19962T, S. humidus LMG 19936T, S. humiferus LMG 20519T, S. katrae LMG 19945T, S. kunmingensis LMG 20521T, S. lanatus LMG 19380T, S. lateritius LMG 19372T, S. laurentii LMG 19959T, S. lienomycini LMG 20091T, S. litmocidini LMG 20052T, S. longisporus LMG 20053T, S. longwoodensis LMG 20096T, S. nojiriensis LMG 20094T, S. novaecaesareae LMG 20069T, S. puniceus LMG 20258T, S. polychromogenes LMG 20287T, S. sporoverrucosus LMG 20314T, S. racemochromogenes LMG 20273T, S. roseofulvus LMG 20263T, S. roseolus LMG 20265T, S. rubrogriseus LMG 20318T, S. sannanensis LMG 20329T, S. spiroverticillatus LMG 20254T, S. spororaveus LMG 20313T, S. subrutilus LMG 20294T, S. tanashiensis LMG 20274T, S. termitum LMG 20289T, S. venezulae LMG 19308T, S. violaceolatus LMG 20293T, S. violaceoruber LMG 20256T, S. virginiae LMG 20534T, S. viridobrunneus LMG 20317T, S. xanthophaeus LMG 21039T, S. yokosukanensis LMG 21040T R 23 S. albosporeus subsp. labilomyceticus LMG 20400T, S. albovinaceus LMG 20402T, S. alboviridis LMG 20403T, S. anulatus LMG 19301T, S. argenteolus LMG 5967T, S. atratus LMG 20420T, S. atroolivaceus LMG 19306T, S. baarnensis LMG 20431T, S. badius LMG 19353T, S. bikiniensis LMG 19367T, S. californicus LMG 19309T, S. canferus LMG 20446T, S. catenulae LMG 20449T, S. cavourensis subsp. washingtonensis LMG 20451T, S. chattanoogensis LMG 19339T, S. chrestomyceticus LMG 20457T, S. chryseus LMG 20458T, S. chrysomallus subsp. chrysomallus LMG 20459T, S. cinereoruber subsp. cinereoruber LMG 20462T, S. citreofluorescens LMG 20475T, S. coelicoflavus LMG 20480T, S. cyaneofuscatus LMG 20493T, S. erumpens LMG 20507T, S. exfoliatus LMG 19307T, S. filamentosus LMG 20512T, S. filipinensis LMG 19333T, S. finlayi LMG 19373T, S. fimicarius LMG 21044T, S. flavofuscus LMG 19900T, S. floridae LMG 19899T, S. fluorescens LMG 8579T, S. fulvorobeus LMG 19901T, S. globisporus subsp. globisporus LMG 8578T, S. griseus subsp. alpha LMG 19953T, S. griseus subsp. cretosus LMG 19946T, S. griseus subsp. griseus LMG 19302T, S. helvaticus LMG 19940T, S. hygroscopicus subsp. angustmyceticus LMG 19958T, S. hygroscopicus subsp. decoyicus LMG 19954T, S. hygroscopicus subsp. glebosus LMG 19950T, S. kanamyceticus LMG 19351T, S. libani subsp. libani LMG 20077T, S. libani s. rufus LMG 20087T, S. lipmanii LMG 20047T, S. lydicus LMG 19331T, S. mediolani LMG 20093T, S. microflavus LMG 19327T, S. mutomycini LMG 20098T, S. nashvillensis LMG 20064T, S. nigrescens LMG 19332T, S. olivoviridis LMG 20057T, S. omiyaensis LMG 20075T, S. parvus LMG 20524T, S. platensis LMG 20046T, S. praecox LMG 20290T, S. pulveraceus LMG 20322T, S. ramulosus LMG 19354T, S. rimosus subsp. paromomycinus LMG 20308T, S. roseosporus LMG 20262T, S. rubiginosohelvolus LMG 20267T, S. setonii LMG 20291T, S. sindenensis LMG 21041T, S. showdoensis LMG 20298T, S. tubercidicus LMG 19361T, S. violaceorectus LMG 20281T, S. wedmorensis LMG 21050T, S. violescens LMG 20272T, S. willmorei LMG 21046T, S. zaomyceticus LMG 19853T R 24 S. albosporeus LMG 19403T, S. aurantiacus LMG 19358T, S. tauricus LMG 20301T R 25 S. diastatochromogenes LMG 20498T, S. scabiei LMG 20323T R 26 S. hygroscopicus subsp. ossamyceticus LMG 19951T, S. ipomoeae LMG 20520T, S. neyagawaensis LMG 20080T, S. torulosus LMG 20305T R 27 S. phosalacineus LMG 20102T, S. setae LMG 20529T R 28 K. cochleatus LMG 20478T, K. paracochleatus LMG 20095T, S. cinereorectus LMG 20461T, S. griseoplanus LMG 19923T R 29 K. kifunensis LMG 19957T, S. aureofaciens LMG 5968T, S. avellaneus LMG 20427T, S. misakiensis LMG 19369T, S. psammoticus LMG 20525T, S. purpureus LMG 19368T, S. xanthocidicus LMG 19370T R 30 K. azaticus LMG 20429T, S. atroaurantiacus LMG 20421T, S. herbaricolor LMG 19929T, S. indigoferus LMG 19930T, S. purpeofuscus LMG 20283T

169 R 31 S. globosus LMG 19896T, S. toxytricini LMG 20269T R 32 S. thermogriseus LMG 20532T, S. thermovulgaris LMG 19342T R 33 S. coeruleoprunus LMG 20483T, S. somaliensis LMG 21049T R 34 S. crystallinus LMG 20490T, S. mauvecolor LMG 20100T, S. noboritoensis LMG 19337T R 35 S. flavoviridis LMG 19881T, S. pilosus LMG 20049T R 36 S. aminophilus LMG 19319T, S. cacaoi subsp. cacaoi LMG 19320T R 37 S. daghestanicus LMG 20496T, S. griseoviridis LMG 19321T R 38 S. fervens subsp. melrosporus LMG 19897T, S. roseiscleroticus LMG 20284T, S. ruber LMG 20285T R 39 S. geysiriensis LMG 19893T, S. plicatus LMG 20288T, S. rochei LMG 19313T, S. vinaceusdrappus LMG 20296T R 40 S. asterosporus LMG 20419T, S. aureorectus LMG 19908T, S. calvus LMG 20442T, S. virens LMG 20316T R 41 S. griseoluteus LMG 19356T, S. recifensis LMG 20261T R 42 S. celluloflavus LMG 21796T, S. kasugaensis LMG 19949T R 43 S. alboflavus LMG 21038T, S. fulvissimus LMG 19310T R 44 S. gardneri LMG 19876T, S. glaucusporus LMG 19907T, S. roseoviridis LMG 20266T, S. narbonensis LMG 20043T R 45 S. fradiae LMG 19371T, S. roseoflavus LMG 20535T R 46 S. flaviscleroticus LMG 19886T, S. minutiscleroticus LMG 20062T R 47 S. caeruleus LMG 19399T, S. niveus LMG 19395T, S. spheroides LMG 19392T R 48 S. aureoversilis LMG 20425T, S. rectiverticillatus LMG 20292T R 49 S. hirsutus LMG 19927T, S. prasinopilosus LMG 19345T, S. prasinus LMG 20259T R 50 S. bacillaris LMG 8585T, S. griseobrunneus LMG 19877T R 51 S. phaeopurpureus LMG 20051T, S. phaeoviridis LMG 20061T R 52 S. chibaensis LMG 20456T, S. corchorusii LMG 20488T, S. regensis LMG 20300T R 53 S. flavogriseus LMG 19887T, S. flavovirens LMG 20516T, S. griseolus LMG 19878T, S. nigrifaciens LMG 20048T, S. olivaceoviridis LMG 19324T R 54 S. misionensis LMG 20063T, S. prasinosporus LMG 19346T, S. umbrinus LMG 20280T, S. viridochromogenes LMG 20260T R 55 S. cellostaticus LMG 20452T, S. curacoi LMG 8588T R 56 S. cuspidosporus LMG 20492T, S. mobaraensis LMG 20086T R 57 S. cinerochromogenes LMG 20466T, S. glaucescens LMG 19330T R 58 S. enissocaesilis LMG 20506T, S. sioyaensis LMG 20531T R 59 S. flaveolus LMG 19328T, S. lavenduligriseus LMG 19943T, S. murinus LMG 10475T

Table 15: Delineation of clusters with closely related species among 463 Streptomyces and Kitasatospora type strains using 16S-ITS RFLP fingerprinting. Bold: sequenced in 16S rRNA gene.

170 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Identification of isolates Description of new Streptomyces species

171171 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

172 142 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

7.1 Screening of large numbers isolates

A last main objective of this work was to use the constructed reference frameworks in BOX-PCR and 16S-ITS RFLP fingerprinting to screen large numbers of isolates with the aim of describing new taxa. Through a collaboration between BCCM / LMG Bacteria Collection and CCCM / Institute of Microbiology of the Chinese Academy of Sciences and CCMM / Faculté des Sciences Semlalia de Marrakech (Morocco), a large collection of around 600 strains was isolated from soil samples of geographically different locations (Annex III). All these strains were preserved and identified to the genus level using chemotaxonomic discriminative tests (see Section 1.3). Bioactivity of all strains was determined by applying (1) a microbial model for screening antibiotics (2) cytochemical-enzyme analysis for screening of enzymes and β-lactamase inhibitors and chitin synthetase inhibitors respectively (3) the spermatogonium as cell model for screening antitumor substances (4) a chemical model for detection of alkaloids.

Of the collection strains, a total number of 173 were analysed in protein profiling (see Annex III). All remaining strains (436), including 4 selected Moroccan strains, were screened in BOX-PCR fingerprinting. Subsequent numerical analysis revealed a wealth in patterns illustrating the high bacterial genomic diversity in soil. The majority of the isolates (75 %) had a unique pattern upon comparison with the BOX- PCR patterns of 473 type strains (see Annex III). Part of this diversity has been investigated in depth (batch FXJ and strains A25, A36; see Annex III). Of the 51 isolates screened, 20 isolates had a unique BOX-PCR pattern and so were analysed in 16S-ITS RFLP for phylogenetic positioning within Streptomyces. Twelve of these 20 strains could be allocated to a phylogenetic cluster and require further taxonomic clarification. Eleven strains i.e. FXJ 2, 4, 10, 12, 14, 22, 23, 39 and 46 and A25, A36 had a unique RFLP pattern or only had a limited number of closest neighbours (FXJ 22, FXJ 46 and A36, see Annex III) after comparison with the reference framework. Polyphasic characterization of already three of these strains i.e. FXJ 14, FXJ 46 and A25 proved their separate position in Streptomyces resulting in the proposal of three new species S. glauciniger, S. jiettaisiensis and S. scopiformis respectively (Huang et al., 2004; He et al., submitted; Li et al., 2002).

142 173 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Regarding the bioactivity of the isolates, 75 % of them produce bioactive compounds of which 37 % are antibacterials, 10 % antifiungal, 10 % anti Fusarium oxysporum, 20 % anti Rhizoctonia solani, 7 % produce β-lactamase inhibitors, 2 % produce inhibitor of chitin synthetase and 3 % produce anthracycline. Respectively 6 % and 2 % of the strains showed an amylase activity and produced alkaloids. Few strains produced aclacinomycin, an antitumor glycoside, cyclopeptide antibiotics and a non typical anthracycline (see Annex III). In general there is no correlation between produced bioactive compound and strain relatedness (Fig. 31).

174 143 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Anthracyclin Aclacinomycin Inhibitor of beta-lactamase Inhibitor of chitin synthetase Alkaloids Cyclopeptide antibiotics Nontypical anthracyclin Antitumor glucoside Unknown: R-5974; R-6185

Fig. 31: Correlation between produced bioactive compound and strain relatedness using protein profiling.

144 175 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

176 155 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

7.2 Description of new Streptomyces species

7.2.1 Streptomyces glauciniger sp. nov., a novel mesophilic streptomycete isolated from soil in South China

Y. Huang, W. Li, L. Wang, B. Lanoot, M. Vancanneyt, C. Rodriguez, L. Zhiheng, J. Swings & M. Goodfellow

Redrafted from Int J Syst Evol Microbiol 54, 2085-2089 (2004)

------SUMMARY A polyphasic study was undertaken to establish the taxonomic status of a soil isolate. The organism, isolate FXJ14T, was found to have chemical and morphological properties characteristic of streptomycetes. Phylogenetic analyses based on an almost complete 16S rDNA sequence of the strain and on the 120 nucleotide variable γ-region of this molecule showed that it formed a distinct phyletic line within the range of variation encompassed by the genus Streptomyces. The sharp separation of the organism from representatives of the genus Streptomyces was underpinned by the fact that it gave BOX-PCR and RFLP of 16S rDNA-ITS fingerprints different from those of over 450 validly described Streptomyces species. The isolate also had a profile of phenotypic properties that readily distinguished it from the genotypically close type strains. It is evident from the combination of genotypic and phenotypic data that the organism should be classified as a new species of the genus Streptomyces, for which the name Streptomyces glauciniger sp. nov. is proposed. The type strain is FXJ14T (= AS 4.1858T = LMG 22082T = JCM 12278T). ------

155177 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

INTRODUCTION

The genus Streptomyces remains a hot centre of systematics, not only because streptomycetes are still the most promising source of commercially significant compounds, but also because current molecular biological methods are having an increasing impact on conventional streptomycete systematics (Williams et al., 1983; Kämpfer et al., 1991) that is based on phenotypic characteristics. While the molecular systematic data show that some groups in this genus are overspeciated (Hatano et al., 2003; Lanoot et al., 2002, 2004), polyphasic studies based on a judicious combination of genotypic and phenotypic features continue to bring us novel species and indicate that the whole genus of Streptomyces is underspeciated (Labeda et al., 1997; S. B. Kim et al., 1998; Al-Tai et al., 1999; B. Kim et al., 2000; Kim & Goodfellow, 2002; Li et al., 2002; Saintpierre et al., 2003). The present study, again, described a distinct mesophilic actinomycete, strain FXJ14T, as a new Streptomyces species by using a polyphasic approach.

MATERIALS AND METHODS

Growth of strain

Strain FXJ14T was isolated on a yeast extract-starch agar (Emerson, 1958) plate supplemented with 50 µg ml-1 cycloheximide, which had been seeded with a soil suspension and incubated at 28 °C for 2 weeks. The soil sample was collected from a willow wood in Nanning city, Guangxi Province, China. The isolate was maintained on Bennett’s agar (Jones, 1949) slopes at 4 °C and as glycerol suspensions (20 %, v/v) at – 20 °C. Biomass for the chemotaxonomic and molecular systematic studies was prepared as described previously (Li et al., 2002)

Morphology and biochemical tests

The arrangement of hyphae and spore chains was observed on modified Bennett’s agar (Jones, 1949) and oatmeal agar (ISP medium 3) after 14 days at 28

178 156 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

°C using the cover-slip technique of Kawato & Shinobu (1959). Spore chain morphology and spore surface ornamentation were observed by examining gold- coated dehydrated specimens with a model FEI QUANTA electron microscope. The cultural characteristics were observed on a number of standard media (Table 16) after 14 days at 28 °C. The tested strain was examined for a range of physiological properties using established procedures described by Williams et al. (1983) and Kämpfer et al. (1991).

The isomers of diaminopimelic acid (A2pm) and whole-organism sugars were analysed according to the procedures developed by Hasegawa et al. (1983) and Lechevalier & Lechevalier (1980). Polar lipids were examined and identified using the method of Minnikin et al. (1984). Menaquinones were extracted and purified following Collins (1985), and then analysed by HPLC (Wu et al., 1989). The fatty acids were extracted, methylated and analysed by GC using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The G+C content of the DNA of the tested strain was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli AS 1.365 as a control.

16S rDNA sequencing

Genomic DNA preparation and PCR amplification of 16S rDNA of strain FXJ14T were performed as described by Chun & Goodfellow (1995). The PCR product was purified and directly sequenced as before (Huang et al., 2001). The resultant sequence was aligned manually using Clustal X version 1·8 program (Thompson et al., 1997) with available almost complete sequences of type strains of the family Streptomycetacea and then with corresponding sequences of representative Streptomyces species; in each case, the reference sequences were retrieved from the DDBJ/EMBL/GenBank databases. Phylogenetic trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms from the PHYLIP package version 3·5c (Felsenstein, 1993). Evolutionary distance matrices were generated after Kimura (1980). The resultant

179157 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

unrooted tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings. A partial sequence covering the variable γ-region (120 nucleotides [nt], positions 158-277) of 16S rDNA sequence of strain FXJ14T was also aligned with corresponding nucleotide sequences of nearly 500 Streptomyces type strains retrieved from GenBank. A phylogenetic tree based on these partial sequences was generated using neighbour- joining algorithm (Saitou & Nei, 1987).

BOX-PCR and RFLP of 16S-ITS fingerprinting

BOX-PCR was carried out following Lanoot et al. (2004). For RFLP of 16S rDNA-ITS, the PCR products that were obtained using universal PCR primers was completely digested with restriction enzymes BstUI and HaeIII, and then separated on 8 % polyacrylamide gel. The fingerprinting patterns were compared with corresponding databases containing more than 450 validly described Streptomyces species using softwarepackage BioNumerics version 2.5.

RESULTS AND DISCUSSION

The morphological and chemical features of strain FXJ14T were consistent with its assignment to the genus Streptomyces (Williams et al., 1989; Mafio et al., 1995). The organism formed an extensively branched substrate mycelium, aerial hyphae which carried smooth-surfaced spores in spiral spore chains, and a greyish aerial spore mass on several standard media (Table 16). It contained major amounts of LL- diaminopimelic acid in whole-organism hydrolysates, hexa- and octahydrogenated

(the most abundant) menaquinones with nine isoprene units (MK-9[H6, H8]) as predominant isoprenologues, and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as typical polar lipids (phospholipid type II sensu Lechevalier et al., 1977), but lacked characteristic sugars and mycolic acids. The fatty acid profile was comprised mainly of saturated straight chain and iso- and anteiso-branched chain fatty acids (fatty acid type 2c sensu Kroppenstedt, 1985).

180 158 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Substrate Agar medium Growth Aerial mycelium mycelium

Modified Bennett’s Abundant Dark greyish brown; abundant Greenish black

Czapek Dox Abundant White / Grey; moderate Yellowish white

Glycerol- asparagine (ISP 5) Poor Greyish brown, moderate Greyish olive Inorganic salts- starch (ISP 4) Moderate Light greyish brown, moderate Yellowish white Nutrient Abundant Greyish white; moderate Yellow Oatmeal (ISP 3) Abundant Greyish brown, abundant Not distinctive

Peptone-yeast extract- iron (ISP 6) Abundant Grey; sparse Yellow

Tyrosine (ISP 7) Abundant Greyish cream; moderate Yellowish cream

Yeast extract-malt extract (ISP 2) Abundant Dark greyish brown, abundant Dark brown

Table 16. Growth and cultural characteristics of strain FXJ14T None soluble pigments were produced on above agars.

159 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

The assignment of strain FXJ14T to the genus Streptomyces was also supported by 16S rDNA data. An almost complete 16S rDNA sequence (1428 nt) was determined for the organism. Preliminary phylogenetic analysis, which included available, almost full sequences of type strains of the family Streptomycetacea showed that strain FXJ14T fell within the evolutionary radiation encompassed by the genus Streptomyces (data not show). Despite that the organism was found to be closely associated with some members of the genera Kitasatospora and Streptacidiphilus in a comparison of 16S rDNA using a standard nucleotide- nucleotide BLAST search (Altschul et al., 1997) against DDBJ/EMBL/GenBank, the specific nucleotide signatures of these two genera (Zhang et al., 1997; Kim et al., 2003) were absent in this strain. It is clear from Fig. 32 that the tested strain forms a distinct phyletic line in the 16S rDNA Streptomyces tree. The 16S rDNA similarity values between strain FXJ14T and its nearest neighbours, namely S. rimosus subsp. rimosus JCM 4667T, S. malaysiensis ATB-11T, S. sparsogenes NRRL 2940T and S. yeochonensis CN 732T, were 97.3 % (39 nt differences at 1428 sites), 97.2 % (40 nt differences at 1415 sites), 97.2 % (40 nt differences at 1425 sites) and 97.2 % (40 nt differences at 1425 sites), respectively. 16S rDNA similarities in this range are well below the range recorded for many validly described Streptomyces species (S. B. Kim et al., 1998; Al-Tai et al., 1999; B. Kim et al., 2000; Sembiring et al., 2000; Kim & Goodfellow, 2002; Li et al., 2002; Saintpierre et al., 2003), nor are the relationships between the tested strain and the type strains supported by good bootstrap levels.

182 160 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

T 0.01 78 S. rutgersensis DSM 40077 (Z76688) * 99 S. gougerotii DSM 40324T (Z76687) * 68 S. intermedius DSM 40372T (Z76686)

61 S. limosus DSM 40131T (Z76679)

74 S. coeruleofuscus ISP 5144T (AJ399473) S. thermocarboxydus DSM 44293T (U94490)

S. purpurascens DSM 40310T (AJ310925) 100 54 S. bellus ISP 5185T (AJ399476)

S. speibonae DSM 41797T (AF452714)

S. thermocarboxydovorans DSM 44296T (U94489) S. mexicanus CH-M-1035T (AF441168)

73 S. thermoviolaceus subsp. apingens DSM 41392T (Z68095) * 88 T 99 S. thermoviolaceus subsp. thermoviolaceus DSM 40443 (Z68096) * S. sparsogenes NRRL 2940T (AJ391817)

S.S. gl glauciniauconiggererFXJ14FXJ14T (AAY314782Y314782)) S. yeochonensis CN 732T (AF101415)

S. malaysiensis ATB-11T (AF117304)

S. indonesiensis A4R2T (AJ391835)

S. griseocarneus DSM40004T (X99943)

56 S. rimosus subsp. rimosus JCM 4667T (AB045883) S. thermolineatus DSM 41451T (Z68097) 99 T * 100 S. megasporus DSM 41476 (Z68100) * S. macrosporus DSM 41449T (Z68099)

Fig. 32: Unrooted neighbour-joining tree (Saitou & Nei, 1987) based on almost complete 16S rDNA sequences showing the position of strain FXJ14T in the Streptomyces tree. The asterisks indicate branches that were recovered using least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) algorithms. The numbers at the nodes indicate the levels of bootstrap support (%) based on a neighbour-joining analysis of 1000 resampled data sets; only values above 50 % are given. The scale bar corresponds to 0·01 substitutions per nucleotide position.

161183 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Phylogenetic analysis based on the 120 nt γ-region of 16S rDNA of strain FXJ14T and nearly 500 Streptomyces type strains showed that the organism loosely clustered with the type strains of S. indigoferus and S. herbaricolor (data not shown). However, BOX-PCR and RFLP of 16S-ITS fingerprints sharply differentiated strain FXJ14T from these two type strains. The fingerprint data also served to distinguish strain FXJ14T from the type strains of over 450 Streptomyces species. The BOX- PCR pattern of strain FXJ14T was most similar to those of S. thermoviolaceus subsp. apingens DSM 41392T and S. thermoviolaceus subsp. thermoviolaceus DSM 40443T (Fig. 33), two thermophilic type strains that were readily distinguished from the tested strain by 16S rDNA analysis (Fig. 33), sharing low 16S rDNA similarities of 96·6 % and 96·5 %, respectively. Strain FXJ14T had a RFLP of 16S-ITS pattern most similar to that of S. cuspidosporus IFO 12378T (Fig. 33), but can be easily separated form the latter by phenotypic properties (Table 17). A number of phenotypic properties also easily distinguished the tested strain from the type strains of the most phylogenetically associated species (Table 17), especially by morphological and pigmentation features.

------rep-PCR ------

. fxj-14 .S. thermoviolaceus subsp. apingens LMG 20307 T .S. thermoviolaceus subsp. thermoviolaceus LMG 19359 T BOX-PCR fingerprints of strain FXJ14T and related reference strains

. fxj-14 ..S. cuspidosporus LMG 20492 T

RFLP of 16S-ITS pattern of strain FXJ14T and its closest neighbour

------

Fig. 33: Closest neighbours of FXJ-14 using the constructed reference frameworks in BOX-PCR and 16S-ITS RFLP fingerprinting

162184 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Character 1 2 3 4 5 6 7 8 Aerial spore mass on oatmeal agar Greyish brown Grey Grey; sparse Grey; sparse Smoky black Yellow/White Grey Grey Spore chain Spiral Spiral Rectiflexibles Rectiflexibles Spiral Spiral Spiral Rectiflexibles Spore surface Smooth Warty Smooth Smooth Rugose Smooth Spiny Smooth Melanin production – – – + + – – – Production of diffusible pigments – + – – + + – – Growth on sole carbon sources (1 %, w/v): L-Arabinose + + + + + + + – D-Fructose + + + – + + + – meso-Inositol + + – – + + d – D-Mannitol + + – – + + + – D-Raffinose + + + – + + + – L-Rhamnose + + – – + – + – D-Sucrose + + + – – – + – D-Xylose + + + d + d + –

Table 17. Phenotypic properties separating strain FXJ14T from related Streptomyces species

Strains: 1, strain FXJ14T; 2, S. cuspidosporus IFO 12378T (= AS 4.1686T); 3, S. herbaricolor ISP 5123T (= ATCC 23922T = JCM 4138T); 4, S. indigoferus ISP 5124T (= T T T ATCC 23924T = JCM 4646T); 5, S. malaysiensis ATB-11 (=DSM 41697 ); 6, S. rimosus subsp. rimosus ISP 5260T (= JCM 4667 = NRRL 2234T); 7, S. sparsogenes T T T ISP 5356T (= DSM 40356T = NRRL 2940T); 8, S. yeochonensis CN 732 (= KCTC 9926 = NRRL B-24245 ). Data for the marker strains are taken from previous studies (Shirling & Gottlieb, 1968, 1969; Williams et al., 1983; Lonsdale, 1985; Al-Tai et al., 1999; Kim et al., 2004). +, Positive; –, negative; d, doubtful.

163151

7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Based on a combination of genotypic and phenotypic data, strain FXJ14T merits recognition as a novel species in the genus Streptomyces. Therefore, we propose the name Streptomyces glauciniger sp. nov. for this new taxon.

Description of Streptomyces glauciniger sp. nov.

Streptomyces glauciniger (glau’ci.ni.ger. L. fem. adj. glaucus greenish-grey; L. adj. niger black; N.L. adj. glauciniger greenish black, referring to the colour of colony reverse on modified Bennett’s agar).

Aerobic, Gram-positive mesophilic actinomycete which forms an extensively branched substrate mycelium and aerial hyphae that differentiate into long spiral spore chains with 15 to 20 cylindrical spores per chain. The spore surface is smooth. Soluble pigments are not produced, nor are melanin pigments formed on peptone- yeast extract-iron or tyrosine agars. Additional cultural characteristics on various agar media are given in Table 16. Growth occurs between 10 and 35 °C, and between pH 5·0 and 10·0, but not at 40 °C or at pH 4·0 or 11·0. Growth also occurs in the presence of phenol (0·1 %, w/v), but not in the presence of NaCl (5 %, w/v), streptomycin (10 µg ml-1) or novobiocin (5 µg ml-1). In addition to the properties listed in Table 17, the organism degrades adenine, casein, hypoxanthine, xanthine and starch, but not cellulose or elastin, and use dextrin, D-galactose, D-glucose (all at 1 %, w/v), sodium acetate and sodium citrate (both at 0·1 %, w/v), but not D-maltose (1 %, w/v), as sole carbon sources for energy and growth. Nitrate is reduced. Gelatin is not liquefied. It shows antimicrobial activity against strains of Bacillus subtilis and Candida albicans, but not against strains of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa or Aspergillus niger. Cell wall type

I, phospholipid type II and menaquinone MK-9(H6, H8). The fatty acid profile is

composed of i-C16 :0 (25·4 %), ai-C17 : 0 (17·4 %), ai-C15 : 0 (16·7 %), i-C15 : 0 (9·5 %),

C16 : 0 (7·7 %), i-C17 : 0 (3·8 %), i-C14 : 0 (3·5 %), i-C17 : 1ω9c (3·3 %), ai-C17 : 1ω9c (3·0

%), i H-C16: 1 (2·9 %), C15 : 0 (2·8 %) and C17 : 0 cyclo (2·7 %). The G + C content of DNA is 67·0 mol %. The type strain is FXJ14T (= AS 4.1858T = LMG 22082T = JCM 12278T), isolated from willow woods soil collected in Nanning city, Guangxi Province, China.

186 187 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Acknowledgements. This study was performed in the frame of the Belgian- Chinese program: ‘Identification and classification of actinomycetes, specifically bioactive Streptomyces strains isolated from Chinese soil’, supported by MOST/NSFC China and OSTC Belgium (BL/02/C10). This work was also supported by the NSFC (grant number 30390004) and through the Royal Society-Chinese Academy of Sciences Exchange Scheme (grant no. Q814). The authors are indebted to Prof. R. M. Kroppenstedt (DSMZ, Germany) and Dr. T. Kudo (JCM, Japan) for providing type cultures, and to Mrs. Yamei Zhang for her selfless help.

187 188 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

188 160 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

7.2.2 Streptomyces jietaisiensis sp. nov.,

isolated from soil in North China.

L. He, L. Wang, W. Li, Z. Liu, B. Lanoot, M. Vancanneyt, J. Swings & Y. Huang

Redrafted from Int J Syst Evol Microbiol, submitted

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SUMMARY An actinomycete, strain FXJ46T, was isolated from cypress forest soil in North China and shown to have chemical and morphological properties consistent with streptomycetes. It developed greyish aerial mycelium and pinkish brown substrate mycelium on oatmeal agar. Phylogenetic analyses based on an almost complete 16S rRNA gene sequence of the organism and on the 120 nucleotide variable γ-region of this molecule showed that it formed a distinct, also closely associated, line with the type strain of Streptomyces griseoaurantiacus in the Streptomyces trees. However, the DNA-DNA relatedness between the two strains was only 48·8 %. A number of phenotypic properties also readily distinguished the isolate from S. griseoaurantiacus and related validly described Streptomyces

186species. It is proposed, therefore, that the organism be classified as a new species of the genus Streptomyces, for which the name Streptomyces jietaisiensis sp. nov. is proposed. The type strain is FXJ46T (= AS 4.1859T = JCM 12279T). ------

160 189 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

INTRODUCTION

In the last decade, the taxonomy of the genus Streptomyces has undergone much improvement, in part due to an increased interest in the identification of these organisms, particularly those from soil environment. The use of molecular taxonomic methods such as 16S rRNA gene sequencing has done much to aid the classification of this complex group and the recognition of novel species (Al-Tai et al., 1999; Kim & Goodfellow, 2002; S. B. Kim et al., 1998, 2004; B. Kim et al., 2000; Li et al., 2002; Saintpierre et al., 2003). However, due to the large number of species, the complete 16S rRNA gene sequences have not been determined for many type strains of this genus. The incomplete sequence data may result in misclassification. (Kataoka et al. 1997; Anderson & Wellington, 2001). In this study, therefore, the partial sequence (120 nucleotides [nt]) data covering the variable γ-region of the 16S rRNA gene of streptomycetes were also phylogenetically analysied to classify strain FXJ46T. While phylogenetic evidence showed that it was closely related to Streptomyces griseoaurantiacus DSM 40430T, polyphasic studies based on a judicious combination of genotypic and phenotypic features brought us a novel species of Streptomyces, Streptomyces jietaisiensis sp. nov.

MATERIALS AND METHODS

Growth of strain

Strain FXJ46T was isolated on a yeast extract-starch agar (Emerson, 1958) plate supplemented with 50 µg ml-1 cycloheximide, which had been seeded with a soil sample suspension and incubated for 14 days at 28 °C. The soil sample was collected from a cypress forest, at Jietaisi, Beijing, China. The isolate was maintained on yeast extract-starch slopes at 4 °C and as glycerol suspensions (20 %, v/v) at – 20 °C.

190 161 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Morphology and biochemical tests

Biomass for the chemotaxonomic and molecular systematic studies was173 prepared as described previously (Li et al., 2002). The morphological characteristic of strain FXJ46T was examined by light- and scanning electron microscope (SEM) of 14 days culture grown on oatmeal agar and inorganic salt-starch agar (ISP medium 4). The cover-slip technique (Kawato, 1959; Zhou et al., 1998) was used to observe the hyphae and spore chain characters by light microscope. Spore chain morphology and spore surface ornamentation were studied by examining gold-coated dehydrated specimens with a model FEI QUANTA electron microscope. The cultural features were observed on a number of standard media (Table 18) after 14 days at 28 °C. The tested strain was examined for a range of biochemical and physiological properties as described by Williams et al. (1983) and Kämpfer et al. (1991).

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Table 18. Comparison of cultural characteristics of strain FXJ46T and Streptomyces griseoaurantiacus DSM 40430T

Aerial mycelium Substrate mycelium Agar medium FXJ 46T DSM 40430T FXJ 46T DSM 40430T

Czapek Dox Grey; moderate White, sparse Pinkish cream Pink

Glycerol- asparagine (ISP 5) Grey, Reddish grey, moderate Grey Greyish yellowish pink

Inorganic slats- starch (ISP 4) Pale grey, abundant Brown, abundant Orange-pink Reddish brown

Nutrient None None Light pinkish brown Light brown

Oatmeal (ISP 3) Smoky grey, abundant Smoky grey, abundant Light pinkish brown Brownish yellow

Peptone-yeast extract- iron (ISP 6) Grey; spares None Yellowish brown Cream

Tyrosine (ISP 7) Pale grey, abundant Greyish black, abundant Whitish grey Brownish black

Yeast extract-malt extract (ISP 2) Mouse grey, abundant White, moderate Orange brown Reddish orange

Yeast entract-starch Grey, abundant Pale grey, abundant Light-vinaceous-purple Light reddish brown

No pigments were formed on the listed agars.

175163 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

The isomers of diaminopimelic acid (DAP) and whole-organism sugars were analysed following the procedures developed by Hasegawa et al. (1983) and Lechevalier & Lechevalier (1980). Menaquinones were extracted and purified according to Collins (1985), and analysed by HPLC. Polar lipids were examined and identified using the method of Minnikin et al. (1984). The fatty acids were extracted, methylated and analysed by GC using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The guanine- plus-cytosine content of the DNA was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli AS 1.365 as a control.

16S rDNA sequencing

Genomic DNA preparation and PCR amplification of 16S rRNA gene of strain FXJ46T were carried out using the procedure of Chun & Goodfellow (1995). The PCR products were sequenced using the method of Huang et al. (2001). Clustal X version 1.8 program (Thompson et al., 1997) was used for multiple alignment with available almost complete sequences of type strains of the family Streptomycetaceae and then with corresponding sequences of representative Streptomyces species; in each case, the reference sequences were retrieved from the DDBJ/EMBL/GenBank databases. The least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms from the PHYLIP package version 3·5c (Felsenstein, 1993) were used to infer the phylogenetic trees. Evolutionary distance matrices were generated as described by Kimura (1980). Tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings. The partial sequence covering the variable γ-region (120 nt, positions 158-277) of 16S rRNA gene sequence of strain FXJ46T was also compared with corresponding nucleotide sequences of 485 Streptomyces type strains retrieved from GenBank. A phylogenetic tree based on these partial sequences was constructed using neighbour-joining algorithm (Saitou & Nei, 1987).

164 193 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

DNA-DNA hybridizations

Levels of DNA-DNA relatedness between strain FXJ46T and S. griseoaurantiacus DSM 40430T were determined according to the micro-well method (Christensen et al., 2000; Ezaki et al., 1989), using a FLUOstar OPTIMA microplate reader (BMG LABTECH).

RESULTS AND DISCUSSION

The organism exhibited a range of chemotaxonomic and phenotypic properties typical of members of the genus Streptomyces. It formed an extensively branched substrate mycelium, aerial hyphae which carried smooth-surfaced spores in rectiflexibiles spore chains (supplementary data in IJSEM online), and a greyish aerial spore mass on several standard media (Table 18). It contained LL- diaminopimelic acid in whole-organism hydrolysates, octa-, hexa-, and a minor of

tetrahydrogenated menaquinones with nine isoprene units (MK-9[H8, H6, H4]) as isoprenologues, and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as major polar lipids (phospholipid type II sensu Lechevalier et al., 1977). The fatty acid profile was comprised mainly of saturated straight chain and iso- and anteiso-branched chain fatty acids (fatty acid type 2c sensu Kroppenstedt, 1985).

An almost complete 16S rRNA gene sequence (1426 nt) was determined for the organism. Primary sequence analysis with those of representatives of the family Streptomycetaceae confirmed that the unknown isolate was closely related to species of the genus Streptomyces. The highest 16S rRNA gene sequence similarity value was found with Streptomyces nogalater JCM 4799T (97·5 %). However, in the phylogenetic tree based on the 120 nt γ-region, the strain showed a very close affinity with type strain of S. griseoaurantiacus, supported by a 97 % bootstrap level, and was loosely related to the others (Fig. 34). So the almost complete 16S rRNA gene sequence (1458 nt) of S. griseoaurantiacus DSM 40430T (= JCM 4763T) was also obtained in this study, and added to 16S rRNA gene analysis. Fig. 35 demonstrates that strain FXJ46T forms a distinct phyletic line with S. griseoaurantiacus DSM

194 165 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

40430T, supported by the results based on three tree-making algorithms and by a 100 % bootstrap value with the neighbour-joining method. The divergence of 16S rRNA gene sequence between the two strains was 0·2 % (3 nt differences at 1421 sites). The isolate also showed moderately low sequence divergences with other Streptomyces type strains, namely Streptomyces nogalater JCM 4799T (2·2 %), Streptomyces ambofaciens ATCC 23877T (2·2 %), Streptomyces rutgersensis subsp. rutgersensis DSM 40077T (2·3 %), Streptomyces intermedius DSM 40372T (2·3 %), Streptomyces gougerotii DSM 40324T (2·4 %) and Streptomyces eurythermus DSM 40014T (2·6 %), respectively. Therefore, the well-selected phenotypic traits and DNA- DNA pairing data are needed (Kim et al., 1998; Labeda, 1988) to clarify the finer relationships between the isolate and the phylogenetically close species.

166 195 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

T 97 S. griseoaurantiacus JCM 4763 (D44342) 0.02 Strain FXJ46T S. spiralis JCM 3302T (D43983) T 99 S. aureoverticillatus JCM4347 (D44066) S. aureomonopodiales JCM4456T (D44153) S. poonensis JCM4815T (D44382) 60 67 S. sclerotialus JCM3039T (D43973) S. fumigatiscleroticus JCM3101T (D43979) S. ruber JCM3131T (AB018203) S. roseiscleroticus JCM4823T (AB018206) S. mashuensis JCM4059T (D43993) S. kishiwadensis JCM4486T (D44180) T 97 S. thermovulgaris JCM4520 (D44208) S. thermonitrificans JCM4841T (D44402) S. echinatus JCM4574T (D44242) S. coerulescens JCM4360T (D44077) S. prasinosporus JCM4816T (D44383) 79 S. chromofuscus JCM4354T (D44072) S. nogalater JCM4799T (D44370)

Fig. 34: Partial neighbour-joining tree (Saitou & Nei, 1987) based on the 120 nt γ -region of 16S rDNA showing the relationships between strain FXJ46T and other Streptomyces species. Bootstrap values (%) of 1000 replication were shown at the nodes of the tree; only values above 50 % are given. The scale bar corresponds to 0·02 substitutions per nucleotide position.

167 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

S. violaceoruber DSM 40049T (AF503492) 55 T 55 S. coelescens DSM 40421 (AF503496) 100 S. coelicolor A3(2) (AL939124) f, m S. cyanogenus DSM 40426T (AF503500) f 76 f f, m, p S. violaceolatus DSM 40438T (AF503497) S. ambofaciens ATCC 23877T (M27245) 90 f S. collinus DSM 40129T (AJ306623) f f, p 81 S. paradoxus DSM 43350T (AJ276570)

53 T f S. eurythermus ATCC14975T (D63870) S. nogalater JCM 4799T (AB045886) S. intermedius DSM 40372T (Z76686) 75 100 f f, m, p S. rutgersensis subsp. rutgersensis DSM 40077T (Z76688) 89 f, m, p S. gougerotii DSM 40324T (Z76687) 84 T f 100 S. jietaisiensis FXJ46 (AY314783) f, m S. griseoaurantiacus DSM 40430T (AY450561) S. scopiformis AS 4.1331T (AF184081) S. mashuensis DSM40221T (X79323)

T 98 S. subrutilus DSM 40445 (X80825) m 98 m, p T 84 100 S. lavendulae subsp. lavendulae IFO 12789 (D85116) m T 94 S. griseus subsp. griseus ISP 5236 (AY094371) m S. griseocarneus DSM40004T (X99943) 79 m S. rimosus subsp. rimosus JCM 4667T (AB045883) 95 T m, p 100 S. violaceusniger NRRL B-1476T (AJ391823) m, p S. hygroscopicus subsp. hygroscopicus NRRL 2387T (AJ391820)

0.01

Fig. 35: Unrooted neighbour-joining tree (Saitou & Nei, 1987) based on almost complete 16S rRNA gene sequences showing the phylogenetic relationships between strain FXJ46T and related Streptomyces species. “f”, “m” and “p” indicate branches that were also recovered when the Fitch-Margoliash (f), maximum-likelihood (m) and maximum- parsimony (p) tree-making algorithms were used respectively. Bootstrap values (%) of 1000 replication were shown at the nodes of the tree; only values above 50 % are given. The scale bar corresponds to 0·01 substitutions per nucleotide position.

168 197 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

The DNA-DNA relatedness between strain FXJ46T and S. griseoaurantiacus DSM 40430T was 48·8 %, which is well below the 70 % cut-off point for recognition of genomic species (Wayne et al., 1987). Thus suggested that the tested strain should be considered as a separate species. Furthermore, the strain was distinguished from S. griseoaurantiacus DSM 40430T in cultural characteristics on a number of standard media (Table 18), in spore chain characters and in utilization of at least two sole carbon sources (Table 19). In addition, unlike S. griseoaurantiacus DSM 40430T, strain FXJ46T could grow at 37 °C and on 5 % NaCl. The organism was also differed from the other moderately close species by comparison of a set of phenotypic properties (Table 19).

Based on the genotypic, chemotaxonomic and phenotypic evidence, strain FXJ46T warrants classification as a new species in the genus Streptomyces, for which the name Streptomyces jietaisiensis sp. nov. is proposed.

198 169 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Character 1 2 3 4 5 6 7 8 Aerial spore mass on oatmeal Yellow or Smoky grey Smoky grey Brownish gray Grey Yellow inadequate Grey agar inadequate Rectiflexibiles to Spore chain Rectiflexibles Spiral Rectiflexibiles Spiral Rectiflexibiles Rectiflexibiles Rectiflexibiles retinaculiaperti Spore surface Smooth Smooth Smooth Smooth to warty Smooth Smooth Smooth Smooth Melanin production – – – – – – – + Production of diffusible pigments – d Red or yellow Yellow or – – Yellow or – – – Growth on sole carbon sources

(1 %, w/v): L-Arabinose + – d + + + – + D-Fructose + + – + + + + + meso-Inositol + + – + – – d d D-Raffinose + d + – – – – d L-Rhamnose + + – + – – – d D-Sucrose – + d d – – – +

Table 19. Phenotypic properties of strain FXJ46T and related Streptomyces species Strains: 1, strain FXJ46T; 2, S. griseoaurantiacus DSM 40430T (= JCM 4763T); 3, S. nogalater JCM 4799T; 4, S. ambofaciens ATCC 23877T; 5, S. rutgersensis subsp. rutgersensis DSM 40077T; 6, S. intermedius DSM 40372T; 7, S. gougerotii DSM 40324T; 8, S. eurythermus DSM 40014T(= ATCC 14975T). Data for reference strains are taken from (Shirling & Gottlieb, 1968a, 1968b, 1969, 1972). +, Positive; –, negative; d, doubtful.

170 182 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Description of Streptomyces jietaisiensis sp. nov.

Streptomyces jietaisiensis (jie.ta.si.en’sis. M. L. adj. jietaisiensis pertaining to Jietaisi, a place in suburb of Beijing, where the organism was isolated). The organism is aerobic, Gram-positive and mesophilic. The colour of aerial and substrate mycelium on various solid media is given in Table 18. Spore chains with 10 to 20 spores are rectiflexibiles. The spore surface is smooth. Diffusible pigments are not produced, nor are melanin pigments formed on peptone-yeast extract-iron or tyrosine agars. Growth occurs between 10 and 40 °C, and between pH 5·0 and 10·0, but not at 45 °C or at pH 4·0 or 11·0. Growth also occurs in the presence of 5 % (w/v) NaCl, but not in the presence of streptomycin (10 µg ml-1) or novobiocin (5 µg ml-1). Adonitol, cellobiose, dextrin, galactose, D-glucose, glycogen, inulin, D-lactose, maltose, mannitol, mannose, D-melezitose, D-ribose, salicin, D-trehalose, xylan, xylose, (all at 1 %, w/v), L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamic acid, L-histidine, L- isoleucine, L-phenylalanine, sodium acetate, sodium citrate, sodium oxalate, sodium pyruvate, L-threonine and L-valine (all at 0.1 %, w/v) are used as sole carbon sources for energy and growth, but not glycerol, glycine, D-sorbitol, xylitol (all at 1 %, w/v), or DL-aminobutyric acid (at 0.1 %, w/v). L-alanine, L-arginine, L-aspartic acid, L- glutamic acid and L-phenylalanine (all at 0.1 %, w/v) are metabolised as sole carbon and nitrogen sources, but not L-isoleucine (at 0.1 %, w/v). Cell wall type I,

phospholipid type II and menaquinone MK-9 (H8, H6, H4). The fatty acid profile is

composed of ai-C15 : 0 (35·7 %), ai-C17 : 0 (18·9 %), i-C16 : 0 (14·8 %), ai-C17 : 1ω9c (8·1

%), C16 : 0 (6·2 %), i-C16 : 1 (4·5 %), i-C15 : 0 (4·3 %), C16 : 1ω7c (2·33 %), i-C17 : 1ω9c

(1·8 %), i-C17 : 0 (1·7 %) and i-C14: 0 (1·7 %). The G + C content of the DNA is 72.3 mol %. Additional characteristics are listed in Table 19. The type strain is FXJ46T (= AS 4.1859T = JCM 12279T), isolated from cypress forest soil collected at Jietaisi, Beijing, China.

Acknowledgements This work was supported by the Natural Science Foundation of China (NSFC, grant number 30370002) and the Belgian Public Science Policy (ref BL/02/C10). The authors are grateful to Prof. R. M. Kroppenstedt (DSMZ, Germany) and Dr. T. Kudo (JCM, Japan) for providing type cultures, and to Mrs. Yamei Zhang for her assistance in isolation.

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7.2.3 Streptomyces scopiformis sp. nov., a novel streptomycete

species with fastigiate spore chains

W. Li, B. Lanoot, Y. Zhang, M. Vancanneyt, J. Swings, & Z. Liu

Redrafted from Int J Syst Evol Microbiol 52, 1629 – 1633 (2002).

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SUMMARY A distinct actinomycete strain was isolated from rhizosphere soil of Tsuga chinensis. The isolate, designated A25T, was assigned to the genus Streptomyces on the basis of morphological and chemotaxonomic criteria, and was examined using a polyphasic approach. An almost complete 16S rDNA sequence of the isolate was determined and compared with representative streptomycetes. The 16S rDNA data not only support classification of the strain in the genus Streptomyces but also showed that it formed a separate phyletic line. DNA-DNA hybridization between strain A25T and close related reference strains confirmed that strain A25T is a new taxon of Streptomyces. It is proposed, therefore that the strain A25T(=AS4.1331T=LMG20251T) is classified in the genus Streptomyces as Streptomyces scopiformis sp. nov. ------

172 201 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

INTRODUCTION

The genus Streptomyces was proposed by Waksman & Henrici (1943) for aerobic, spore-forming actinomycetes and classified in the family Streptomycetaceae on the basis of morphological and cell wall chemotaxonomic characters. The taxon currently accommodates aerobic gram-positive bacteria that have high DNA G+C% content (69-78 mol%), present LL-diaminopimelic acid and absence of characteristic sugars in the cell wall (Cell wall type I, according to Lechevalier & Lechevalier, 1970), and that produce extensive branching substrates mycelia and aerial hyphae (Williams, et al. 1983, 1989; Embley & Stackebrandt, 1994). With more than 500 validly described species and subspecies, the taxon currently contains the largest number of species in the domain Bacteria (Hain, et al. 1997). Molecular systematic methods, notably 16S rDNA phylogenetic analysis, are giving increasing impact on Streptomyces systematic (Kim, 1998). Due to the large number of species, the complete 16S rDNA sequences have not been determined for many type strains. The incomplete sequence data may result in misclassification. It had been reported that the γ variable region of 16S rDNA could be used to resolve inter- and intraspecies relationships within the streptomycetes (Kataoka et al. 1997; Anderson & Wellington, 2001). Hence the partial sequence covering γ region (from 158-276) of 485 streptomycetes had been sequenced and deposited in GenBank. Phylogenetic analysis by comparing with those sequence data can provide useful information for classification and diminish wrong identification. The isolate A25T was reported previously as an invalid new taxon of “Streptomyces scopiformis” producing distinct broom-like spore chains (Liu & Zhang, 1996). In the present study, strain A25T was subjected to a polyphasic taxonomic analysis, an almost complete 16S rRNA gene (rDNA) was sequenced and the phylogenetic relationships were examined using different tree-making methods. DNA- DNA hybridization of isolate A25T and its closest neighbours were studied in order to reveal whether the isolate represents a new taxon within the genus Streptomyces.

202 173 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

METHODS Organisms and cultural conditions. Strain A25T was isolated on glycerol-asparagine agar plate (glucose, 10 g; L- asparagine, 0.5 g; agar, 20 g; distilled water 1000 ml; pH 7.2-7.4) seeded with a soil sample suspension and incubated for 14 day at 28 °C. The soil sample was collected from rhizosphere of Chinese hemlock (Tsuga chinensis) in Lushan Mountain, Jiangxi190 province, China. The isolate and reference strains were maintained on Bennett’s agar (Williams et al., 1983) slopes at 4 °C and glycerol suspension (20 %, v/v) at –20 °C. Biomass for the chemotaxonomic and molecular studies were prepared by growing all of the organisms in 250 ml flasks containing 80 ml of Bennett’s medium (prepared by 0.01 M phosphate buffer, pH 7.0) and shaken for 4 d at 28 °C. The flasks were inoculated by transferring the 4 d old culture on Bennett’s agar slopes (prepared in 0.01 M phosphate buffer, pH 7.0) with 2 ml 0.1M phosphate buffer (pH 7.0). The cells for chemical studies were washed in distilled water and freeze-dried; those for molecular systematic investigations were washed twice in distilled water and Tris-EDTA buffer (0.03 M Tris, 0.1 M EDTA, pH 8.0) and stored at –20 °C until required.

Cultural and morphological properties. The aerial hyphae, substrate mycelium and spore chain morphology was examined by light- and scanning electron microscope (SEM) of 14 d culture grown on Bennett’s agar and inorganic salt/starch agar (ISP medium 4). The coverslip technique (Zhou et al., 1998; Kawato, 1959) was used for observing the hyphae and spore chain characters by light microscope. Spore morphology was studied by examining gold-coated dehydrated specimens with a model HITACHI-570 electron microscope. The cultural characteristics were observed on a number of medium for 14 d at 28 °C.

Biochemical and physiological properties. The test strain were examined for a broad range of biochemical and physiological characteristics as described by Kämpfer et al. (1991), Williams et al. (1983) and Al- Tai et al. (1999).

174 203 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Chemotaxonomy. The diagnostic isomers of diaminopimelic acid (DAP) and whole-organism sugars of the test strain were analyzed following the procedures developed by Hasegawa et al. (1983) and Lechevalier & Lechevalier (1977). Menaquinones and phospholipids were extracted from freeze-dried biomass and purified according to Collins (1985) and Lechevalier and Lechevalier (1970) and analyzed by HPLC (Warwick, 1994). The resultant fatty acid was prepared and analyzed using the procedure described191 by Minnikin et al. (1980).

DNA G+C mol % composition. The G+C content of the DNA of the test strains was determined using the thermal denaturation method of Marmur & Doty (1962) with Escherichia coli AS1.365 as control.

16S rDNA sequencing . Genomic DNA of test strains was isolated using the procedure of Chun & Goodfellow (1995). PCR amplification of 16S rDNA was carried out as described previously (Kim et al. 1996). The PCR products were sequenced using the method of Lu et al. (2001) on an Applied Biosystems DNA Sequencer (model 373A) and the software provided by the manufacturer.

Phylogenetic analysis The almost complete 16S rDNA sequence of strain A25T was aligned manually with the available streptomycetes nucleotide sequences that retrieved from the EMBL/GenBank and RDP (Ribosomal Database Project, Maidak et al., 1997) using Clustal X1.8 program (Thompson et al. 1997). Phylogenetic trees were inferred by using three tree-making algorithms, namely, the Neighbour-Joining (Saitou & Nei, 1987), Fitch-Margoliash (Fitch & Margoliash, 1967), Maximum-likelihood (Felsenstein, 1981) treeing algorithms from the PHYLIP package (Felsenstein, 1993). Evolutionary distance matrices were generated as described by Kimura (1980). Tree topologies were evaluated by carrying out bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset using the SEQBOOT and CONSENSE program provided by the PHYLIP package (Felsenstein, 1993).

204 175 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

The partial sequence covering γ region (120 bp) from 16S rDNA sequence of strain A25T was compared with all available sequences in GenBank. The partial sequence was also aligned with γ region partial sequences of 485 streptomycetes and used for construction Neighbour-Joining tree.

DNA-DNA relatedness studies Levels of DNA-DNA relatedness between strain A25T and the type strains of S. ambofaciens AS4.1528T, S. coerulescens JCM 4358T, S. caelestris JCM 4218T, S. nogalater JCM 4799 T, S. intermedius JCM 4483T, S. albidoflavus JCM 4446T and S. lateritius JCM 4389T were determined according to the thermal renaturation method (De Ley et al., 1970; Huβet al. 1983; Yassin et al., 1993), using a UV-1206 spectrophotometer (SHIMADZU) equipped with a TB-85 thermo bath.

RESULTS AND DISCUSSION

Almost complete 16S rDNA sequences of strain A25T (1402nt) were determined. The sequence data clearly showed that strain A25T is a member of the genus Streptomyces. The chemotaxonomic data confirmed the generic assignment. The presence of LL-DAP and absence of characteristic sugars in whole-cell hydrolysates showed that strain A25T had a wall type I (Lechevalier & Lechevalier, 1970). The strain also contained tetra- and hexahydrogenated menaquinone with nine isoprene unit as the predominant isoprenologue. The phospholipid pattern is type PII (containing phosphatidylethanolamine and diphosphatidylglycerol). Strain A25T showed a typical fatty acid profile of Streptomyces (type IIc according to Kroppenstedt, 1985) with iso-C15:0 (21.5 %), anteiso-C15:0 (13.2%), iso-C16:0 (18.9 %) and C16:0 (13.5 %) as predominant compositions. The DNA G+C mol% composition of strain A25T is 71 %. The phenotypic properties are also consistent with the classification of strain A25T in the genus Streptomyces. Strain A25T grew poorly on inorganic media, while grew well on organic media, producing abundant branching mycelium. The substrate mycelium is light blue/Berlin blue to brown blue, consists of septate and swollen elements and not fragment. The aerial mycelium tends to blue grey/light blue grey and differentiates into long, rectiflexibles spore chains with spiny surface. The spore

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chains are derived from the point of a sporosphores and often arranging in fastigiate structure (Figure 36). No verticillate structure is detected. The fastigiate structure of strain A25T is distinct from that of other streptomycetes. Verticillate streptomycetes also producing similar structure, but the verticillate spore chains formed regularly with smooth spore surface, whereas the fastigiate spore chains produced random and the spore surface is spiny. The diagnostic characteristics of strain A25T and related strains are listed in Table 20.

Strain A25T S. ambofaciens S. coerulescens S. coelicolor Characteristic AS 4.1331T AS 4.1528 T JCM 4389T DSM 40233 T

Spore chain RF RF SP RF morphology

Spore surface Spiny Smooth Spiny Smooth

Melanoid pigment - - + -

Diffusible pigment - - + +

pH indicator + - - +

D-Glucose + + + +

D-Xylose + + + +

L-Arabinose + + + +

L-Rhamnose + + + -

D-Fructose + + + +

Raffinose - - + -

D-Galactose + + + +

D-Mannitol - + + +

i-Inositol + + + -

Sucrose + D + -

Starch + + - +

NO3 reduce + - + +

Growth at 45 ºC + + - -

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Table 20: Diagnostic characteristics for strain A25T and related strains.

Abbreviations: RF, rectiflexibiles; SP, spirales; +, Positive; -, negative. D, doubt.

Fig. 36. Scanning electron micrograph of strain A25T grown on Bennett’s agar for 14-day at 28 °C.

It is clear from the phylogenetic tree (Fig. 37) that A25T forms a separate single strain branch from representatives of the genus Streptomyces, notably from its nearest neighbours Streptomyces ambofaciens (97.75 %), “Streptomyces coelicolor” A3(2) (97.56 %), and Streptomyces coerulescens (97.55 %). The branch was supported by 74 % bootstrap value and by the results of Fitch-Margoliash and Maximum-likelihood methods.

178 207 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

Streptomyces sampsonii ATCC25495T (D63871) Streptomyces felleus DSM 40130T (Z76681) Streptomyces limosus DSM 40131T (Z76679) Streptomyces coelicolor DSM 40233T (Z76678) Streptomyces canescens DSM 40001T (Z76684) 100 Streptomyces odorifer DSM 40347T (Z76682) m,f Streptomyces albidoflavus DSM 40455 T (Z76676 ) 99 m,f Streptomyces intermedius DSM 40372T (Z76686) 90 Streptomyces rutgersensis DSM 40077T (Z76688) 83 m,f Streptomyces gougerotii DSM 40324T (Z76687) 65 Streptomyces coeruleofuscus ISP 5144T (AJ399473) 77 Streptomyces pallidus ISP 5531T (AJ399492) Streptomyces fumanus ISP 5154T (AJ399463) 100 Streptomyces coerulescens ISP 5146 T (AJ399462) Streptomyces caelestris NRRL 2418T (X80824) 64 Streptomyces afghaniensis ISP 5228 T (AJ399483) Streptomyces steffisburgensis JCM 4833T (AB045889) Streptomyces nogalater JCM 4799T (AB045886) T 74 m,f Streptomyces nodosus ATCC14899 (AF114035) Streptomyces eurythermus ATCC14975T (D63870) Streptomyces ambofaciens ATCC 23877T (M27245) 93 m,f “Streptomyces coelicolor “ A3(2) (AL79345) m,f Streptomyces tendae ATCC19812T (D63873) Strain A25T AS4.1331T (AF184081) 75 Streptomyces albofaciens JCM 4342T (AB045880) Streptomyces lydicus ATCC 25470T (Y15507) S. rimosus subsp. rimosus JCM 4667T (AB045883) Streptomyces albulus ISP 5492T (AB024440) T ( 56 Streptomyces griseocarneus DSM40004 X99943) Streptomyces cattleya JCM 4925T (AB045871) Streptomyces mashuense DSM 40221T (X79323) Streptomyces lanatus ISP 5090T (AJ399469) T 75 Streptomyces echinatus ISP 5013 (AJ399465) 99 Streptomyces bobili JCM 4624T (AB045876) 61 f,m Streptomyces galilaeus JCM 4757T (AB045878) 87 Streptomyces chartreusis ISP 5085T (AJ399468) 50 85 Streptomyces pseudovenezuelae ISP 5212T (AJ399481) 75 Streptomyces griseorubiginosus ISP 5469T (AJ399488) f Streptomyces clavuligerus JCM 4710T (AB045869) 69 68 Streptomyces lavendulae IFO 14028T (D85114) m,f Streptomyces virginiae IFO 12827T (D85123) Streptomyces peucetius JCM 9920T (AB045887) 100 m,f Streptomyces lateritius JCM 4389T (AF454764) Streptomyces venezuelae JCM 4526 T (AB045890) 0.01 m,f 100 m,f Streptomyces setonii ATCC25497 T (D63872) Streptomyces. caviscabies ATCC51928 T (AF112160)

Fig. 37: Neighbour-Joining tree based on nearly complete 16S rDNA sequences of 45 streptomycetes. “f” and “m” indicate branches that were also recovered when the Fitch-Margoliash and maximum-likelihood methods were used. The numbers at the nodes indicate the level of bootstrap support based on a neighbour-joining analysis of 1000 re-sampled dataset. Only values that were >50 % are given. The scale bar indicates 0.01 substitutions per nucleotide position.

208 179 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

It has been shown that the discriminative power of 16S rRNA sequencing is limited when closely related organisms are being inspected (Yassin et al. 1997), especially for members of the genus Streptomyces. The well-selected phenotypic traits and DNA-DNA pairing data are therefore needed to differentiate Streptomyces species sharing very similar 16S rDNA sequences (Kim et al., 1998; Labeda, 1988). To clarify the finer relationships, DNA-DNA association studies were carried out between strain A25T and close related strains selected on the basis of their 16S rDNA sequence similarities and phylogenetic positions. The low DNA-DNA homology between A25T and S. ambofaciens AS4.1528T (13.1 %), S. coerulescens JCM 4358T (34.3 %), S. caelestris JCM 4218T (23.4 %), S. nogalater JCM 4799 T (31.4 %), S. intermedius JCM 4483T (26.3 %) and S. albidoflavus JCM 4446T (19.6 %) confirmed that strain A25T can be considered as a new taxon. This is also supported by phenotypic data as at least five differences in phenotypic properties (see Table 20) were observed between strain A25T and its closest neighbours S. ambofaciens, S. coerulescens and S. coelicolor. The analysis of γ region partial sequence showed that strain A25T was grouped into a branch with S. lateritius JCM 4389Tand “Streptomyces flavochromogenes” JCM 4752, but the almost complete 16S rDNA sequence analysis showed that strain S. lateritius JCM 4389T fell into a distinct branch with S. venezulae from strain A25T. The low DNA-DNA homology value (17.4 %) between S. lateritius and strain A25T also confirmed they are different species. The genotypic, chemotaxonomic and phenotypic data showed that strain A25T form a new taxon within the genus Streptomyces. Therefore, it is proposed that this organism be recognized as a new species of the genus Streptomyces, i.e. Streptomyces scopiformis sp. nov.

Description of Streptomyces scopiformis sp. nov. Streptomyces scopiformis (sco. pi. formis L.n.f. scopa, a broom; l.n.f. forma, form. Scopiformis, broom form, referring to the spore chains structure). The organism is aerobic, mesophilic, Gram positive. Substrate hyphae extensive branching, septate and swollen, Rectiflexibles chains of roundish spiny-surfaced spores (0.7-0.8 µm) are arranged in fastigiate form. Spore mass

180 209 7. IDENTIFICATION OF STREPTOMYCETES – DESCRIPTION OF NEW TAXA

is grey or blue grey, the reverse is blue to grey-blue. Diffusible pigments are not formed. Cell wall type I, phospholipid PII and MK9 (H4,6) of menaquinone are detected. The fatty acid are type II. Mycolic acids are not present. The guanine- plus-cytosine ratio of the DNA is 71 %. Strain A25T growths in presence of pencillin G, biomycin, phenol, ethanol, 20-45 °C, pH 5-10 but not in bacteracin, lysozyme, sodium azide, methyl violet nor at 10 °C, 50 °C, pH 4.0, pH 11.0. It utilizes L-arabinose, D-fructose, D- galactose, D-glucose, D-maltose, L-rhamnose, D-xylose, D-sucrose, dulcitol, meso-Inositol, melibiose, trehalose, sodium acetata, sodium citrate as sole carbon source but not D-mannitol, D-raffinose, adonitol, α-methyl-glucoside, I- erythrol and inulin. The test for esculin, starch, dextrin, elastin, nitrate reduction and gelatine are positive and for hippurate, cellulose, lipolysis are negative. The type strain is Streptomyces scopiformis AS 4.1331T (Chinese General Microbiological Collection Center) = LMG 20251T (BCCM/LMG, Belgian Co- ordinated Collections of Micro-organisms / Laboratorium voor Microbiologie, University Gent).

Acknowledgements. This study was performed in the frame of the Belgian-Chinese program: ‘Identification and classification of actinomycetes, specifically bioactive Streptomyces strains isolated from Chinese soil’, supported by MOST/NSFC P.R. China and OSTC Belgium (BL/02/C10), and by the NSFC (grant number 39970001). We also thank Dr. D. E. Minnikin (Newcastle University) for the determination of fatty acid contents. The authors are also indebted to Prof. R. M. Kroppenstedt (DSMZ, Germany) and Dr. T. Kudo (JCM, Japan) for providing cultures.

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PART 3

Conclusions & perspectives

211

212 8. CONCLUSIONS & PERSPECTIVES

Conclusions & perspectives

In this chapter the research challenges as stated in the “Aims and conceptual framework” are discussed in the light of the obtained results (Chapters 4 to 7). Finally, perspectives regarding further taxonomic improvements are discussed.

8.1 Detection and reclassification of synonymous species

8.1.1 Evaluation of a fast and reliable screening method

► SDS-PAGE of whole-cell proteins (Chapter 4.1)

This chemotaxonomic technique was selected because of its reported differentiation at species level and good correlation with DNA-DNA homology studies (Manchester et al., 1990). In this study, we investigated a subset of 93 representative Streptomyces species with this technique. Visually aided numerical analysis of protein profiles yielded five clusters containing strains with visually identical patterns. Within each cluster DNA-DNA homology values above 70 % were detected and led to the reclassification of 4 synonymous species and 1 subspecies. Despite the promising results, the methodology was considered as old-fashioned and priority was given to a genotypic approach.

► Rep-PCR fingerprinting (Chapters 4.2 & 4.3)

Compared to SDS-PAGE of whole-cell proteins advantages and disadvantages became clear. Advantages are its high reproducibility, ease of application, fastness, data on the whole-genome and not influenced by cultivation conditions (Versalovic et

213 al., 1994; Rademaker & de Bruijn, 1997, Rademaker et al., 2000; Masco et al., 2003, Gevers et al., 2001, Lanoot et al., 2004; 2005a), making this technique a good tool to screen large number of strains. Preliminary studies on streptomycetes (Clark et al., 1998; Sadowsky et al., 1996) and our study showed that robust and reproducible fingerprints were obtained by BOX-PCR, a methodology finally selected for overall screening. Numerical analysis of the BOX-PCR patterns of 473 Streptomyces type strains showed that the majority (74 %) had a unique BOX pattern. In addition, 30 clusters were delineated each comprising species with nearly identical patterns. Relationships between twelve of the latter clusters were subsequently investigated by DNA-DNA hybridizations. It was shown that nearly identical rep patterns were correlated with DNA-DNA homology values above 70 %. However, DNA-DNA relatedness levels above 70 % were also found among different patterns and indicates that BOX-PCR fingerprinting is differentiating between strain and species level. The latter may be seen as a disadvantage compared to SDS-PAGE of whole-cell proteins.

It is clear from these results that both protein profiling and BOX-PCR fingerprinting are reliable tools for detecting synonyms among streptomycetes.

8.1.2 Proposed reclassifications (Chapter 4)

The following reclassifications of species were proposed based on DNA-DNA homology values above 70 %, as recommended by Wayne et al. (1987) (see Chapter 4; Lanoot et al., 2002; 2004; 2005a).

214 8. CONCLUSIONS & PERSPECTIVES

Emended species Subjective synonym

Using protein profiling S. violatus S. varsoviensis S. aurantiacus S. albosporeus subsp. albosporeus S. cacaoi subsp. cacaoi S. aminophilus S. caeruleus S. niveus and S. spheroides Using BOX-PCR fingerprinting S. cinereorectus S. cochleatus S. fradiae S. roseoflavus S. tricolor S. roseodiastaticus S. filamentosus S. roseosporus S. colombiensis S. distallicus S. vinaceus S. arabicus S. phaeopurpureus S. phaeoviridis S. flavovirens S. nigrifaciens S. anulatus S. citreofluorescens, S. chrysomallus subsp. chrysomallus and S. fluorescens S. corchorusii S. chibaensis S. minutiscleroticus S. flaviscleroticus S. microflavus S. lipmanii , S. willmorei, S. griseus subsp. cretosus and S. griseus subsp. alpha

8.2 Intra-species genotypic relatedness of S. virginiae strains and correlation with phenotypic traits (Chapter 5)

S. virginiae was selected because of its economical relevance as an antibiotic producer, the existence of a unique collection of variants of one particular strain and the availability of several additional strains of the species. A total number of five S. virginiae strains, including the type strain, were screened using BOX-PCR, resulting in 4 different fingerprint types. Subsequent DNA- DNA hybridizations confirmed the genomic heterogeneity of this species although phenotypically homogeneous.

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On the other hand, morphology of colony and color of aerial and substrate mycelium were highly variable among 19 variants of S. virginae strain 899 provided by the company Phibro Animal Health Belgium. The latter heterogeneity was however not reflected at genomic level using BOX-PCR fingerprinting. Nearly identical patterns of the variants were correlated with DNA-DNA homology values above 70 % confirming their close relationship, as expected. Even by applying high resolution AFLP fingerprinting, and 6 different primer combinations, only two bands of difference were found among the patterns of WT strain 899 and one mutant (PD 170). This proves that over 40 years of mutational pressure on S. virginiae strain 899, either forced or natural, did not cause huge chromosomal distortions and are most likely limited to base substitutions. From these results it is clear that true relationships are only revealed through a genotypic approach, the major aim of this thesis. BOX-PCR fingerprinting was proved to be a convenient tool for species delimitation in Streptomyces. More research is needed regarding the stability of morphological traits in other species prior to their use for defining minimal standards for the description of Streptomyces species.

8.3 Phylogenetic grouping using 16S-ITS RFLP fingerprinting as a valuable alternative for 16S rRNA gene sequencing (Chapter 6)

At the start of this project, almost 6 years ago, complete 16S rRNA gene sequences were only publicly available for a minority of the validly described Streptomyces species. Seen the high number of species to be screened and the unavailability of high-throughput sequencing technologies, an alternative technique was searched to construct a phylogenetic framework. PCR aided amplification of the 16S rRNA gene of 78 representative Streptomyces type strains and subsequent combined digestion with four restriction enzymes (BstUI, NIaIIII, TaqI and RsaI) resulted in fingerprints differentiating at intra-genus level. In order to enhance the discriminative power to species level, the hypervariable 16S-23S ITS region was included in RFLP. Careful examination of restriction sites in both fragments, using the GeneCompar software (Applied Maths, Belgium) resulted in the selection of two restriction enzymes i.e. BstUI and HaeIII yielding highly reproducible and diverse

216 8. CONCLUSIONS & PERSPECTIVES fingerprints. This combination was finally applied on 463 Streptomyces and Kitasatospora type strains. In order to validate our data, all publicly available complete 16S rRNA gene sequences and 69 sequences determined in the present study were aligned. It was shown that 16S-ITS RFLP fingerprints have a higher level of differentiation compared to 16S rRNA gene sequencing, regardless of the 16S similarity cut-off level applied (97 or 98 %). A total number of 59 stable groups were delineated, each comprising closely related strains with highly reproducible and similar patterns. The cluster delineation obtained is phylogenetically valid and is supported by sequencing information, with few exceptions. Awaiting the completion of the complete 16S rRNA gene based phylogenetic tree, 16S-ITS RFLP fingerprinting it is a valuable alternative to delineate phylogenetically related species. In the case of new species descriptions it minimizes the set of species for DNA-DNA hybridisations studies.

8.4 Identification of streptomycetes from Chinese and Moroccan soil and description of new taxa (Chapter 7)

Another main objective of this work was to use the constructed reference frameworks in protein profiling, BOX-PCR and 16S-ITS RFLP fingerprinting to screen large numbers of isolates with the aim to assign them to one of the described species or to describe new taxa.

► Chinese strains

Twenty-three percent of the 592 isolates screened in protein profiling and BOX- PCR had an identical pattern to one or more of the 473 type strains and are hence identified (see Annex III). Six percent of the isolates had a BOX pattern similar to a type strain indicating some relationship that needs to be clarified in DNA-DNA hybridizations. Part of the unidentified strains might still be highly related to a reference strain seen the quite high taxonomic resolution of BOX-PCR fingerprinting. Subsequent 16S-ITS RFLP fingerprinting of non-identified isolates, in particular those belonging to batch FXJ and two other isolates, allowed the selection of in total

217

11 strains which had a unique 16S-ITS RFLP pattern or were grouping with a limited number of reference strains. The latter selection of strains were further characterized using a polyphasic approach i.e. 16S rDNA sequence analysis, phenotypic characterisation and subsequent DNA-DNA hybridizations. Three isolates were found to hold a separate position and were described as new species in Streptomyces, as predicted by our 16S-ITS RFLP data (see Section 8.5).

► Moroccan strains

Among the 13 strains screened, respectively three and four of them could be identified in protein profiling and BOX-PCR fingerprinting (see Annex III).

From our results it is clear that the genus Streptomyces may contain a considerable number of novel species, confirming earlier reports (Rondon et al., 1999).

8.5 Description of new Streptomyces species

Three novel species have been proposed.

►Streptomyces glauciniger sp. nov., a novel mesophilic streptomycete isolated from soil in South China (Chapter 7.2.1).

Huang Y, Li W, Wang L, Lanoot B, Vancanneyt M, Rodriguez Z, Liu Z & Swings J. (2004). Int J Syst Evol Microbiol 54, 2085-2089.

Streptomyces glauciniger was isolated from willow woods soil collected in Nanning city, Guangxi Province, China. It could be clearly differentiated from its closest phylogenetic neighbours i.e. S. rimosus subsp. rimosus, S. malaysiensis, S. sparsogenes and S. yeochonensis on basis of phenotypic and BOX-PCR data. The strain shows antimicrobial activity against strains of Bacillus subtilis and Candida

218 8. CONCLUSIONS & PERSPECTIVES albicans, but not against strains of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa or Aspergillus niger. The type strain is FXJ14T (= AS 4.1858T = LMG 22082T = JCM 12278T).

► Streptomyces jietaisiensis sp. nov., isolated from soil in North China (Chapter 7.2.2).

He L, Wang L, Li W, Liu Z, Lanoot B, Vancanneyt M, Swings J & Huang Y. Int J Syst Evol Microbiol, submitted.

Streptomyces jietaisiensis had S. griseoaurantiacus and S. nogalater as closest neighbours after alignment with publicly available 120nt long and almost complete 16S rRNA gene sequences. DNA-DNA hybridization between strain FXJ46 and S. griseoaurantiacus revealed a DNA-DNA homology value below 70 %, proving its novelty. Latter observation is supported by differences in cultural characteristics on a number of standard media. The type strain is FXJ46T (= AS 4.1859T = JCM 12279T) and has been isolated from a soil sample in Jietaisi, a place in suburb of Beijing.

► Streptomyces scopiformis sp. nov., a novel streptomycete with fastigiate spore chains (Chapter 7.2.3).

Li W, Lanoot B, Zhang Y, Vancanneyt M, Swings J & Liu Z. (2002). Int J Syst Evol Microbiol 52, 1629 – 1633.

Streptomyces scopiformis formed a separate single strain branch from representatives of the genus Streptomyces, notably from its nearest neighbours S. ambofaciens, “S. coelicolor”, S. lateritius JCM 4389T, “S. flavochromogenes” and S. coerulescens. Low DNA-DNA homology values were shared among strain A25 and all the latter closest neighbours proving its novelty. This is also supported by at least five differences in phenotypic properties. The soil sample was collected from rhizosphere of Chinese hemlock (Tsuga chinensis) in Lushan Mountain, Jiangxi province, China. The type strain is AS 4.1331T= LMG 20251T.

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8.6 General conclusions

►A genotypic backbone is provided for a more transparent and easier classification of streptomycetes as a replacement to the unreliable phenotypy based approach. At least 10 % synonymous species are present within Streptomyces, part of which have been reclassified in this study.

►Color of aerial and substrate mycelium were found to be highly variable among variants of strain S. virginiae 899 despite their high genomic homogeneity.

►The constructed BOX-PCR and 16S-ITS RFLP databases allow to identify Streptomyces’ isolates in an objective way compared to the current approach.

►Minimal standards for the description of new species should be primarily based on genotypic information.

► Chinese soil shows a high genomic streptomycete diversity, harboring many new species (potentially producing novel bioactive compounds) due to extreme climatological conditions in this vast country.

8.7 Future perspectives

Future research should focus on

(1) a further reduction in the number of species (2) completion of the 16S rDNA sequence tree and its exploitation for the description of novel taxa (3) the evaluation of the production of bioactive compounds as taxonomic marker and the discovery of novel bioactive compounds

The genus Streptomyces is still the largest genus in number of species despite numerous taxonomic improvements last decade to lower this number. In our study, BOX-PCR fingerprinting analysis allowed the delineation of 30 clusters comprising

220 8. CONCLUSIONS & PERSPECTIVES strains with nearly identical patterns. This insight indicates the existence of synonymous species. Of this number, 14 clusters need to be further taxonomically clarified using DNA-DNA hybridisations. In addition, the exact meaning of similar BOX patterns should be investigated at DNA-DNA homology level as this category of patterns counts for 10 % of all strains examined. A prerequisite to improve the taxonomy of Streptomyces significantly is 16S rRNA gene sequencing of the remaining type strains (about 70 %) in order to complete the 16S rDNA tree. Although not complete, the current tree already revealed the delineation of several groups of species clearly forming separate lineages in Streptomyces, supported by high bootstrap values e.g. the S. violaceoruber, S. hygroscopicus and S. albidoflavus species groups. A taxonomic survey is needed to verify whether phenotypic, chemotaxonomic and genotypic information can prove a low relatedness with other streptomycetes. In this case a split off of these particular groups from the genus (M. Goodfellow, personal communication) should be considered. Streptomycetes have the intrinsic feature to produce strain specific secondary metabolites. By using the high-throughput HPLC methodology all produced metabolites can be fingerprinted. Construction of databases would allow to understand to which extent the chemical diversity in secondary metabolites correlates with the taxonomic diversity. While quite a number of established Streptomyces species are clearly synonyms of previously described species many new species still need to be discovered seen the high genomic diversity found in soil samples and the low identification rates.

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222 SUMMARY - SAMENVATTING

Summary

The taxonomy of the genus Streptomyces has a complex history and remains such nowadays. After being lifted from the genus Actinomyces in 1943 by Henrici and Waksman, much attention has been given to streptomycetes because of their feature to produce secondary metabolites e.g. antibiotics (Chapter 1). Hence, a high number of novel species have been described (over 300) during the 1950s to 1970s on basis of morphological and physiological traits. Chemotaxonomic and genotypic data challenge the use of these traits which still dominate contemporary classification systems. Despite several successive taxonomic improvements the last decennia (Chapter 2), mainly through the work of Williams et al. (1983), the number of species still remains extremely high (over 500) and is a major bottleneck to establish a genomically based alternative for revealing true relationships among species. A first goal of this study is the detection and reclassification of synonymous species (Chapter 4). Prior to overall screening, a chemotaxonomic or genomic fingerprint technique was evaluated and selected from an array of available techniques. A pilot study, using a representative set of Streptomyces species, revealed that the rep-PCR technique is to be preferred for screening all species over protein profiling because of being fast, easy to apply, low-cost, highly reproducible and differentiating around species level. Numerical analysis of BOX primed PCR reactions assigned the majority of the 473 screened type strains in the category of unique patterns (74 %). Sixteen percent of the strains analysed grouped in 30 clusters, each containing multiple taxa showing nearly identical patterns. Twelve of these groups were taxonomically further clarified using DNA-DNA hybridizations.

223 SUMMARY - SAMENVATTING

Regarding the latter technique, an improved protocol was developed to extract pure and non-fragmented DNA on large scale. On basis of the results obtained, a good correlation was found between rep-PCR groupings and hybridization data; strains with nearly identical BOX patterns shared a DNA-DNA homology value above 70 % (level accepted for species delineation). Emendation of 16 species has already been proposed (see Section 8.1.2). A second goal of this study was to tackle the link between phenotypy and genotypy at the intra-species level. Herefore intra-species relationship were determined of strains and variants of one particular species i.e. S. virginiae (Chapter 5). Genotypical analysis of variants of S. virginiae strain 899 clearly showed that in particular the morphological traits color of aerial and substrate mycelium are susceptible to mutational pressure and thus are unstable for the classification of S. virginiae strains. It was shown that virginiamycin producing strain S. virginiae 899 is not belonging to the latter species. In addition, genotypic analysis of S. virginiae strains, using BOX-PCR fingerprinting and DNA-DNA hybridizations showed that the species S. virginiae is genomically heterogeneous despite its phenotypical homogeneity. These results underpin the value of the establishment of a genotypy based system for classifying streptomycetes. A third goal was to tackle the currently applied subjective approach to describe and identify Streptomyces’ isolates (Chapter 6). Following internationally accepted recommendations, a complete phylogenetic framework is needed to determine all closest neighbours of a potentially new species. However, due to the fact that in the first years of this work 75 % of all validly described Streptomyces’ species still needed to be sequenced in their 16S rRNA gene forced us to develop an alternative tool for grouping closely related species i.e. 16S-ITS RFLP fingerprinting. The latter technique combines information derived from the 16S rRNA gene and hypervariable 16S-23S spacer in one fingerprint using both the PCR and RFLP technology. Upon analysis with nearly complete 16S rRNA sequence data, partly determined in this study, it was concluded that 16S-ITS RFLP fingerprinting allows to delineate stable groups of closely related species at a higher taxonomic level compared to 16S rRNA gene sequencing. Applied on 463 Streptomyces and Kitastospora species, a total number of 59 phylogenetic groups were delineated. Although 16S-ITS RFLP fingerprinting does not generate absolute data, it is a valuable approach for species-

224 SUMMARY - SAMENVATTING

group discrimination. A polyphasic comparison of the latter taxa and new members of the group allow to identify them or provide evidence for the description of potentially new species. In a last part of this work large numbers of Chinese and Moroccan isolates were screened (Chapter 7), against the constructed BOX-PCR and 16S-ITS RFLP fingerprinting reference frameworks. Major goal was the delineation of new species in Streptomyces. BOX-PCR fingerprinting analysis and protein profiling revealed a high diversity among the 600 bioactive isolates. More in depth analysis of selected isolates resulted in the delineation of 11 strains with a unique 16S-ITS RFLP pattern or closely related to few reference strains. Already three of these strains were shown to hold a separate position in Streptomyces and have been described as new species.

225 SUMMARY - SAMENVATTING

226 223 SUMMARY - SAMENVATTING

SAMENVATTING

De taxonomie van het genus Streptomyces heeft een bewogen historiek en is heden nog steeds gecompliceerd. Na de afsplitsing van Streptomyces van het genus Actinomyces in 1943 door Henrici en Waksman kreeg Streptomyces grote aandacht vanuit de industrie wegens de eigenschap belangrijke secondaire metabolieten te vormen zoals antibiotica (Hoofdstuk 1). Hierdoor werd een groot aantal species beschreven merendeel in de jaren 1950 tot ’70 (> 300), voornamelijk op basis van morfologische en fysiologische kenmerken. Hoewel nog steeds dominant in huidige classificatiesystemen stellen chemotaxonomische en genotypische data het gebruik van deze kenmerken in vraag voor speciesdifferentiatie. Ondanks successieve taxonomische verbeteringen (Hoofdstuk 2), ondermeer via het baanbrekend werk van Williams et al. (1983), blijft het aantal species nog steeds hoog (> 500) wat een belangrijke hinderpaal is om over te stappen naar een polyfasische (genomisch) gebaseerde aanpak om meer gefundeerde verwantschappen tussen species op te sporen. Een eerste doelstelling van deze studie spitste zich toe op het reduceren van het aantal species door herclassificeren van synonieme species (Hoofdstuk 4). Hiervoor werd een chemotaxonomische of genomische fingerprinttechniek geëvalueerd en geselecteerd uit een waaier van beschikbare technieken om verwante species te groeperen. Na een verkennende studie op een beperkte set van representatieve species en op basis van literatuurdata werd finaal de rep-PCR techniek geprefereerd voor een screening van alle species boven eiwitprofilering omdat die voldoet aan de volgende criteria: snel, eenvoudig, goedkoop, hoog

227 SUMMARY - SAMENVATTING

reproduceerbaar en differentiërend rond species niveau. Numerieke analyse van de BOX primer gegenereerde profielen catalogeerde de grote meerderheid van de 473 type stammen als uniek (74 %). Zestien percent van de stammen groepeerden in 30 clusters, elk met meerdere taxa die nagenoeg identische patronen vertoonden. Twaalf van deze groepen werden verder taxonomisch onderzocht gebruik makende van de gouden standaard voor species delineatie in de bacteriële systematiek nl. DNA-DNA hybridisaties. Om deze techniek succesvol te kunnen toepassen werd een verbeterd protocol uitgewerkt voor de bereiding van voldoende zuiver en niet gefragmenteerd DNA op grote schaal. Op basis van de bekomen resultaten werd een goede correlatie gevonden tussen rep-PCR groepering en hybridisatiedata; stammen met nagenoeg identische patronen waren steeds gecorreleerd met DNA-DNA homologie waarden boven de 70 % (niveau geaccepteerd voor aflijning van bacteriële species). Op deze wijze werden reeds respectievelijk 18 en 4 synonieme species en subspecies geherclassificeerd. In een tweede luik werd de link tussen fenotypie en genotypie onderzocht op intra-species niveau door het taxonomisch onderzoeken van een species nl. S. virginiae (Hoofdstuk 5). Genotypische analyse van mutanten van stam S. virginiae 899 toonde aan dat in het bijzonder de kleur van lucht- en substraatsmycelium gevoelig is aan mutationele druk en bijgevolg onstabiel zijn voor het classificeren van S. virginiae stammen. Verder werd aangetoond dat de virginiamycine producerende stam S. virginiae 899 niet tot het laatst genoemde species behoort. Onze studie bewees dat het species S. virginiae heterogeen is op basis van BOX-PCR en DNA- DNA hybridisaties, in tegenstelling tot de verwantschappen bepaald via hun fenotype. Deze resultaten tonen het belang aan van een genomisch gebaseerd classificatiesysteem. In een derde luik werd ingegaan op de huidige niet echt gefundeerde aanpak voor beschrijving en identificatie van nieuwe Streptomyces isolaten (Hoofdstuk 6). Volgens internationaal aanvaarde aanbevelingen is hiervoor een compleet fylogenetisch referentiekader nodig om alle nauwverwante species van een isolaat te kunnen bepalen. Het feit dat in de eerste jaren van dit onderzoek voor 75 % van alle valied beschreven species nog geen 16S rRNA gen sequentie publiek voorhanden was, leidde ons tot het op punt stellen en toepassen van een alternatieve techniek nl. 16S rDNA-ITS RFLP fingerprinting. Deze techniek combineert informatie van het 16S

228 225 SUMMARY - SAMENVATTING

rRNA gen en van de hypervariabele 16S-23S spacer in eenzelfde fingerprint gebruik makende van de PCR en de RFLP technologie. Na vergelijking met nagenoeg volledige 16S rRNA sequentiegegevens, deels bepaald in deze studie, kon besloten worden dat 16S-ITS RFLP toelaat om op een snelle en relatief goedkope manier stabiele groepen van nauwverwante species af te bakenen op een fijner taxonomisch niveau dan 16S rRNA sequenering. Toegepast op 463 Streptomyces en Kitasatospora type stammen konden in totaal 59 fylogenetische groepen afgebakend worden, ondersteund door beschikbare 16S rRNA informatie. Alhoewel 16S-ITS RFLP fingerprinting geen absolute data genereert is het een waardevolle aanpak voor species-groep discriminatie en laat toe, voor beschrijving van nieuwe taxa, een goed overwogen minimale set van species voor DNA-DNA hybridisatie studies en bijkomende polyfasische analyses af te bakenen. In een laatste luik werd de diversiteit in streptomyceten nagegaan in Chinese bodemstalen (Hoofdstuk 7) gebruik makende van de geconstrueerde referentiedatabanken in BOX-PCR en 16S-ITS RFLP fingerprinting. BOX-PCR fingerprinting van 600 bioactieve isolaten toonde een hoge diversiteit aan. Een meer diepgaande studie van een geselecteerde reeks isolaten resulteerde in de aflijning van negen stammen met een uniek 16S-ITS RFLP patroon waarvan reeds drie al polyfasisch onderzocht werden en voorgesteld werden als nieuw species.

226 229

230 BIBLIOGRAPHY

BIBLIOGRAPHY

Abd-Allah, E.F. (2001). Streptomyces plicatus as a model biocontrol agent. Folia Microbiol (Praha) 46(4), 309-14.

Abou-Zeid, A.Z., Khalili, A. El-G. & Rabei, M. (1980). Spiramycin, a macrolide antibiotic. Zentralbl Bakteriol Naturwiss 135(5), 443-53.

Al-Jawadi, M., Wellington, E.M.H., Calam, T. (1985). Identification of some streptomycetes producing oxytetracycline. J Gen Bacteriol 131, 2241-2244.

Al-Tai, A., Kim, B., Kim, S. B., Manfio, G. P. & Goodfellow, M. (1999). Streptomyces malaysiensis sp. nov., a new streptomycete species with rugose, ornamented spores. Int J Syst Bacteriol 49, 1395-1402.

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang. J., Zhang. Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-410.

Ambujom, S. (2001). Studies on composition and stability of a large membered bacterial consortium degrading phenol. Microbiol Res 156(4), 293-301.

Amir, H. & Pineau, R. (1998). Influence of plants and cropping on microbiological characteristics of some New-Caledonian ultramafic soils. Aust J Soil Res 36, 457-471.

Ammar, Y.B., Matsubara, T., Ito, K., Iizuka, M., Limpaseni, T., Pongsawasdi, P., & Minamiura, N. (2002). New action pattern of a maltose-forming alpha-amylase from Streptomyces sp. and its possible application in bakery. J Biochem Mol Biol 30, 35(6), 568- 75.

231 Amoroso, M.J., Schubert, D., Mitscherlich, P. Schumann, P. & Kothe, E. (2000). Evidence for high affinity nickel transporter genes in heavy metal resistant Streptomyces spec. J Basic Microbiol 40(5-6), 295-301.

Anadon, A. & Reeve-Johnson, L. (1999). Macrolide antibiotics, drug interactions and microsomal enzymes: implications for veterinary medicine. Res Vet Sci 66(3), 197-203.

Anderson A. S. & Wellington, E. M. H. (2001). The taxonomy of Streptomyces and related genera. Int J Syst Evol Bacteriol 51, 797-814.

Andersson, M.A., Mikkola, R., Kroppenstedt, R.M., Rainey, F.A., Peltola, J., Helin, J., Sivonen, K. & Salkinoja-Salonen, M.S. (1998).The mitochondrial toxin produced by Streptomyces griseus strains isolated from an indoor environment is valinomycin. Appl Environ Microbiol 64(12), 4767-73.

Angehrn, P., Buchmann, S., Funk, C., Goetschi, E., Gmuender, H., Hebeisen, P., Kostrewa, Link, H., Luebbers, T., Masciadri, R., Nielsen, J., Reindl, P., Ricklin, F., Schmitt-Hoffmann & Theil, F.P. (2004). New antibacterial agents derived from the DNA gyrase inhibitor cyclothialidine. J Med Chem 47(6), 1487-513.

Antonopoulos, V.T., Hernandez, M., Arias, M.E., Mavrakos, E.& Ball. A.S. (2001). The use of extracellular enzymes from Streptomyces albus ATCC 3005 for the bleaching of eucalyptus kraft pulp. Appl Microbiol Biotechnol 57(1-2), 92-7.

Anzai, Y., Okuda, T. & Watanbe, J. (1994). Application of the random amplified polymorphic DNA using the polymerase chain reaction for efficient elimination of duplicate strains in microbial screening. J Antibiot 47, 183-193.

Arai, M., Karasawa, K., Nakamura, S., Yonehara, H. , Umezawa, H. (1958). Studies on mikamycin. Int J Antibiot 9, 14-21.

Arai, M., Serizawa, N., Terahara, A., Tsujita, Y., Tanaka, M., Masuda, H. & Ishikawa, S. (1988). Pravastatin sodium (CS-514), a novel cholesterol-lowering agent which inhibits HMG- CoA reductase. Annu Rep Sankyo Res Lab 40, 1-38.

Archuleta, J. G. & Easton, G.D. (1981). The cause of deep-pitted scab of potatoes. Am Potato J 58, 385-392.

232 BIBLIOGRAPHY

Aretz, W., Koller, K.P. & Riess G. (1989). Proteolytic enzymes from recombinant Streptomyces lividans TK24. FEMS Microbiol Lett 53(1-2), 31-5.

Ashy, M.A., Khalili, A.E. & Abou-Zeid, A.A. (1980). Carbomycin, a macrolide antibiotic. Zentralbl Bakteriol Naturwiss 135(6), 541-51.

Baath, E., Diaz-Ravina, M., Frostegard, A. & Campbell, C.D. (1998). Effect of metal-rich sludge amendments on the soil microbial community. Appl Environ Microbiol 64, 238-245.

Baldacci, E., Spalla, C. & Grein, A. (1954). The classification of the Actinomyces species (=Streptomyces). Archiv für Mikrobiologie 20, 347-357.

Ball, A.S. (1991). Degradation by Streptomyces viridosporus T7A of plant material grown under elevated CO2 conditions. FEMS Microbiol Lett 15, 68(2), 139-42.

Bandlish, R.K., Hess, J., Epting, K.L., Vieille, C. & Kelly, R.M. (2002). Glucose-to-fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and two hyperthermophilic Thermotoga species. Biotechnol Bioeng 20,80(2), 185- 94.

Bang, H.O. (1979a). Studies on potato russet scab. I. Characterisation of different isolates from northern Sweden. Acta Agric Scand 29, 145-150.

Bansal, A.K. & Meyer, T.E. (2002). Evolutionary analysis by whole-genome comparisons. J Bacteriol 184 (8), 2260-2272.

Barry,T., Coleran G., Lennon M., Dunican, L.K. & Gannon (1991). The 16S/23S ribosomal spacer region as a target for DNA probes to identify Eubacteria. PCR methods and Applications, 51, 51-56.

Bax, R.P., Anderson, R., Crew, J., Fletcher, P., Jonhson, T., Kaplan, E., Knaus, B., Kristinsson, K., Malek, M. & Strandberg, L. (1998). Antibiotic resistance - what can we do ? Nat med 4, 545-546.

Bendl, B.J., Mackey, D., Al-Saati, F., Sheth, K.V., Ofole, S.N. & Bailey, T.M. (1987). Mycetoma in Saudi Arabia. J Trop Med Hyg 90(2), 51-9.

233 Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O'Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J. & Hopwood, D.A. (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 9, 417(6885), 141-7.

Berdy, J. (1974). Recent developments of antibiotic research and classification of antibiotics according to chemical structure. In: Advances in Applied Microbiology, pp. 08-406, Edited by D. Perlman. New York: Academic Press.

Berger, F., Fischer, G., Kyriacou, A., Decaris, B. & Leblond, P. (1996). Mapping of the ribosomal operons on the linear chromosomal DNA of Streptomyces ambofaciens DSM 40697. FEMS Microbiol Lett 143, 167-173.

Bernal, A., Ear, U., Kyrpides, N. (2001). Genomes OnLine Database (GOLD): a monitor of genome projects world-wide. Nucl Acids Res 29, 126-127.

Berrocal, M., Ball, A.S., Huerta, S., Barrasa, J.M., Hernandez, M., Perez-Leblic M.I. & Arias, M.E. (2000). Biological upgrading of wheat straw through solid-state fermentation with Streptomyces cyaneus. Appl Microbiol Biotechnol 54(6),764-71.

Bertasso, M., Holzenkampfer, M., Zeeck, A., Stackebrandt, E., Beil, W. & Fiedler, H.P. (2003). Ripromycin and other polycyclic macrolactams from Streptomyces strain Tu 6239: taxonomy, fermentation, isolation and biological properties. J Antibiot (Tokyo) 56(4), 364-71.

Beyazova, M. & Lechevalier, M.P. (1993). Taxonomic utility of restriction endonuclease fingerprinting of large DNA fragments from Streptomyces strains. Int J Syst Bacteriol 43, 674- 682.

Biot, A. (1984). Virginiamycin: properties, biosynthesis, and fermentation. In: Biotechnology of Industrial antibiotics. E.J. Vandamme (ed.). Marcel Dekker Inc..

234 BIBLIOGRAPHY

Bode, H.B., Kerkhoff, K. & Jendrossek, D. (2001). Bacterial degradation of natural and synthetic rubber. Biomacromolecules 2(1), 295-303.

Bouchek-Mechiche, K., Gardan, L., Normand, P. & Jouan, B. (2000). DNA relatedness among strains of Streptomyces pathogenic to potato in France: description of three new species, S. europaeiscabiei sp. nov. and S. stelliscabiei sp. nov. associated with common scab, and S. reticuliscabiei sp. nov. associated with netted scab. Int J Syst Evol Microbiol 50 (1), 91-9.

Brenner, D.J., Fanning, G.R., Rake, A.V. & Johnson, K.E. (1969). Batch procedure for thermal elution of DNA from hydroxyapatite. Analytical Biochemistry 28, 447-459.

Brim, H., Heuer, H., Krogerrcklenfort, E., Mergeay, M. & Smalla, K. (1999a). Characterization of the bacterial community of a zinc-polluted soil. Can J Microbiol 45, 326- 338.

Brim, H., Heyndrickx, M., de Vos, P., Wilmotte, A., Springael, D., Schlegel, H.G. & Mergeay, M. (1999b). Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs. Syst Appl Microbiol 22, 258-268.

Brown, J.W. (1999). The ribonuclease P database. Nucleic Acids research, 27, 314.

Bruggink, A. & Roy, P.D. (2001). Industrial synthesis of semi-synthetic antibiotics. In: Bruggink, A. (Ed.), Synthesis of β-lactam antibiotics, Chemistry biocatalysis and process integration. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 13-56.

Caugant, D.A. (2001). From multi locus enzyme electrophoresis to multi locus sequence typing, pp. 299-349. In: Dijkshoorn, K.J. Towner and Struelens (Eds.), New approaches for the generation and analysis of microbial typing data. Elsevier. Amsterdam.

Cevallos, A. & Guerriero, A. (2003). Isolation and structure of a new macrolide antibiotic, erythromycin G, and a related biosynthetic intermediate from a culture of Saccharopolyspora erythraea. J Antibiot (Tokyo) 56(3), 280-8.

235 Chamberlain, K. & Crawford, D.L. (2000). Thatch biodegradation and antifungal activities of two lignocellulolytic Streptomyces strains in laboratory cultures and in golf green turfgrass. Can J Microbiol 46(6), 550-8.

Chater, K.F., Lomovskarya, N.D., Voeykova, T.A., Sladkova, I.A., Mkrtumian, N.M. & Muravnik, G.L. (1986). Streptomyces ΦC31-like phages: cloning vectors, genome changes and host range. In: Biological, Biochemical and Biomedical aspects of actinomycetes, pp. 45- 54. Edited by G. Szabo, S. Biro & M. Goodfellow. Budapest: Akademiai Kiado.

Christensen, H., Angen, O., Mutters, R., Olsen, J. E. & Bisgaard, M. (2000). DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA. Int J Syst Evol Microbiol 50, 1095-1102.

Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequence. Int J Syst Bacteriol 45, 240-245.

Chun, J., Youn, H.D., Yim, Y.I., Lee, H., Kim, M.Y., Hah, Y.C. & Kang, S.O. (1997). Streptomyces seoulensis sp. nov. Int J Syst Bacteriol 47(2), 492-8.

Ciferri, O. (1999). Microbial degradation of paintings. Appl Environ Microbiol, 65, 879-885.

Clark, C.A., Chen, C., Ward-Rainey, N. & Pettis, G.S. (1998). Diversity within Streptomyces ipomoeae based an inhibitory interactions, rep-PCR, and plasmid profiles. Phytopathology 88(11), 1179-1186.

Clarke, S.D., Ritchie, D.A. & Williams, S.T. (1993). Ribosomal DNA restriction fragment analysis of some closely related Streptomyces species. Syst Appl Microbiol 16, 256-260.

Coates, A., Hu, Y., Bax, R. & Page, C. (2002). The future challenges facing the development of new antimicrobial drugs. Nat Rev Drug Discov 1, 895-910.

Cocito, C. (1979). Antibiotics of the virginiamycin family, inhibitors which contain synergistic components. Microbiol Rev 43(2), 145-198.

Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J. R. W., Kersters K. & P. Vandamme (1999). Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov. Int J Syst Bacteriol 49, 405-413.

236 BIBLIOGRAPHY

Cole, J.R., Chai, B., Marsh, T.L., Farris, R.J., Wang, Q., Kulam, S.A., Chandra, S., McGarrell, D.M., Schmidt,,T.M., Garrity, G.M. & Tiedje, J.M. (2003). The Ribosomal Database project (RDP-II) : previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids research, 31, 442-443.

Collins, M. D. (1985). Isoprenoid quinone analysis in classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267-287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.

Conrads, G., Claros, M.C., Citron, D.M., Tyrell, K.L., Merriam, V. & Goldstein, E. J. C. (2002). 16S-23S internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Fusobacterium. Int J Syst Bacteriol 52, 493-499.

Cooper, J.E. & Feil, E.J. (2004). Multilocus sequence typing - what is resolved? Trends Microbiol 12(8), 373-7.

Corbaz, R. (1964). Etude des streptomycetes provoquant la gal commune de la pomme de terre. Phytopathol Z 51, 351-360.

Crawford, L., Stepan, A.M., McAda, P.C., Rambosek, J.A., Conder, M.J., Vinci, V.A. & Reeves, C.D. (1995). Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Bio Technol 13, 58-62.

Crosa, J.H., Brenner, D.J. & Falkow, K.B. (1973). Use of a single-strand-specific nuclease for analysis of bacterial and plasmid deoxyribonucleic acid homo- and heteroduplexs. J Bacteriol 115, 904-911.

Cross, T., Lechevalier, M.P. & Lechevalier, H.A. (1963). A new genus of the Actinomycetales : Microellobosporia gen. nov. J of General Microbiol 31, 421-429.

Davey, P.G., Malek, M.M. & Parker, S.E. (1992). Pharmaeconomics of antibacterial treatment. Pharmaeconomics 1(6), 409-37.

De Bruijn, F.J. (1992). Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus) sequences and the polymerase chain

237 reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol 58, 2180-2187.

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133-142.

De Somer P. & Van Dijck, P. (1955). A preliminary report on antibiotic number 899, a streptogramin-like substance. Antibiotics and Chemotherapy 5, 632 –639.

Demain, A.L. (2002). Prescription for an ailing pharmaceutical industry. Nat Biotechnol 20, 331.

Descheemaeker, P., Pot, B., Ledeboer, A.M., Verrips, T. & Kersters K. (1994). Comparison of the Lactococcus lactis differential medium (DCL) and SDS-PAGE of whole- cell protein extracts for the identification of lactococci to subspecies level. Syst Appl Microbiol 17, 459-466.

Desjardin, V., Bayard, R., Huck, N., Manceau, A. & Gourdon, R. (2002). Effect of microbial activity on the mobility of chromium in soils. Waste Manag 22(2),195-200.

Dewhirst, F. E., Fox, J. G. & On, S. L. W. (2000). Recommended minimal standards for describing new species of the genus Helicobacter. Int J Syst Evol Microbiol 50, 2231-2237.

Dey, S. & Agarwal, S.O. (1999). Characterization of a thermostable alpha-amylase from a thermophilic Streptomyces megasporus strain SD12. Indian J Biochem Biophys 36(3),150-7.

Diels, L., De Smet, M., Hooyberghs, L. & Corbisier, P. (1999). Heavy metals bioremediation of soil. Mol Biotechnol 12,149-158.

Doering-Saad, C., Kämpfer, P., Manulis, S., Kritzman, G., Schneider, J., Zakrzewska- Czerwinska, J., Schrempf, H. & Barash, I. (1992). Diversity among Streptomyces strains causing potato scab. Appl. Environ Microbiol 58, 3932-3940.

Donato, P., Lo Passo, C., La Cono, V., Albertano, P. & Urzi, C. (2000). Biodiversity of Streptomyces strains isolated from Roman hypogea, pp. 88-89. In: protection and conservation of the cultural heritage of the Mediterranean cities (Eds. E. Galan, P. Aparicio,

238 BIBLIOGRAPHY

A. Miras). Lectures, short papers and enlarged abstracts of the 5th International Symposium on the Conservation of Monuments in the Mediterranean basin. Seville.

Duraes Sette, S.L., Mendonca Alves Da Costa, L.A., Marsaioli A.J. & Manfio, G.P. (2004). Biodegradation of alachlor by soil streptomycetes. Appl Microbiol Biotechnol 64(5), 712-7. Epub 2004 Jan 16.

Dutkiewicz, J., Krysinska-Traczyk, E., Skorska, C., Sitkowska, J., Prazmo, Z. & Golec, M. (2001). Exposure to airborne microorganisms and endotoxin in herb processing plants. Ann Agric Environ Med, 8(2), 201-11.

Egan, S., Wiener, P., Kallifidas, D. & Wellington, E.M.H. (1998). Transfer of streptomycin biosynthesis gene clusters within streptomycetes isolated from soil. Appl Environ Microbiol 64, 5061-5063.

Eisen, J.A. (1995). The RecA protein as a model molecule for molecular systematic studies of bacteria: comparison of trees of recAs and 16S rRNAs from the same species. J Molec Evol 41, 1105-1123.

Embley, T.M. & Stackebrandt, E. (1994). The molecular phylogeny and systematics of the actinomycetes. Annu Rev Microbiol 48, 257-289.

Emerson, R. (1958). Mycological organization. Mycologia 50, 589-621.

Eriksson, K.E.L., Blanchette, R.A. & Ander, P. (1992). Microbial and enzymatic degradation of wood and wood components, Ch. 2.6: Cellulose degradation by bacteria, pp. 137-158, Berlin, Springer Verlag.

Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid – deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224-229.

Falcäo, D.E., Morais, J.O., Batista, A.C. & Massa, D.M.G. (1966). Elytrosporangium : a new genus of the Actinomycetales. Mycopathologia et Mycologia applicata 30, 161-171.

239 Faucher, E., Otrysko, B., Paradis, E., Hodge, N.C., Stall, R.E. & Beaulieu, C. (1993). Characterisation of streptomycetes causing russet scab in Quebec. Plant Dis 77, 1217-1220.

Faucher, E., Paradis, E., Goyer, C., Hodge, N.C., Hogue, R., Stall, E. & Beaulieu, C. (1995). Characterization of streptomycetes causing deep-pitted scab of potato in Quebec, Canada. Int J Syst Bacteriol 45, 222-225.

Felsenstein, J. (1981). Evolutionary trees from DNA sequence: a maximum likelihood approach. J Mol Evol 17, 368-376.

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.

Felsenstein, J. (1993). PHYLIP (phylogenetic inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.

Ferguson, E.V., Ward, A.C., Sanglier, J.J. & Goodfellow, M. (1997). Evaluation of Streptomyces species-groups by pyrolysis mass spectrometry. Zentralblatt fur Bakteriologie – Int J Medical Microbiol Virol Parasit Infect Dis 285, 169-181.

Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees: a method based on mutation distances as estimated from cytochrome c sequences is of general applicability. Science 155, 279–284.

Fox, G.E., Wisotzkey, J.D. & Jurtshuk, P. Jr. (1992). How close is close, 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42, 166-170.

Frandberg, E., Petersson, C., Lundgren, L.N. & Schnurer, J. (2000). Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1. Can J Microbiol 46(8),753-8.

Fujii, I & Ebizuka,Y. (1997). Anthracycline biosynthesis in Streptomyces galileus. Chemical Reviews 97, 2511-2523.

240 BIBLIOGRAPHY

Gadkari, D., Schricker, K., Acker, G., Kroppenstedt, R.M. & Meyer, O. (1990).

Streptomyces thermoautotrophicus sp. nov., a thermophilic CO-and H2-oxidizing obligate chemolithotroph. Appl Environ Microbiol 56, 3727-3734.

Garcia-Martinez, J., Bescos, I., Rodriguez-Sala, J.J. & Rodriguez-Valera, F. (2001). RISSC : a novel database fro ribosomal 16S-23S RNA genes spacer regions. Nucl Acids Research 29(1), 178-180.

Gauze, G.F., Preobrazhenkaya, T.P., Kudrina, E.S., Blinov, N.O., Ryabova, I.D. & Sveshnikova, M.A. (1957). Problems of classification of actinomycetes antagonists. Government Publishing House of Medical Literature (Russian), Medzig: Moscow.

Georghiou, P., Doggett, A., Kielhofner, M., Stout, J., Watson, D.A., Lupski, J.R. & Jamill, R.J. (1994). Molecular fingerprinting of Legionella species by repetitive element PCR. J Clin Microbiol 32, 2989-2994.

Gevers, D., Huys, G. & Swings, J. (2001). Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 27, 205(1), 31-6.

Gherardi, G., Del Grosso, M., Scotto D'Abusco, A., D'Ambrosio, F., Dicuonzo, G., Pantosti, A. (2003). Phenotypic and genotypic characterization of two penicillin-susceptible serotype 6B Streptococcus pneumoniae clones circulating in Italy. J Clin Microbiol 41(7), 2855-61.

Giacobini, C., De Cicco, M.A., Tiglie, I. & Accardo, G. (1988). Actinomycetes and biodeterioration in the field of fine art, pp. 418-423. In: Biodeterioration (Eds. D.R. Houghton, R.N. Smith, H.O.W. Eggins) vol. 7., Elsevier Applied Science, New York, NY.

Gillespie, D. & Spiegelman, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J of Molecular Biology 12, 829-842.

Gillis, M., De Ley, J. & De Cleene, M. (1970). The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur J of Biochemistry 12, 143-153.

Glaus, M.A., Heijman, C.G., Schwarzenbach, R.P. & Zeyer, J. (1992). Reduction of nitroaromatic compounds mediated by Streptomyces sp. exudates. Appl Environ Microbiol 58(6),1945-51.

241 Godoy, D., Randle, G., Simpson, A.J., Aanensen, D.M., Pitt, T.L., Kinoshita, R. & Spratt, B.G. (2003). Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei. J Clinical Microbiol 41, 2068-2079.

Goncalves, E.R. & Rosato, Y.B. (2002). Phylogenetic analysis of Xanthomonas species based upon 16S-23S r DNA intergenic spacer sequences. Int J Syst Evol Microbiol 52, 355- 361.

Goodfellow, M. & O’Donnell, A. G. (1993). Root of bacterial systematics. In: Handbook of New Bacterial Systematics, pp. 3-54, Edited by M. Goodfellow & A.G. O’Donnell. London: Academic Press.

Goodfellow, M. & Williams, S.T. (1983). Ecology of actinomycetes. Ann Rev Microbiol 37,189-216.

Goodfellow, M. (2000). Microbial systematics: background and uses, p. 1-18. In: F.G. Priest and M. Goodfellow (Eds.), Apllied microbial systematics. Kluwer Academic Publishers. Dordrecht.

Goodfellow, M., Manfio, G.P. & Chun, J. (1997). Towards a practical species concept for cultivable bacteria, in: M.F. Claridge, H.A. Dawah, M.R. Wilson (Eds.), Species: The units of biodiversity, Chapman and Hall, London.

Goodfellow, M., Williams, S.T. & Alderson, G. (1986a). Transfer of Actinosporangium violaceum Krasilnikov & Yuan, Actinosporangium vitaminophilum Shomura et al. and descriptions of the species. Syst Appl Microbiol 8, 61-64.

Goodfellow, M., Williams, S.T. & Alderson, G. (1986b). Transfer of Chainia species to the genus Streptomyces with emended description of species. Syst Appl Microbiol 8, 55-60.

Goodfellow, M., Williams, S.T. & Alderson, G. (1986c). Transfer of Kitasatoa purpurea Matsumae & Hata to the genus Streptomyces as Streptomyces purpureus comb. nov. Syst Appl Microbiol 8, 65-66.

Goodfellow, M., Williams, S.T. & Alderson, G. (1986d). Transfer of Elytrosporangium brasiliense Falcão de Morais et al., Elytrosporangium carpinense Falcão de Morais et

242 BIBLIOGRAPHY

al.,Elytrosporangium spirale Falcão de Morais et al., Microellobosporia cinerea Cross et al., Microellobosporia flavea Cross et al., Microellobosporia grisea (Konev et al.), Pridham and Microellobosporia violacea (Tsyganov et al.) Pridham to the genus Streptomyces, with emended description of the species. Syst Appl Microbiol 8, 48-54.

Gordon, R.E. & Horan, A.C. (1968). A piecemeal description of Streptomyces griseus (Krainsky) Waksman and Henrici. J Gen Microbiol 50, 223-233.

Goris, J., De Vos, P., Caballero-Mellado, J., Park, J., Falsen, E., Quensen, J.F., Tiedje, J.M. & Vandamme, P. (2004). Classification of the biphenyl- and polychlorinated biphenyl- degrading strain LB400T and relatives as Burkholderia xenovorans sp. nov. IJSEM Papers in Press, published online 9 July 2004. doi:10.1099/ijs.0.63101-0.

Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridisation method compared with initial renaturation method. Can J Microbiol 44, 1148-1153.

Gottlieb, D. & Shirling, E.B. (1967). Cooperative description of type cultures of Streptomyces.I. The International Streptomyces project. Int J Syst Bacter 17, 315-322.

Gottlieb, D. (1961). An evaluation of criteria and procedures used in the description and characterisation of the streptomycetes. A co-operative study. Appl Microbiol 9, 55-65.

Gottlieb, D. (1963). Recommendations for description of some Actinomycetales appearing in patent applications. Int Bulletin of Bacteriological Nomenclature and Taxonomy 13, 159-160.

Goyer, C. & Beaulieu, C. (1997). Host range of streptomycete strains causing common scab. Plant Dis 81, 901-904.

Goyer, C., Otrysko, B. & Beaulieu, C. (1996). Taxonomic studies on streptomycetes causing potato common scab – A review. Can J of Plant Pathology 18, 107-113.

Grabski, A.C., Forrester, I.T., Patel, R. & Jeffries, T.W. (1993). Characterization and N- terminal amino acid sequences of beta-(1-4)endoxylanases from Streptomyces roseiscleroticus: purification incorporating a bioprocessing agent. Protein Expr Purif 4(2), 120-9.

243 Gravius, B., Bezmalinovic, T., Hranueli, D. & Cullum, J. (1993). Genetic instability and strain degeneration in Streptomyces rimosus. Appl Environ Microbiol 59(7), 2220-2228.

Grimont, F. & Grimont, P.A. (1986). Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann Inst Pasteur Microbiol 137B, 165-75.

Groth, I., Vettermann, R., Schuetze, B., Schumann, P. & Saiz-Jimenez, C. (1999). Actinomycetes in karstic caves of northern Spain (Altamira and Tito Bustillo). J Microbiol Methods 36, 115-122.

Grundy, W.E., Whitman, A.L., Rdzok, E.G., Rdzok, E.J., Hanes, M.E., Sylvester, J.C. (1952). Actithiazic acid I. Microbiological studies. Antibiot Chemother 2, 399-408.

Gugnani, A.C., Unaogu, I.C. & Emeruwa, C.N. (1993). Pulmonary infection due to Streptomyces griseus. J Commun Dis 25(1), 38-40.

Gupta, R., Saxena, R.K., Chaturvedi, P. & Virdi, J.S. (1995). Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis. J Appl Bacteriol 78(4), 378-83.

Gürtler V. & Stanisich V. A. (1996). New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiology 142, 3-16.

Hain, T., Ward-Rainey, N., Kroppenstedt, R.M., Stackebrandtet, E. & Rainey, F. A. (1997). Discrimination of Streptomyces albidoflavus strains based on the size and number of 16S-23S ribosomal DNA intergenic spacers. Int J Syst Bacteriol 47, 202-206.

Hall. T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT, Nucleic Acids Symp Ser 41, 95-98.

Harchand, R.K. & Singh, S. (1997). Extracellular cellulase system of a thermotolerant streptomycete: Streptomyces albaduncus. Acta Microbiol Immunol Hung 44(3), 229-39.

Harrison, M.D. (1962). Potato russet scab, its cause and factors affecting its development. American Potato Journal 39, 368-387.

Hasegawa, T., Takizawa, M., & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319-322

244 BIBLIOGRAPHY

Hatano, K., Nishii, T. & Kasai, H. (2003). Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA-DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. rev. Int J Syst Evol Microbiol 53, 1519-1529.

Hattori, H. (1992). Influence of heavy metals on soil microbial activities. Soil Sci Plant Nutr 38, 93-100.

He, L., Wang, L., Li, W., Liu, Z., Lanoot, B., Vancanneyt, M., Swings, J. & Huang, Y. Streptomyces jietaisiensis sp. nov., isolated from soil in North China. Submitted to Int J Syst Evol Microbiol.

Helgason, E., Tourasse, N.J., Meisal, R., Caugant, D.A., Kolsto, A.B. (2004). Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl Environ Microbiol 70(1), 191-201.

Herr, R.R., Janhnke, H.K. & Argoudelis, A.D. (1967). The structure of streptozotocin. J of the American Chemical Society 89, 4808-4809.

Hesseltine, C.W., Bennedict, R.G. & Pridham, T.G. (1954). Useful criteria for species differentiation in the genus Streptomyces. Annals of New York. Academy of Science 60, 136- 151.

Heyndrickx, M., Vauterin, L., Vandamme, P., Kersters, K. & De Vos, P. (1996). Applicability of combined amplified ribosomal DNA restriction analysis (ARDRA) patterns in bacterial phylogeny and taxonomy. J Microbiol Meth 26, 2477-259.

Heyrman J, Mergaert J, Denys R, Swings J. (1999). The use of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present on three mural paintings showing severe damage by microorganisms. FEMS Microbiol Lett 1, 181(1), 55-62.

Heyrman, J. & Swings, J. (2001). 16S rDNA sequence analysis of bacterial isolates from biodeteriorated mural paintings in the Servilia tomb (Necropolis of carmona, Seville, Spain). Syst Appl Microbiol 24(3), 417-22.

Hopwood, D.A. (1999). Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology 145, 2183-2202.

245 Horikoshi, K. (1999). Alkaliphiles: some applications of their products for biotechnology. Microbiol Mol Biol Rev 63(4), 735-50.

Hoshiko, S., Makabe, O., Nojiri, C., Katsumata, K., Satoh, E. & Nagaoka, K. (1987). Molecular cloning and characterization of the Streptomyces hygroscopicus alpha-amylase gene.J Bacteriol 169(3),1029-36.

Huang, Y., Qi, W., Lu, Z., Liu, Z. & Goodfellow, M. (2001). Amycolatopsis rubida sp. nov., a new Amycolatopsis species from soil. Int J Syst Evol Microbiol 51, 1093-1097.

Huang, Y., Li, W., Wang, L., Lanoot, B., Vancanneyt, M., Rodriguez, Z., Liu, Z. & Swings, J. (2004). Streptomyces glauciniger sp. nov., a novel mesophilic streptomycete isolated from soil in south China. Int J Syst Evol Microbiol 54, 2085-2089.

Huβ, V.A.R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184-192.

Hyvarinen, A., Reponen, T., Husman, T. & Nevalainen, A. (2001). Comparison of the indoor air quality in mould damaged and reference buildings in a subarctic climate. Cent Eur J Public Health 9(3), 133-9.

Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M. & Omura, S. (2003). Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21(5), 526-31. Epub 2003 Apr 14.

Janssen, P., Coopman, R., Huys, G., Swings, J., Bleeker, M., Vos, P., Zabeau M. & Kersters, K. (1996). Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. Microbiology 142(7), 1881-1993.

Jensen, H.L. (1930). Actinomycetes in Danish soil. Soil Science 30, 59-77.

Jersek, B., Gilot, P., Gubina, M., Klun, N., Mehle, J., Tcherneva, E., Rijpens, N. & Herman, L. (1999). Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR. J Clin Microbiol 37(1), 103-9.

246 BIBLIOGRAPHY

Johansen, J.E.& Binnerup, S.J. (2002). Contribution of Cytophaga-like Bacteria to the potential of turnover of carbon, nitrogen, and phosphorus by bacteria in the rhizosphere of barley (Hordeum vulgare L.) Microb Ecol 45, 298-306.

Johnson, A.P., Sheppard, C.L., Harnett, S.J., Birtles, A., Harrison, T.G., Brenwald, N.P., Gill, M.J., Walker, R.A., Livermore, D.M. & George, R.C. (2003). Emergence of a fluoroquinolone-resistant strain of Streptococcus pneumoniae in England. J Antimicrob Chemother 52(6), 953-60. Epub 2003 Oct 29.

Jones, K. L. (1949). Fresh isolates of actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. J Bacteriol 57, 141-145.

Judd, A.K., Schneider, M., Sadowsky, M.J. & De Bruijn, F.J. (1993). Use of repetitive sequences and the polymerase chain reaction technique to classify genetically related Bradyrhizobium japonicum serocluster 123 strains. Appl Environ Microbiol 59, 1702-1708.

Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.

Kämpfer, P. & Kroppenstedt, R.M. (1991). Probabilistic identification of Streptomyces using miniaturized physiological test. J Gen Microbiol 137, 1893-1902.

Kämpfer, P., Kroppenstedt, R. M. & Dott, W. (1991). A numerical classification of the genera Steptomyces and Streptoverticillium using miniaturized physiological tests. J Gen Microbiol 137,1831-1891.

Kaneko, T., Saito, K., Kawamura, Y. & Takahashi, S. (2001). Molecular cloning of acid- stable glucose isomerase gene from Streptomyces olivaceoviridis E-86 by a simple two-step PCR method, and its expression in Escherichia coli. Biosci Biotechnol Biochem 65(5), 1054- 62.

Kansoh, A.L. & Nagieb, Z.A. (2004). Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp. Antonie Van Leeuwenhoek 85(2), 103-14.

247 Karpovich-Tate, N. & Rebrikova, N.L. (1990). Microbial communities on damaged frescoes and building materials in the cathedral of the nativity of the virgin in the Pafnutii-Borovskii monastery, Russia. Int Biodeterior 27, 281-296.

Kasai, H., Watanabe, K., Gasteiger, E., Bairoch, A., Isono, K., Yamamoto, S. & Harayama, S. (1998). Construction of the gyrB database for the identification and classification of bacteria. Genome Informatics 9, 13-21.

Kataoka, M., Ueda, K., Kudo, T., Seki, T. & Yoshida, T. (1997). Application of the variable region in 16S rDNA to create an index for rapid species identification in the genus Streptomyces. FEMS Microbiol Lett 151, 249-255.

Kawase, T., Saito, A., Sato,T., Kanai, R., Fujii, T., Nikaidou, N., Miyashita, K. & Watanabe, K. (2004). Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria. Appl Environ Microbiol 70(2), 1135-44.

Kawato, M. & Shinobu, R. (1959). On Streptomyces herbaricolor sp. nov., supplement: a single technique for microscopical observation. Mem Osaka Unit Lib Arts Educ B Nat Sci 8, 114-119.

Keim P., Kalif A., Schupp J., Hill K., Travis S.E., Richmond K., Adair D.M., Hugh-Jones M., Kuske C.R. & Jackson P. J. (1997). Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J Bacteriol 179(3), 818-24.

Kersters, K. (1985). Numerical methods in the classification of bacteria by protein electrophoresis. In Computer-assisted Bacterial Systematics, pp. 337-68. Edited by M. Goodfellow, D. Jones & F.G. Priest. Academic Press (London)

Kim, B (1999). Polyphasic taxonomy of thermophilic actinomycetes. PhD thesis, University of Newcastle upon Tyne, UK.

Kim, B., Al-Tai, A. M., Kim, S. B. Somasundaram, P. & Goodfellow, M. (2000). Streptomyces thermocoprophilus sp. nov., a cellulase-free endo-xylanase-producing streptomycete. Int J Syst Evol Microbiol 50, 505-509.

248 BIBLIOGRAPHY

Kim, B., Sahin, N., Minnikin, D. E., Zakrzewska-Czerwinska, J., Mordarski, M. & Goodfellow, M. (1999). Classification of thermophilic streptomycetes including the description of Streptomyces thermoalcalitolerans sp. nov. Int J Syst Bacteriol 49, 7- 17.

Kim, B.J., Kim, C.J., Chun, J., Koh, Y.H., Lee, S.H., Hyun, J.W., Cha, C.Y. & Kook, Y.H. (2004). Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase -subunit gene (rpoB) sequences. Int J System Evol Microbiol 54(2), 593-598.

Kim, B.J., Lee, S.H., Lyu, M.A., Kim, S.J., Bai, G.H., Chae, G.T., Kim, E.C., Cha, C.Y.& Kook, Y.H. (1999). Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 37(6), 1714-20.

Kim, D., Chun, J., Sahin, N., Hah, Y.C. & Goodfellow, M. (1996). Analysis of thermophilic clades within the genus Streptomyces by 16S ribosomal DNA sequence comparisons. Int J Syst Bacteriol 46, 581-587.

Kim, E., Kim, H., Hong, S.P., Kang, K.H., Kho, Y.H. & Park, Y.H. (1993). Gene organisation and primary structure of a ribosomal RNA gene cluster from Streptomyces griseus subsp. griseus. Gene 132, 21-31.

Kim, S. B., & Goodfellow, M. (2002). Streptomyces avermitilis sp. nov., nom. rev., a taxonomic home for the avermectin-producing streptomycetes. Int J Syst Evol Microbiol 52, 2011-2014.

Kim, S. B., Falconer, C., Williams, E. & Goodfellow, M. (1998). Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately thermophilic carboxydotrophic species from soil. Int J Syst Bacteriol 48, 59-68.

Kim, S. B., Lonsdale, J., Seong, C.-N. & Goodfellow, M. (2003). Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici 1943AL) emend. Rainey et al. 1997. Antonie Leeuwenhoek 83, 107-116.

249 Kim, S. B., Seong, C. N., Jeon, S. J., Bae, K. S. & Goodfellow, M. (2004). A taxonomic study on neutrotolerant acidophilic actinomycetes isolated from soil and description of Streptomyces yeochonesnsis sp. nov. Int J Syst Evol Microbiol 54, 1, 211-4.

Kim, S., Falconer, C., Williams, E. & Goodfellow, M. (1998). Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately hermophilic carboxydotrophic species from soil. Int J Syst Bacteriol 48, 59-68.

Kim, S., Lonsdale, J., Seong, C-N. & Goodfellow, M. (2003). Streptacidiphilus gen.nov., acidophilic actinomycetes with cell wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici (1943)AL emend. Rainey (1997). Antonie van Leeuwenhoek 83(2),107-16.

Kim, S.B. & Goodfellow, M. (2002). Streptomyces thermospinisporus sp. nov., a moderately thermophilic carboxydotrophic streptomycete isolated from soil. Int J Syst Evol Microbiol 52, 4, 1225-8.

Kim, W (2004). The Streptomyces violaceoruber clade: a model system for defining a post- genomic species concept for Streptomyces. PhD thesis, University of Newcastle upon Tyne, UK.

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.

Klokke, A.H., Swamidasan, G., Anguli, R. & Verghese, A. (1968). The causal agents of mycetoma in South India. Trans R Soc Trop Med Hyg 62, 509-516.

Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1-32.

Ko, K. S., Kim, J.M., Kim, J.W., Jung, B.Y., Kim, W., Kim, I.J. & Kook, Y.H. (2003). Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR. J Clinical Microbiol 41, 2908-2914.

Koga, T., Shimada, Y., Kuroda, M., Tsujita, Y., Hasegawa, K. & Yamazaki, M. (1990). Tissue-selective inhibition of cholesterol synthesis in vivo by pravastatin sodium, a specific inhibitor of HMG-CoA reductase. Biochim Biophys Acta 1045, 115-120. 250 BIBLIOGRAPHY

Korn-Wendisch, F. & Kutzner, H.J. (1992). The family Streptomycetaceae. In the Prokaryotes. A handbook on the Biology of Bacteria: Ecophysiology, isolation, identification, application 2nd edn., vol.1 pp. 921-995, Edited by A. Balows, H.G. Truper, M. Dworkin, W. Harder & K.H. Schleifer. New York: Springer-Verlag.

Korn-Wendisch, F. & Schneider, J. (1992). Phage typing – a useful tool in actinomycete systematics. Gene 115, 243-247.

Kozdroj, J. & VanElsas, J.D. (2001). Structural diversity of microorganisms in chemically pertubated soil assessed by molecular and cytochemical approaches. J Microbiol Methods 43,197-212.

Krainsky, A. (1914). Die Aktinomyceten und ihren Bedeutung in der Natur. Zentralblatt für bakteriologie Parasitenkunde infektionskrankheiten und Hiegiene. Abteilung II, 41, 649-688.

Krassilnikov, N.A. & Yuan, C.S. (1961). Actinosporangium, a new genus of the family Actinoplanaceae. Izvestiya Akademii Nauk SSSR Seriya Bioloicheskaya 8, 113-116.

Kristijansson, J.K. (1989). Thermophilic bacteria as a source of thermostable enzymes. Trends in biotechnology 7, 349-353.

Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Bacterial Systematics, pp. 173-199. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.

Kubota, T., Miyamoto, K., Yasuda, M., Inamori, Y. & Tsujibo, H. (2004). Molecular characterization of an intracellular beta-N-acetylglucosaminidase involved in the chitin degradation system of Streptomyces thermoviolaceus OPC-520.Biosci Biotechnol Biochem 68(6),1306-14.

Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S.C., Bron, S., Brouillet, S., Bruschi, C.V., Caldwell, B., Capuano, V., Carter, NM., Choi, S.K., Codani, J.J., Connerton, I.F., Cummings, N.J., Daniel, R.A., Denizot, F., Devine, K.M., Düsterhöft, A., Ehrlich, S.D., Emmerson, P.T., Entian, K.D., Errington, J., Fabret, C., Ferrari, E., Foulger, D., Fritz, C., Fujita, Y., Fuma, S., Galizzi, A. Galleron, N., Ghim, S.-Y., Glaser, P., Goffeau, A., Golightly, E.J., Grandi, G., Guiseppi,

251 G., Guy, B.J., Haga, K., Haiech, J., Harwood, C.R., Hénaut, A., Hilbert, H., Holsappel, S., Hosono, S., Hullo, M.-F., Itaya, M., Jones, L., Joris, B., Karamata, D., Kasahara, Y., Jklaerr-Blanchard, M., Klein, C., Kobayashi, Y., Koetter, P., Koningstein, G., Krogh, S., Kumano, M., Kurita, K., Lapidus, A., Lardinois, S., Lauber, J., Lazarevic, V., Lee, S.-M., Levine, A., Liu, H., Masuda, S., Mauël, C., Médigue, C., Medina, N., Mellada, R.P., Mizuno, M., Moest, D., Nakai, S., Noback, M., Noone, D., O’reilly, M., Ogawa, K., Ogiwara, A., Oudega, B., Park, S.-H., Parro, V., Pohl, T.M., Portetelle, D., Porwollik, S., Prescott, A.M., Presecan, E., Pujic, P., Purnelle, B., Rapoport, G., Rey, M., Reynolds, S., Rieger, M., Rivolta, C., Rocha, E., Roche, B., Rose, M., Sadaie, Y., Sato, T., Scanlan, E., Schleich, S., Schroeter, R., Scoffone, F., Sekiguchi, J., Sekowska, A., Seror, S.J., Serror, P., Shin, B.-S., Soldo, B., Sorokin, A., Tacconi, E., Takagi, T., Takahashi, H., Takemaru, K., Takeuchi, M., Tamakoshi, A., Tanaka, T., Terpstra, P., Tognoni, A., Tosato, V., Uchiyama, A., Vandenbol, M., Vannier, F., Vassarotti, A., Viari, A., Wambutt, R., Wedler, E., Wedler, H., Weitzenegger, T., Winters, P., Wipat, A., Yamamoto, H., Yamane, K., Yasumoto, K., Yata, K., Yoshida, K., Yoshikawa, H.-F., Zumstein, E., Yoshikawa, H. & Danchin, A. (1997). The complete genome sequence of the Gram positive bacterium Bacillus subtilis. Nature 390, 249-256.

Kurtboke, I., Hayakawa, M., Terekhova, L. & Okazaki, T. (2003). Selective isolation of rare actinomycetes. Edited by I. Kurtboke. University of the Sunshine Coast, Queensland, Australia. National Press of Australia.

Kurylowicz, W. & Gyllenberg, H.G. (1988). Problems of taxonomy of streptomycetes. In Biology of actinomycetes ’88, pp. 11-18. Edited by Y. Okami, T. Beppu, & H. Ogawara. Tokyo: Japan Scientific Societies Press.

Küster, E. (1959). Outline of a comparative study of criteria used in the classification of actinomycetes. Int Bulletin of Bacteriology, Nomenclature and Taxonomy 9, 97-104.

Küster, E. (1961). Results of a comparative study of criteria used in the classification of actinomycetes. Int Bulletin of Bacteriology, Nomenclature and Taxonomy 11, 91-98.

Kutzner, H. J. (1981). The family Streptomycetateae, The Prokaryotics (vol II), Mortimer P. Starr, Heinz Stolp, Hans G. Trüper, Albert Balows, Hans G. Schlegel, Springer-verslag, p. 2029-2058.

252 BIBLIOGRAPHY

La Farina, M., Stira, S., Mancuso, R. & Grisanti, C. (1996). Characterisation of Streptomyces venezulae ATCC 10595 rRNA gene clusters and cloning of rrnA. J Bacteriol 178 (5), 1480-1483.

Labeda, D. P. & Lyons, A. J. (1992). DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948. Appl Environ Microbiol 58, 532-535.

Labeda, D. P. (1988). Kitasatosporia mediocidica sp. nov. Int J Syst Bacteriol 38, 287-290.

Labeda, D. P. (1993). DNA relatedness among strains of the Streptomyces lavendulae phenotypic cluster group. Int J Syst Bacteriol 822-825.

Labeda, D. P. (1998). DNA relatedness among the Streptomyces fulvissimus and Streptomyces griseoviridis phenotypic cluster group. Int J Syst Bacteriol 48, 829-832.

Labeda, D. P., Lechevalier, M. P. & Testa, R. T. (1997). Streptomyces stamineus sp. nov., a new species of the verticillate streptomycetes. Int J Syst Bacteriol 47, 747-753.

Labeda, D.P. & Lyons, A.J. (1991a). Deoxyribonucleic acid relatedness among species of the Streptomyces cyaneus cluster. Syst Appl Microbiol 14, 158-164.

Labeda, D.P. & Lyons, A.J. (1991b). The Streptomyces violaceusniger cluster is heterogeneous in DNA relatedness among strains. Emendation of the descriptions of S. violaceusniger and S. hygroscopicus. Int J Syst Bacteriol 41, 398-401.

Labeda, D.P. (1992). DNA-DNA hybridisation in the systematics of Streptomyces. Gene 115, 249-253.

Labeda, D.P. (1996). DNA relatedness among verticilliate-forming Streptomyces species (formerly Streptoverticillium species). Int J Syst Bacteriol 46, 699-703.

Lallai, A. & Mura, G. (2004). Biodegradation of 2-chlorophenol in forest soil : effect of inoculation in aerobic sewage sludge. Environ Toxicol Chem 23(2), 325-30.

Lambert, D.H. & Loria, R. (1989a). Streptomyces acidiscabies sp. nov. Int J Syst Bacteriol 39, 393-396. 253 Lambert, D.H. & Loria, R. (1989b). Streptomyces scabies sp. Nov. Int J Syst Bacteriol 39, 387-392.

Lan, R. & Reeves, P.R. (1998). Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae. Microbiology 144, 1213-1221.

Langham, C.D., Williams, S.T., Sneath, P.H.A. & Mortimer, A.M. (1989). New probability matrices for identification of Streptomyces. J of General Microbiology 135, 121-133.

Lanoot, B., Vancanneyt, M., Cleenwerck, I., Wang, L., Li, W., Liu, Z. & Swings, J. (2002). The search for synonyms among streptomycetes by using SDS-PAGE of whole-cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violaceus. Int J Syst Evol Microbiol 52(3), 823- 829.

Lanoot, B., Vancanneyt, M., Dawyndt, P., Cnockaert, M.C., Zhang, J., Huang, Y., Liu, Z. & Swings, J. (2004). BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol 27(1), 84-92.

Lanoot, B., Vancanneyt, M., Van Schoor, A., Liu, Z. & Swings, J. (2005a). Reclassification of Streptomyces nigrifaciens as a later synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as later synonyms of Streptomyces anulatus; Streptomyces chibaensis as later synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as later synonym of S. minutiscleroticus; Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as later synonyms of Streptomyces microflavus. Int J Syst Evol Microbiol, in press.

Lanoot, B., Vancanneyt, M., Hoste, B., Vandemeulebroecke, K., Cnockaert, M.C., Dawyndt, P., Liu, Z. & Swings, J. Phylogenetic grouping of streptomycetes using 16S-ITS RFLP fingerprinting. Submitted to Research in Microbiology.

Lanoot, B., Vancanneyt, M., Hoste, B. , Cnockaert, M.C., Piecq, M., Gosselé, F. & Swings, J. (2005b). Phenotypic and genotypic characterisation of mutants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae. System Appl Microbiol, in press.

254 BIBLIOGRAPHY

Lapwood, D.H. (1973). Streptomyces scabies and potato scab disease, pp. 253-260. In: Sykes, G., Skinner, F.A. (eds), Actinomycetales: characteristics and practical importance. London, New York: Academic Press.

Lawrence, C.H., Clark, M.C. & King, R.R. (1990). Induction of common scab symptoms in aseptically cultured potato tubers by the vivotoxin thaxtomin. Phytopathology 80, 606-608.

Lazzarini, A., Cavaletti, L., Toppo, G. & Marinelli, F. (2000). Rare genera of actinomycetes as potential producers of new antibiotics. Antonie van Leeuwenhoek 78, 399-405.

Leblond, P., Demuyter, P., Simonet, J.M. & Decaris, B. (1991). Genetic instability and associated genome plasticity in Streptomyces ambofaciens: pulsed-field gel electrophoresis evidence for large DNA alterations in a limited genomic region. J Bacteriol 173(13), 4229-33.

Lechevalier, H. A. & Lechevalier, M. P. (1980). The chemotaxonomy of actinomycetes. In Actinomycete Taxonomy, Special Publication 6, pp. 277-284. Edited by A. Dietz & D. W. Thayer. Arlington, VA: Society of Industrial Microbiology.

Lechevalier, M. P., De Biévre, C. & Lechevalier, H. A. (1977). Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem Syst Ecol 5, 249–260.

Lechevalier, M.P., & Lechevalier, H.A. (1970). Chemical composition as a criterion in the classification of aerobic actinomyces. Int J Syst Bacteriol 20, 435-443.

Lemee, L., Dhalluin, A., Pestel-Caron, M., Lemeland, J.F. & Pons, J.L. (2004). Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. J Clin Microbiol 42(6), 2609-17.

Li, W., Lanoot, B., Zhang, Y., Vancanneyt, M., Swings, J. & Liu, Z. (2002). Streptomyces scopiformis sp. nov., a novel streptomycete with fastigiate spore chains. Int J Syst Evol Microbiol 52, 1629-1633.

Li, W.-J., Zhang, L.-P., Xu, P., Cui, X.-L., Lu, Z.-T., Xu, L.-H. & Jiang, C.-L. (2002). Streptomyces beijiangensis sp. nov., a psychrotolerant actinomycete isolated from soil in China. Int J Syst Evol Microbiol 52, 1695-1699.

255 Liu, Z.H. & Zhang, Y.M. (1996). Streptomyces scopiformis sp. nov. Actinomycetes 7(1), 23- 27.

Locci, R., Rogers, J., Sardi, G., & Schofield, G.M. (1981). A preliminary numerical study on named species of the genus Streptoverticillium. Annali di Microbiologia 31, 115-121.

Locci, R.E. & Schofield, G. (1989). Genus Streptoverticillium Baldacci 1958, 15, emend. Mut.char. Baldacci, Farina and Locci 1966, 168 AL, pp. 2492-2504. In: Bergey’s Manual of systematic bacteriology, 4 (Williams, S.T., Sharpe, M.E., Holt, J.G. eds.). Baltimore, Williams and Wilkins 1989.

Lonsdale, J. T. (1985). Aspects of the biology of acidophilic actinomycetes. PhD thesis, University of Newcastle, Newcastle upon Tyne, UK.

Loria, R., Bukhalid, R.A., Fry, B.A. & King, R. R. (1997). Plant pathogenicity in the genus Streptomyces. Plant Dis 81, 836-846.

Louws, F.J., Fulbright, D.W., Stephens, C.T. & De Bruijn, F.J. (1994). Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl Environ Microbiol 60, 2286-2295.

Louws, F.J., Rademaker, J.L.W. & de Bruijn, F.J. (1999). The three D’s of PCR-based genomic analysis of phytobacteria: diversity, detection and disease diagnosis. Ann Rev Phytopath 37, 85-125.

Lu, Z.H., Liu, Z.H., Wang, L.M., Zhang, Y.M., Qi, W.H. & Goodfellow, M. (2001). Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., novel actinomycetes from soil. Int J Syst Evol Bacteriol 51, 319-325.

Lupski, J.R. & Weinstock, G.M. (1992). Short, interspersed repetitive DNA sequences in prokaryotic genome. J Bacteriol 174, 4525-4529.

Madigan M.T., Martinko, J. M. & Parker, J. (2003). Brock Biology of Microorganisms, 10th edition, Prentice-Hall, New Jersey, USA.

Magnuson TS, Roberts MA, Crawford DL & Hertel G. (1991). Immunologic relatedness of extracellular ligninases from the actinomycetes Streptomyces viridosporus T7A and

256 BIBLIOGRAPHY

Streptomyces badius 252. Appl Biochem Biotechnol 28-29, 433-43.

Mahe, A., Develoux, M., Lienhardt, C., Keita, S. & Bobin, P. (1996). Mycetomas in Mali: causative agents and geographic distribution. Am J Trop Med Hyg. 54(1),77-9.

Maidak, B.L., Cole, J.R., Parker, C.T., Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, J.M. & Woese, C.R. (1999). A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27, 171-173.

Maidak, B.L., Olsen, G.L., Larsen, N., Overbeek, R., McCaaughey, M.J. & Woese, C.R. (1997). The Ribosomeal Database Project (RDP). Nucleic Acids Res 25, 109-111.

Maiden, M.C., Bygraves, J.A., Feil, E., Morelli, G., Russell, J.E., Urwin, R., Zhang, Q., Zhou, J., Zurth, K., Caugant, D.A., Feavers, I.M., Achtman, M. & Spratt, B.G. (1998). Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microroganisms. Proc Natl Acad Sci USA 95, 3140-3145.

Manchester, L., Pot, B., Kersters, K. & Goodfellow, M. (1990). Classification of Streptomyces and Streptoverticillium species by numerical analysis of electrophoretic protein patterns. Syst Appl Microbiol 13, 333-337.

Manfio, G. P., Zakrzewska-Czerwinska, J., Atalan, E. & Goodfellow, M. (1995). Towards minimal standards for the description of Streptomyces species. Biotechnologia 7-8, 242-253.

Manning, G., Dowson, C.G., Bagnall, M.C., Ahmed, I.H., West, M. & Newell, D.G. (2003). Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni. Appl Environ Microbiol 69(11),6370-9.

Mansouri, K. & Piepersberg, W. (1991). Genetics of streptomycin production in Streptomyces griseus: nucleotide sequence of five genes, strFGHIK, including a phosphatase gene. Mol Gen Genet 228(3), 459-69.

Marmur, J. & Doty, P. (1962). Determination of base composition of deoxyribonucleic acid from its denaturation temperature. J Mol Biol 5, 109-118.

257 Martin, P., Dary, A., Andre, A. & Decaris, B. (2000). Identification and typing of Streptomyces strains: evaluation of interspecific, intraspecific and intraclonal differences by RAPD fingerprinting. Res Microbiol 151(10), 853-64.

Masco, L., Huys, G., Gevers, D., Verbrugghen, L. & Swings, J. (2003). Identification of Bifidobacterium species using rep-PCR fingerprinting. Syst Appl Microbiol 26(4), 557-63.

Mata-Sandoval, J.C. (2001). Influence of rhamnolipids and triton X-100 on the biodegradation of three pesticides in aqueous phase and soil slurries. J Agric Food Chem 49(7), 3296-303.

Matsumae, A.M., Ohtani, M. & Hata, T. (1968). A new genus of Actinomycetales: Kitasatoa gen. Nov J of Antibiotics 21, 616-625.

Mayama, M. (1959). A consideration on the classification of streptomycetes. Annual Report of Shionogi Research Laboratories (Japan) 9, 1185-1212.

McCarthy, B.J. & Bolton, E.T. (1963). An approach to the measurement of genetic relatedness among organisms. Proceedings of the National Academy of Sciences of the United States of America, 50, 156-164.

Mengoni, A., Barabesi, C., Gonnelli, C., Galardi, F., Gabbrielli, R. & Bazzicalupo, M. (2001). Genetic diversity of heavy metal-resistant populations in Selene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis. Mol Ecol 10, 1909-1916.

Mergaert, J., Wouters, A. & Swings, J. (1994). Estimation of the intrinsic biodiversity among poly(3-hydroxyalkanoates) degrading streptomycetes using gas chromatographic analysis of fatty acids. System Appl Microbiol 17, 601-612.

Mergeay, M., Nies, D., Schlegel, H.G., Gerits, J., Charles, P. & van Gijsegem, F. (1985). Alcaligenes eutrophus CH34 is a facultative chemolitotroph with plasmid-bound resistance to heavy metals. J Bacteriol 162, 328-334.

Meyers, P.R., Porter, D.S., Omorogie, C., Pule, J.M. & Kwetane, T. (2003). Streptomyces speibonae sp. nov., a novel streptomycete with blue substrate mycelium isolated from South African soil. Int J Syst Evol Microbiol 53, 801 - 805.

258 BIBLIOGRAPHY

Mikami, Y., Miyashita, K. & Arai, T. (1982). Diaminopimelic acid profiles of alkalophilic and alkaline-resistant strains of actinomycetes. J Gen Microbiol 128,1709-1712.

Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233-241.

Minnikin, D.E., Hutchinson, I.G., Caldicott, A.B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic acid containing bacteria. J Chromatogr 188, 221-233.

Miyajima, K., Tanaka, F., Takeuchi, T. & Kuninage, S. (1998). Streptomyces turgidiscabies sp. nov. Int J Syst Bacteriol 48, 495-502.

Mlynarski, J., Ruiz-Caro, J. & Furstner, A. (2004). Total synthesis of macroviracin D (BA- 2836-4). Chemistry 3,10(9), 2214-22.

Mochizuki, S., Hiratsu, K., Suwa, M., Ishii, T., Sugino, F., Yamada, K. & Kinashi, H. (2003). The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organisation for secondary metabolism. Mol Microbiol 48, 1501-1510.

Monson, A.M., Bradley, S.G., Enquist, L.W. & Cruces, G. (1969). Genetic homologies among Streptomyces violaceoruber strains. J Bacteriol 99, 702-706.

Mordarski, M., Goodfellow, M. & Sneath, P.H.H. (1986). Evaluation of species groups in the genus Streptomyces. In Biological, Biochemical and Biomedical Aspects of actinomycetes. Pp. 517-525, Edited by G. Szabó, S. Biró & M. Goodfellow. Budapest: Akadémiai Kiadó.

Mossad S.B., Tomford J.W., Stewart R., Ratliff N.B. & Hall G.S. (1995). Case report of Streptomyces endocarditis of a prosthetic aortic valve. J Clin Microbiol 33(12), 3335-7.

259 Murakami, S., Harada, S., Takahashi, Y., Naganawa, H., Takeuchi, T. & Aoyagi, T. (1996). Piperastatin B: a new selective serine carboxypeptidase inhibitor from Streptomyces lavendofoliae MJ908-WF13. J Enzyme Inhib 11(1), 51-66.

Nakagaito, Y., Yokota, A. & Hasegawa, T. (1992). Three new species of the genus Streptomyces: Streptomyces cochleatus sp.nov., Streptomyces paracochleatus sp.nov. and Streptomyces azaticus sp. nov. J Gen Appl Microbiol 38, 105-120.

Nakano, T., Miyake, K., Ikeda, M., Mizukami, T. & Katsumata, R. (2000). Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens. Appl Environ Microbiol 66(4),1400-4.

Natsume, M., Komiya, M., Koyanagi, F., Kawaide, H. & Tashiro, N. (2003). Phytotoxin produced by Streptomyces cheloniumii causing potato russet scab. 13th International Symposium on the Biology of actinomycetes, Melbourne, Australia.

Neuzil J., Kristufek, V. & Blumauerova, M. (1988). Enzymic degradation of bromoxynil by cell-free extracts of Streptomyces felleus. Folia Microbiol (Praha) 33(5),349-54.

Nevalainen, A., Pasanen, A.-L., Niininen, M., Reponen, T., Kalliokoski, P. & Jantunen, M.J. (1991). The indoor air quality in Finnish homes with mold problems. Environ Int 17, 299- 302.

Nguyen, J., Francou, F., Virolle, M.J. & Guerineau, M. (1997). Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene. J Bacteriol 179(20), 6383-90.

Nick, G., Jussila, M., Hoste, B., Niemi, M., Kaijalainen, S., De Lajudie, P., Gillis, M., De Bruijn, F.J. & Lindstrom, K. (1999). Rhizobia isolated from root nodules of tropical leguminous trees characterized using DNA-DNA dot-blot hybridisation and rep-PCR genomic fingerprinting. System Appl Microbiol 22, 287-299.

Nour, M. (1998). 16S-23S and 23S-5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis. Res Microbiol 149, 433-448.

260 BIBLIOGRAPHY

O’Donnell, A.G., Goodfellow, M. & Hawksworth, D.L. (1994). Theoretical and practical aspects of the quantification of biodoiversity among microorganisms. Philosophical Transactions of the Royal Society of London Series B, 345, 65-73.

Ochi K. (1989). Heterogeneity of ribosomal proteins among Streptomyces species and its application to identification. J Gen Microbiol 135(10), 2635-42.

Ochi, K. & Hiranuma, H. (1994). A taxonomic review of the genera Kitasatosporia and Streptoverticillium by analysis of ribosomal protein AT-L30. Int J Syst Bacteriol 44(2), 285- 292.

Ochi, K. (1992a). Electrophoretic heterogeneity of ribosomal protein AT-L30 among actinomycete genera. Int J Syst Bacteriol 42(1), 144-50.

Ochi, K. (1992b). Polyacrylamide gel electroforese analysis of ribosomal protein: a new approach for actinomycete taxonomy. Gene 115, 261-265.

Ochi, K. (1995). A taxonomic study of the genus Streptomyces by analysis of ribosomal protein AT-L30. Int J Syst Bacteriol 45(3), 507-14.

Okami, Y., Tazaki, T., Katumata, S., Honda, K., Suzuki, M. & Umezawa, H. (1959). Studies on Streptomyces kanamyceticus, producer of kanamycin. J Antibiot (Tokyo), 12, 252-6.

Okeke, B.C. & Frankenberger, W.T. Jr. (2003). Biodegradation of methyl tertiary butyl ether (MTBE) by a bacterial enrichment consortia and its monoculture isolates. Microbiol Res 158(2), 99-106.

Omura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M., Takahashi, Y., Horikawa, H., Nakazawa, H., Osonoe, T., Kikuchi, H., Shiba, T., Sakaki, Y. & Hattori, M. (2001). Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.Proc Natl Acad Sci U S A. 9, 98(21), 12215-20. Epub 2001 Sep 25.

Omura, S., Takahashi, Y. & Iwai, Y. (1989). Genus Kitasatosporia Omura, Takahashi, Iwai and Tanaka 1983a, 672VP, p. 2594-2598. In: S.T. Williams, M.E. Sharpe and J.G. Holt (ed), Bergey’s Manual of systematic bacteriology, vol.4, Williams & Wilkins, Baltimore.

261 Omura, S., Takahashi, Y., Iwai, Y. & Tanaka, H. (1982). Kitasatosporia, a new genus of the order Actinomycetales. J Antibiot 35, 1013-1019.

Park, D.H., Kim, J.S., Kwon, S.W., Wilson, C., Yu, Y.M., Hur, J.H. & Lim, C.K. (2003). Streptomyces luridiscabiei sp. nov., Streptomyces puniscabiei sp. nov. and Streptomyces niveiscabiei sp. nov., which cause potato common scab disease in Korea. Int J Syst Evol Microbiol 53, 2049-2054.

Pearson, W.R. & Lipman, D.J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci USA, 85, 2444-2448.

Peltola, J., Andersson, M.A., Haahtela, T., Mussalo-Rauhamaa, H., Rainey, F.A., Kroppenstedt, R.M., Samson, R.A. & Salkinoja-Salonen, M.S. (2001). Toxic-metabolite- producing bacteria and fungus in an indoor environment. Appl Environ Microbiol 67(7), 3269- 74.

Pernodet, J.L., Boccard, F., Alegre, M.T., Gagnat, J. & Guerineau, M. (1989). Organisation and nucleotide sequence analysis of a ribsomal RNA gene cluster from Streptomyces ambofaciens. Gene 79, 33-46.

Person, L.H. & Martin, W.J. (1940). Soil rot of sweet potatoes in Lousiana. Phytopathology, 30, 913-926.

Petrosyan, P., García-Varela, M. Luz-Madrigal, A., Huitrón, C & Flores, M.E. (2003). Streptomyces mexicanus sp. nov., a xylanolytic micro-organism isolated from soil. Int J Syst Evol Microbiol 53, 269 - 273.

Pitcher, D.D., Saunders, N.A. & Owen, R.J. (1989). Rapid extraction of bacterial genomic DNA with guanidinium thiocyanate. Lett Appl Microbiol 8, 151-156.

Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Modern Microbial Methods. Chemical Methods in Prokaryotic Systematics, pp. 493-521. Edited by M. Goodfellow & A.G. O’Donnell. Chichester: Wiley.

Preud’homme, J., Belloc, A, Carpentié, Y., Tarridec, P. (1965). Un antibiotique formé de deux groupes de composants à synergie d’action : la pristinamycine. C.R. Acad Sci Paris 260, 1309-1312.

262 BIBLIOGRAPHY

Pridham, T.G. & Tresner, H.D. (1974a). Family VII. Streptomycetaceae Waksman & Henrici 1943, 339AL In Bergey’s Manual of Determinative Bacteriology, 8th edition, pp. 747-748. E. Buchanan & N.E. Gibbons. Baltimore: Williams 7 Wilkins Co.

Pridham, T.G. & Tresner, H.D. (1974b). Genus I. Streptomyces Waksman & Henrici 1943, 339AL In Bergey’s Manual of Determinative Bacteriology, 8th edition, pp.748-829. E. Buchanan & N.E. Gibbons. Baltimore: Williams 7 Wilkins Co.

Pridham, T.G., Hesseltine, C.W. & Bennedict, R.G. (1958). A guide for the classification of Streptomyces according to selected groups. Applied Microbiol 6, 52-79.

Projan, J. (2003). Why is big Pharma getting out of antibacterial drug discovery? Current opinion in Microbiology 6, 427-430.

Quintana, E.T., Hamid, M.E., Fahal, A.H. & Goodfellow, M. (2003). Are the causal agents of actinomycetoma underspeciated? 13th International Symposium on the Biology of actinomycetes, Melbourne, Australia.

Rademaker J.L.W. & de Bruijn, F.J. (1997). Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis. In: G. Caetano- Anolles & P.M. Gresshoff (Eds.), DNA markers: Protocols, Applications and Overviews. Pp. 151-171. J. Wiley & Sons, Inc. new York, N.Y.

Rademaker J.L.W., Hoste, B., Louws, F.J., Kersters, K., Swings, J., Vauterin, L., Vauterin, P. & de Bruijn, F.J. (2000). Comparison of AFLP and rep-PCR genomic fingerprinting with DNA-DNA homology studies: Xanthomonas as a model system. Int J Syst Evol Microbiol 50, 665-677.

Rademaker, J.L., Louws, F.J., De Bruijn, F.J. (1998). Characterization of the diversity of ecological important microbes by rep-PCR genomic fingerprinting. In : Akkermans A.D.L., van Elsas, J.D. and de Bruijn, F.J. Molecular Ecology Manual, Kluwer Academic Publishers, Dordrecht, Suppl. 3, Chapter 3.4.3, pp. 1-26.

Radwan, S.S., Sorkhoh, N.A., Fardoun, F., al-Hasan, R.H. (1995). Soil management enhancing hydrocarbon biodegradation in the polluted Kuwaiti desert. Appl Microbiol Biotechnol 44, 265-70.

263 Raes, J., Y. Van de Peer (1999). ForCon: a software tool for the conversion of sequence alignments, http://www.ebi.ac.uk/embnet.news/vol6_1/ForCon/body_forcon.html.

Raykovska, V., Dolashka-Angelova, P., Paskaleva, D., Stoeva, S, Abashev, J., Kirkov, L.& Voelter, W. (2001). Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86. Biochem Cell Biol 79(2), 195-205.

Rezanka, T., Vancurova, I., Kristufek, V., Koza, T., Caslavska, J., Prikrylova, V., Blumauerova, M.: Taxonomic studies of Streptomyces virginiae mutants overproducing virginiamycin M1. Folia Microbiol 37, 105-110 (1992).

Ridell, M., Wallerstrom, S. & Williams, S.T. (1986). Immunodiffusion analysis of phonetically defined strains of Streptomyces, Streptoverticillium and Nocardiopsis. Syst Appl Microbiol 8, 24-27.

Rintala H, Hyvarinen A, Paulin L, Nevalainen A. (2004). Detection of streptomycetes in house dust--comparison of culture and PCR methods. Indoor Air 14(2), 112-9.

Rintala, H., Nevalainen, A. & Suutari, M. (2002). Diversity of streptomycetes in water- damaged building materials based on 16S rDNA sequences. Lett Appl Microbiol 34(6),439- 43.

Roane, T.M. & Kellogg, S.T. (1996). Characterisation of microbial communities in heavy metal contaminated soils. Can J Microbiol 42, 593-603.

Rondon, M.R., Goodman, R.M. & Handelsman, J. (1999). The Earth’s bounty: assessing and accessing soil microbial diversity. Trend Biotechnol 17, 403-409.

Rosselló-Mora, R. & Amann, R. (2001). The species concept for Prokaryotes. FEMS Microbiology Reviews 25, 39-67.

Ryckeboer J, Mergaert J, Vaes K, Klammer S, DeClercq, D., Coosemans, J., Insam, H. & Swings, J. (2003). A survey of bacteria and fungi occuring during composting and self- heating processes. Annals of Microbiology 53, 349-410.

Saddler, G. S., O’Donnel, A. G., Goodfellow, M. & Minnikin, D. E. (1987). SIMCA pattern recognition in the analysis of streptomycete fatty acids. J Gen Microbiol 133, 1137-1147.

264 BIBLIOGRAPHY

Sadowsky, M.J., Kinkel, L.L., Bowers, J.H., Schottel, J.L. (1996). Use of repetitive intergenic DNA sequences to classify pathogenic and disease-suppressive Streptomyces strains. Appl Environ Microbiol 62(9), 3489-93.

Saintpierre, D. (2001). Identification de souches originales d’actinomycetes isolées de sols ultramafiques de Nouvelle-Calédonie. Caractérisation chimique de quelques antibiotiques produits. PhD thesis. Institut National Polytechnique de Toulouse, France.

Saintpierre, D., Amir, H., Pineau, R., Sembiring, L. & Goodfellow, M. (2003). Streptomyces yatensis sp. nov., a novel bioactive streptomycete isolated from a New- Caledonian ultramafic soil. Antonie Leeuwenhoek 83, 21-26.

Saito, A. & Schrempf, H. (2004). Mutational analysis of the binding affinity and transport activity for N-acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader Streptomyces olivaceoviridis. Mol Genet Genomics 271(5), 545-53. Epub 2004 May 18.

Saitou, N. & Nei, M. (1987). The neighbor joining method: a new method for constructing phylogenetic tree. Mol Biol Evol 4, 406-425.

Salanoubat, M., Genin, S., Artiguenave, F., Gouzy, J., Mangenot, S., Arlat, M., Billault, A., Brottier, P., Camus, J.C., Cattolico, L., Chandler, M., Choisne, N., Claudel-renard, C., Cunnac, S., Demange, N., Gaspin, C., Lavie, M., Moisan, A., Robert, C., Saurin, W., Schiex, T., Siguier, P., Thebault, P., Whalen, M., Wincker, P., Levy, M., Weissenbach, J. & Boucher, C.A. (2002). Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415, 497-502.

Saleh, E.A., Mahmoud, S.A., el-Haddad, M.E. & Abdel-Fatah, M.K. (1985). Purification and identification of Streptomyces aureofaciens ID13 antibiotic. Zentralblatt für Mikrobiologie 140, 325-332.

Sandaa, R.A., Enger, O. & Torsvik, V. (1999a). Abundance and diversity of Archaea in heavy metal contaminated soils. Appl Environ Microbiol 65, 3293-3297.

Sanglier, J.J., Haag, H., Huck, T.A. & Fehr, T. (1996). Review of actinomycetes compounds 1990-1995. Expert Opinion on Investigational Drugs 5, 207-223.

265 Sanglier, J.J., Whitehead, D., Saddler, G.S., Ferguson, E.V. & Goodfellow, M. (1992). Pyrolysis mass spectrometry as a method for the classification, identification and selection of actinomycetes. Gene 115, 235-242.

Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. Technical Note 101. Newark, DE: Microbial ID.

Sawabe, T., Thompson, F.L., Heyrman, J., Cnockaert, M., Hayashi, K., Tanaka, R., Yoshimizu, M., Hoste, B., Swings, J. & Ezura, Y. (2002). Fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting reveal host- specific genetic diversity of Vibrio halioticoli-like strains isolated from the gut of Japanese abalone. Appl Environ Microbiol 68, 4140-4144.

Schleifer, K.H. & Ludwig, W. (1994). Molecular taxonomy: classification and identification, In: F.G; Priest, A; Ramos-Cormenzana & Tindall, B.J. (eds.) bacterial Diversity and Systematics; Planum Press, New York, 1-15.

Schlosser, A., Jantos, J., Hackmann, K. & Schremph, H. (1999). Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli. Appl Environ Microbiol 65(6), 2636-43.

Schmidt, T., Stoppel, R.D. & Schlegel, H.G. (1991). High-level resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KT02. Appl Environ Microbiol 57, 3301-3309.

Schrempf, H., Kessler, A., Bronneke, V. Dittrich, W. & Betzler, M. (1989). Genetic instability in streptomycetes. In: Microbiology ’89, pp. 133-40, Edited by C.L. Hershberger, St. W. Queener & G. Hegeman. Washington DC: American Society for Microbiology.

Schumann, P., Rainey, F.A., Wilting, R., Schaefer, T., Brambilla-Rosenberger, E.M., Steffen, M., Kaiser, V., Kroppenstedt, R.M. & Stackebrandt, E. (2001). Phenotypic versus molecular characterization addressing the problem of species delineation in the genus Streptomyces. 12th International Symposium on the Biology of actinomycetes, Vancouver, Australia.

Sebek, O.K., Whaley, H.A. (1971). Lipoxamycin and process for preparing same. U.S. Patent 3629402.

266 BIBLIOGRAPHY

Sembiring, L., Ward, A. C. & Goodfellow, M. (2000). Selective isolation and characterisation of members of the Streptomyces violaceusniger clade associated with the roots of Paraserianthes falcataria. Antonie Leeuwenhoek 78, 353-366.

Semedo, L.T.A.S., Gomes, R.C., Linhares, A.A., Duarte, G.F., Nascimento, R.P., Rosado, A.S., Margis-Pinheiro, M., Margis, R., Silva, K.R.A., Alviano, C.S., Manfio, G.P., Soares, R.M.A., Linhares, L.F. & Coelho, R.R.R. (2004). Streptomyces drozdowiczii sp. nov., a novel cellulolytic streptomycete from soil in Brazil. Int J Syst Evol Microbiol 54, 1323- 1328.

Serizawa, N. (1996). Biochemical and molecular approaches for production of pravastatin potent cholesterol-lowering drug. Biotechnol.Annu Rev 2, 373-89.

Shirling, E. B. & Gottlieb, D. (1968a). Cooperative description of type cultures of Streptomyces. II. Additional species description from first and second studies. Int J Syst Bacteriol 18, 69-189.

Shirling, E. B. & Gottlieb, D. (1968b). Cooperative description of type cultures of Streptomyces. III. Additional species description from first and second studies. Int J Syst Bacteriol 18, 279-392.

Shirling, E. B. & Gottlieb, D. (1969). Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int J Syst Bacteriol 19, 391-512.

Shirling, E.B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313-340.

Shirling, E.B. & Gottlieb, D. (1972). Cooperative description of type cultures of Streptomyces V. Additional descriptions. Int J Syst Bacteriol 22, 265-394.

Shirling, E.B. & Gottlieb, D. (1976). Retrospective evaluation of International Streptomyces Project taxonomic criteria, p. 9-41. In: Arai (ed), Actinomycetes - the boundary microorganisms. Toppan Co. Ltd., Tokyo.

267 Skerman, V.B.D., McGowan, V. & Sneath, P.H.A. (1980). Approved lists (AL) of Bacterial names. Int J Syst Bacteriol 30, 225-420.

Smejkal, C.W., Vallaeys, T., Seymour, F.A., Burton, S.K. & Lappin-Scott, H.M. (2001). Characterisation of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid-degrading Alcaligenes sp. CS1 and Ralstonia sp. CS2 isolated from agricultural soils. Environ Microbiol 3(4), 288-93.

Sneath, P.H.A. (1970). Application of numerical taxonomy to Actinomycetales: problems and prospects. In The Actinomycetales, pp. 371-377, Edited by H. Prauser. Jena: Gustav Fischer Verlag.

Sneath, P.H.A. (ed.).(1992). The international code of nomenclature of bacteria 1990 revision, ASM, Washington, USA.

Sommer, P., Bormann, C. & Gotz, F. (1997). Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus. Appl Environ Microbiol 63(9), 3553-60.

Song J, Lee SC, Kang JW, Baek HJ, Suh JW. (2004). Phylogenetic analysis of Streptomyces spp. isolated from potato scab lesions in Korea on the basis of 16S rRNA gene and 16S-23S rDNA internally transcribed spacer sequences. Int J Syst Evol Microbiol 54, 203-9.

Stackebrandt, E. & Goebel, B.M. (1994). Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846-849.

Stackebrandt, E., Frederiksen, W., Garrity, G., Grimont, P.A.D., Kämpfer, P., Maiden, M.C.J., Nesme, X., Rosselló-Mora, R., Swings, J., Trűper, H.g., Vauterin, L., Ward, A.C. & Whitman, W.B. (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043-1047.

Stackebrandt, E., Liesack, W. & Witt, D. (1992). Ribosomal RNA and rDNA sequence analyses. Gene 15, 115(1-2), 255-60.

268 BIBLIOGRAPHY

Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classic nov. Int J Syst Bacteriol, 479-491.

Stackebrandt, E., Witt, D., Kemmerling, C., Kroppenstedt, R. & Liesack, W. (1991). Designation of streptomycete 16S and 23S rRNA-base target region for oligonucleotide probes. Appl Environ Microbiol 57, 1468-1477.

Steingrube, V.A., Wilson, R.W., Brown, B.A., Jost, K.C., Blacklock, J.Z., Gibson, J.L. & Wallace, R.J. (1997). Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis. J Microbiol 35 (4), 817-822.

Stoppel, R.D. & Schlegel, H.G. (1995). Nickel-resistant bacteria from anthropogenically nickel-polluted and naturally nickel-percolated ecosystems. Appl Environ Microbiol 61, 2276- 2285.

Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J., Brinkman, F.S., Hufnagle, W.O., Kowalik, D.J., Lagrou, M., Garber, R.L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L., Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier, M.H., Hancock, R.E., Lory, S. & Olsen, M.V. (2000). Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959- 964.

Sukesan, S. & Watwood, M.E. (1998). Effects of hydrocarbon enrichment on trichloroethylene biodegradation and microbial populations in finished compost. J Appl Microbiol 85(4), 635-42.

Suzuki, Y., Nagata, A., Ono, Y. & Yamada, T. (1988). Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J Bacteriol 170, 2886- 2889.

Swofford, D.L. & Olsen, G.J. (1990). Phylogeny reconstruction. In Molecular systematics, pp. 411-501. Edited by D.M. Hillis and C. Moritz. Sunderland: Sinauer Associates.

269 Taber, W.A. (1959). Identification of an alkaline-dependent Streptomyces as Streptomyces caeruleus Baldacci and characterization of the species under controlled conditions. Can J of Microbiol 5, 335-344.

Taber, W.A. (1960). Evidence for the existence of acid-sensitive actinomycetes in soil. Can J of Microbiol 6, 503-514.

Takeuchi, T., Sawada, H., Tanaka, F. & Matsuda, I. (1996). Phylogenetic analysis of Streptomyces spp. causing potato scab based on 16S rDNA sequences. Int J Syst Bacteriol 46, 476-479.

Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed- phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125-128.

Tamura, T., Nagy, I., Lupas, A., Lottspeich, F., Cejka, Z., Schoofs, G., Tanaka, K., De Mot, R. & Baumeister, W. (1995). The first characterisation of a eubacterial proteasome: the 20S complex of Rhodococcus. Current Biology 5, 766-774.

Thakur, M.S., Prapulla, S.G., Jaleel, S.A., Prasad, M.S., Ghildya, l. N.P., Lonsane, B.K. (1988). Cultural stability of Streptomyces fradiae in the production of xylose isomerase: studies in shake flasks. Folia Microbiol (Praha) 33(1), 21-8.

Thaxter, R. (1892). Potato scab. Conn Agric Exp Stn Rep 1891, 153-160.

Thirumalachar, M.J. (1955). Chainia, a new genus of the Actinomycetales. Nature 176, 934- 935.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 25, 4876-4882.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 25, 4876-4882.

Thykaer, J. & Nielsen, J. (2003). Metabolic engineering of β-lactam production. Metabolic engineering 5, 56-69. 270 BIBLIOGRAPHY

Totevova, S., Prouza, M., Burkhard, J., Demnerova, K. & Brenner, V. (2004). Characterisation of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia. Folia Microbiol (Praha) 17(3), 247-54.

Trejo, W.H. (1970). An evaluation of some concepts and criteria used in the speciation of streptomycetes. Transactions of New York Academy of Science Series 2, 32, 989-997.

Tresner, H.D. & Backus, E.J. (1963). System of color wheels for streptomycete taxonomy. Appl Microbiol 11, 335-338.

Trujillo, M.E. & Goodfellow, M. (2003). Numerical phenetic classification of clinically significant aerobic sporoactinomycetes and related organisms. Antonie van Leeuwenhoek 84(1), 39-68.

Truper, H.G. & Schleifer, K.H. (1991). Principles of characterization and identification of prokaryotes. In: Balows, A., Truper, H.G., Dworkin, M., Harder, W. and Schleifer, K.H. (eds). The prokaryotes. Second edition, 126-148, Springer Verlag, New York.

Uchida, K. & Seino, A. (1997). Intra-and intergeneric relationships of various actinomycete strains based on the acyl types of the muramyl residue in cell wall peptidoglycans examined in a glycolate test. Int J Syst Bacteriol 47, 182-190.

Ueda, K., Seki, T., Kudo, T., Yoshida, T. & Kataoka, M. (1999). Two distinct mechanisms cause heterogeneity of 16S rRNA. J Bacteriol 181, 78-82.

Urwin, R. & Maiden, M.C. (2003). Multi-locus sequence typing: a tool for global epidemiology.Trends Microbiol 11(10), 479-87.

Uyeda, M., Mizukami, M., Yokomizo, K. & Suzuki, K. (2001). Pentalenolactone. I and hygromycin A, immunosuppressants produced by Streptomyces filipinensis and Streptomyces hygroscopicus. Bioscience, Biotechnology and Biochemistry 65, 1252-1254.

Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: A software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Computer Application for Biological Sciences 10, 569-570.

271 Vancanneyt, M., Torck, U., Dewettinck, D., Vaerewijck, M. & Kersters, K. (1996). Grouping of pseudomonads by SDS-PAGE of whole-cell proteins. Syst Appl Microbiol 19, 556-568.

Vandamme, E. (1984). Antibiotic search and production: an overview. In: Biotechnology of Industrial Antibiotics. E.J. Vandamme (ed.). Marcel Dekker Inc.

Vandamme, P., Pot, B., Gillis, M, De Vos, P., Kersters, K. & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60, 407-438.

Vaneechoutte, M. (1996). DNA fingerprinting techniques for microorganisms. A proposal for classification and nomenclature. Molecular Biotechnology 6, 115-142.

Versalovic, J., Koeuth, T. & Lupski, J.R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 25, 19(24), 6823-31.

Versalovic, J., Schneider, M., De Bruijn, F.J. & Lupski, J.R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in molecular and cellular biology 5, 25-40.

Vertesy, L., Beck, B., Bronstrup, M., Ehrlich, K., Kurz, M., Muller, G., Schummer, D.& Seibert, G. (2002).Cyclipostins, novel hormone-sensitive lipase inhibitors from Streptomyces sp. DSM 13381. II. Isolation, structure elucidation and biological properties. J Antibiot (Tokyo). 55(5), 480-94.

Vigal, T., Martin, J.F. & Gil, J.A. (1994). Expression of the Streptomyces griseus alpha- amylase gene in Escherichia coli. FEMS Microbiol Lett. 15,118(3), 259-63.

Virolle, M.J. & Bibb, M.J. (1988). Cloning, characterization and regulation of an alpha- amylase gene from Streptomyces limosus. Mol Microbiol. 2(2), 197-208.

Virolle, M.J., Long, C.M., Chang, S. & Bibb, M.J. (1988). Cloning, characterisation and regulation of an alpha-amylase gene from Streptomyces venezuelae.Gene 30, 74(2), 321-34.

272 BIBLIOGRAPHY

Volff, J.N. & Altenbuchner, J. (2000). A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution. FEMS Microbiol Lett 15,186(2), 143-50.

Volff, J.-N. & Altenbuchner, J. (1998). Genetic instability of the Streptomyces chromosome. Molecular Microbiology 27, 239-246.

Vujaklija, D., Schroder, W., Abramic, M., Zou, P., Lescic, I., Franke, P. & Pigac, J. (2002). A novel streptomycete lipase: cloning, sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene. Arch Microbiol 178(2),124-30. Epub 2002 May 16.

Vukelic, B., Ritonja, A., Renko, M., Pokorny, M. & Vitale, L. (1992). Extracellular alpha- amylase from Streptomyces rimosus. Appl Microbiol Biotechnol 37(2), 202-4.

Waksman, S. & Henrici, A. (1943). The nomenclature and classification of the actinomycetes. J Bacteriol 46, 337-341.

Waksman, S.A. & Curtis, R.E. (1916). The Actinomyces of the soil. Soil Science 1, 99-134.

Waksman, S.A. & Henrici, A.T. (1943). The nomenclature and classification of the actinomycetes. J Bacteriol 46, 337-341.

Waksman, S.A. & Henrici, H.T. (1948). Family Actinomycetaceae Buchanan and Family Streptomycetaceae Waksman & Henrici. In Bergey’s Manual of Determinative Bacteriology. 6th edition. Edited by R.S. Breed, E.G.D. Murray & A.P. Hitchens. Baltimore: Williams & Wilkins.

Waksman, S.A. & Woodruff, H.B. (1941). Actinomyces antibioticus, a new soil organism antagonistic to pathogenic and non-pathogenic bacteria. J Bacteriol 42, 231-249.

Waksman, S.A. (1919). Cultural studies of species Actinomyces. Soil Science 8, 71-215.

Waksman, S.A. (1957). Family III. Streptomycetaceae Waksman & Henrici. In Bergey’s Manual of Determinative Bacteriology 7th edition, pp. 744-825. Edited by R.S. Breed, E.G.D. Murray & N.R. Smith: Williams & Wilkins.

273 Waksman, S.A. (1961). The actinomycetes. Classification, Identification and Descriptions of Genera and Species, volume 2. Baltimore: Williams & Wilkins.

Wannet, W.J., Spalburg, E., Heck, M.E., Pluister, G.N., Willems, R.J., De Neeling, A.J. (2004). Widespread dissemination in The Netherlands of the epidemic berlin methicillin- resistant Staphylococcus aureus clone with low-level resistance to oxacillin. J Clin Microbiol 42(7), 3077-82.

Warwick, S.T., Bowen, T., McVeigh, H. & Embley, T.M. (1994). A phylogenetic analysis of the family Pseudonocardiaceae and the genrea Actinokineospora and the genera Actinokineospora and Saccharothrix with 16S rRNA sequences and proposal to combine the genera Amycolata and Pseudonocardia in an emended genus Pseudonocardia. Int J Syst Bacteriol 44, 293-299.

Watanabe, I., Nara, F. & Serizawa, N. (1995). Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from S. carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene 163, 81-85.

Watve, M.G., Tickoo, R., Jog, M.M. & Bhole, B.D. (2001). How many antibiotics are produced by the genus Streptomyces? Archives of Microbiology 176, 386-390.

Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D., Kandler, P., Krichevsky, M.I., Moore, L.H., Murray, R.G.E, Stackebrandt, E., Starr, M.P. & Truper, H.G. (1987). Report on the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bact 37, 463-464.

Weber, T., Welzel, K., Pelzer, S., Vente, A. & Wohlleben, W. (2003). Exploiting the genetic potential of polyketide producing streptomycetes. J of Biotechnol 106, 221-232.

Weil, J., Miramonti, J. & Ladisch, M.R. (1995). Cephalosporin C : mode of action and biosynthetic pathway. Enzyme Microb Tech 17, 85-87.

Weirich, G. (1989). Untersuchungen über Mikroorganismen von Wandmalereien. Mat Org 24, 139-159.

Wellington, E. M. H., Stackebrandt, E., Sanders, D., Wolstrup, J. & Jorgensen, N. O. G. (1992). Taxonomic status of Kitasatosporia, and proposed unification with Streptomyces on

274 BIBLIOGRAPHY

the basis of phenotypic and 16S analysis and emendation of Streptomyces Waksman and Henrici 1943. Int J Syst Bacteriol 42(1),156-160.

Wenner, T., Roth, V., Decaris, B. & Leblond, P. (2002). Intragenomic and intraspecific polymorphism of the 16S-23S rDNA internally transcribed sequences of Streptomyces ambofaciens. Microbiology 148, 633-642.

Werner, G., Hagenmaier, H., Drautz, H., Baumgartner, A & Zähner, H. (1984). Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity, J Antibiotics 37, 110-117.

Whittam, T.S. & Bumbaugh, A.C. (2002). Inferences from whole-genome sequences of bacterial pathogens. Current Opinion in Genetics & Development 12, 719-725.

Williams, S. T., Goodfellow, M. & Alderson, G. (1989). Genus Streptomyces Waksman and Henrici, 1943, 339AL. In Bergey’s Manual of Systematic Bacteriology, Volume 4, pp. 2452-2492. Edited by S. T. Williams, M. E. Sharpe and J. G. Holt. Baltimore, Williams and Wilkins.

Williams, S. T., Goodfellow, M., Alderson G., Wellington, E. M. H., Sneath, P. H. A. & Sackin, M. J. (1983a). Numerical classification of Streptomyces and related genera. J Gen Microbiol 129, 1743-1813.

Williams, S. T., Goodfellow, M., Wellington, E. M. H., Vickers J. C., Alderson, G., Sneath, P. H. A., Sackin, M. J. & Mortimer, A. M. (1983b). A probability matrix for identification of some streptomycetes. J Gen Microbiol 129, 1815-1830.

Williams, S.T., Goodfellow, M. & Alderson, G. (1989). Streptomyces and related genera. Bergey’s Manual of Systematic Bacteriology (volume IV), Stanley T. Williams, M. Elisabeth Sharpe, John G. Holt, Williams & Wilkins, Section 29.

Williams, S.T., Vickers, J.C. & Goodfellow, M. (1985). Application of new theoretical concepts to the identification of streptomycetes. In: Computer-assisted bacterial systematics. M. Goodfellow, D. Jones and F.G. Priest (Eds), Academic Press, London.

275 Wilson, C.R., Eyles, A., Wilson, A.J., Luckman, G.A. & Tegg, R.S. (2003). In vitro development of common scab resistance in processing potatoes. 13th International Symposium on the Biology of actinomycetes, Melbourne, Australia.

Winker, S. & Woese, C.R. (1991). A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics. Syst Appl Microbiol 14, 305-310.

Wirth, S. & Ulrich, A. (2002). Cellulose-degrading potentials and phylogenetic classification of carboxymethyl-cellulose decomposing bacteria isolated from soil. Syst Appl Microbiol 25, 584-591.

Witt, D. & Stackebrandt, E. (1990). Unification of the genera Streptoverticillium and Streptomyces, and emendation of Streptomyces Waksman and Henrici 1943, 339AL. Syst Appl Microbiol 13, 361-371.

Woese, C.R. (1987). Bacterial evolution, Microbiological reviews 51, 221-271.

Woese, C.R., Kandler, O. & Wheelis, M.L. (1990). Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A 87(12), 4576-9.

Woodruff, H.B. (1999). Natural products from microorganisms, an odyssey revisited. Actinomycetologica 13, 58-67.

Wu, C., Lu, X., Qin., M., Wang, Y. & Ruan, J. (1989). Analysis of menaquinone compound in microbial cells by HPLC. Microbiology (English translation of Mikrobiologiya) 16, 176-178.

Wuertz, S. & Mergeay, M. (1997). The impact of heavy metals on soil microbial communities and their activities. In modern Soil Microbiology. Vanelsas, J.D., Trevors, J.T., and Wellington, E.M.H. (Eds.) New York: Marcel Dekker, pp. 607-639.

Xu, L.-H., Tiang, Y.-Q., Zhang, Y.-F., Zhao, L.-X. & Jiang, C.-L. (1998). Streptomyces thermogriseus, a new species of the genus Streptomyces from soil, lake and hot-spring. Int J Syst Bacteriol 48, 1089-1093.

276 BIBLIOGRAPHY

Yao, T. (2002). Bioinformatics for the genomic sciences and towards systems biology. Japanese activities in the post-genome era. Progress in Biophysics & Molecular Biology 80, 23-42.

Yassin, A.F., Galinski, E.A., Wohlfarth, A., Jahnke, K.-D., Schaal, K.P. & Trüper, H.G. (1993). A new actinomycete species, Nocardiopsis lucentensis sp. nov. Int J Syst Bacteriol 43, 266-271.

Yassin, A.F., Rainey, F.A., Burghardt, J., Gierth, D., Ungerechts, J., Lux, I., Seifert, P., Bal, C. & Schaal, K.P. (1997). Description of Nocardiopsis synnemataformans sp. nov., elevation of Nocardiopsis alba subsp. prasina to Nocardiopsis prasina comb. nov., and designation of Nocardiopsis antarctica and Nocardiopsis alborubida as later subjective synonyms of Nocardiopsis dassonvillei. Int J Syst Bacteriol 47, 983-988.

Yoon H.J., Lee S.T., Park Y.H. (1998). Genetic analyses of the genus Nocardioides and related taxa based on 16S-23S rDNA internally transcribed spacer sequences. Int J Syst Bacteriol 48 (3), 641-50.

Zhang Z., Wang Y. & Ruan J. (1997). A proposal to revive the genus Kitasatospora (Omura, Takahashi, Iwai, and Tanaka 1982). Int J Syst Bacteriol 47(4), 1048-1054.

Zhang, Q., Li, W-J., Cui, X.-L., Li, M.-G., Xu, L.-H. & Jiang, C.-L. (2003). Streptomyces yunnanensis sp. nov., a mesophile from soils in Yunnan, China. Int J Syst Evol Microbiol 53, 217 - 221.

Zhang, Z., Kudo, T., Nakajima, Y. & Wang, Y. (2001). Clarification of the relationship between the members of the family Thermomonosporaceae on the basis of 16S rDNA, 16S- 23S rRNA internal transcribed spacer and 23S rDNA sequences and chemotaxonomic analyses.Int J Syst Evol Microbiol 51, 373-383.

Zhang, Z., Wang, Y. & Ruan, J. (1997). A proposal to revive the genus Kitasatospora (Omura, Takahashi, Iwai, and Tanaka 1982). Int J Syst Bacteriol 47, 1048-1054.

Zhou, Z.H., Liu, Z.H., Qian, Y.D., Kim, S.B. & Goodfellow. M. (1998). Saccharopolyspora spinosporotrichia sp. nov., a novel actinomycete from soil. Int J Syst Bacteriol 48, 53-58.

277 Zouboulis A.I., Matis K.A., Rousou E.G. & Kyriakldis D.A. (2001). Biosorptive flotation for metal ions recovery. Water Sci Technol 43(8), 123-9.

278

ANNEXES

279

280 ANNEX I.

List of standard media used for cultivation and description of Streptomyces spp. (Shirling & Gottlieb, 1966)

Bennett’s medium ISP 2 medium (Yeast-Malt extract) yeast extract 1.0 g yeast extract 4.0 g beef extract 1.0 g 0.8 g* malt extract 1.0 g casein hydrolysate 2.0 g dextrose 4.0 g glucose / glycerol * 10.0 g 10.0 g* agar 20.0 g agar 20.0 g 12.0 g* final pH 7.3 / 7.0*

*: modified Bennett’s medium (Jones, 1949) final pH 7.2

ISP 3 (Oatmeal agar) 20 g Oatmeal (Quaker White Oats) 1.8 % agar

Adjust pH to 7.2. Cook or steam 20.0 g in 1000.0 ml distilled water for 20 min. Filter through cheese cloth. Add 18.0 g agar and make up to 1000.0 ml. Add 1 ml of trace salts solution.

Trace salt solution FeSO4 x 7 H2O 0.1 g MnCl2 x 4 H2O 0.1 g ZnSO4 x 7 H2O 0.1 g Distilled water 100.0 ml

ISP medium 4 (Inorganic salts-Starch) Solution I: Soluble starch, 10.0 g. Make a paste of the starch with a small amount of cold distilled water and bring to a volume of 500 ml.

Solution II: CaCO3 2.0 g K2HPO4 (anhydrous) 1.0 g MgSO4 x 7 H2O 1.0 g NaCl 1.0 g (NH4)2SO4 2.0 g Distilled water 500.0 ml Trace salt solution 1.0 ml pH between 7.0 and 7.4. Mix solutions I and II together. Add 20.0 g agar.

ISP medium 5 (Glycerol-Asparagine) L-asparagine (anhydrous basis) 1.0 g Glycerol 10.0 g K2HPO4 (anhydrous basis) 1.0 g Distilled water 1000.0 ml Trace salts solution 1.0 ml Agar 20.0 g

Final pH 7.0-7.4. ISP medium 6 (Peptone-yeast extract-iron) Peptone 36.0 g Yeast Extract 1.0 g Distilled water 1000.0 ml Agar 20.0 g

Final pH 7.0-7.2

ISP medium 7 (Tyrosine) Glycerol 15.0 g L-tyrosine 0.5 g L-asparagine 1.0 g K2HPO4 (anhydrous) 0.5 g MgSO4.7H2O 0.5 g NaCl 0.5 g FeSO4.7H2O 0.04 g Distilled water 1000.0 ml Trace salt solution 1.0 ml Agar 20.0 g

Final pH 7.2-7.4

Czapek Dox agar Sucrose 30.00 g NaNO3 3.00 g K2HPO4 1.00 g MgSO4 x 7 H2O 0.50 g KCl 0.50 g FeSO4 x 7 H2O 0.01 g Yeast extract 2.00 g Peptone 5.00 g Agar 15.00 g Distilled water 1000.00 ml

Adjust pH to 7.3

ANNEX II. List of reference strains used in this study

Screened in DNA-DNA Formerly Protein hybridisations (> 16S Species subspecies classified BOX-PCR ** ARDRA # 16S-ITS RFLP α AFLP β Number ε profiling * 70% related to sequencing $ in genus other strain) S. abikoensis Streptoverticillium 6 20386T + (j) cl 12 S. aburaviensis 19305T + + cl 3 + S. achromogenes achromogenes 20387T + cl 22 S. achromogenes rubradiris 20388T + + S. acidiscabies 19856T + + S. acrimycini 21798T + cl 5 S. aculeolatus 19906T + + S. afghaniensis 20390T + AJ399483 cl 1 S. alanosinicus 20391T + cl 12 S. albaduncus 20392T + cl 5 S. albidoflavus 21791T + + (e) (k) Z76676 cl 6 cl 8 S. albireticuli Streptoverticillium 6 20393T + (j) cl 13 S. albofaciens 20394T + AB045880 cl 10 S. alboflavus 21038T + + cl 43 S. albogriseolus 20395T + + S. albolongus 20396T + + S. alboniger 20397T + + S. albospinus 20398T + + S. albosporeus albosporeus 19403T cl 1 + (c ) AJ781327 cl 5 cl 24 S. albosporeus labilomyceticus 20400T + cl 23 S. alboverticillatus Streptoverticillium 6 20401T + (j) cl 17 S. albovinaceus 20402T cl 16 cl 23 S. alboviridis 20403T + cl 23 S. albulus 20404T + AB024440 + S. albus albus 20405T cl 20 (e) + cl 18 S. albus albus 19318 cl 2 - S. albus albus 19389 cl 11 - S. albus pathocidicus 20406T + cl 7 S. almquistii 21307T cl 20 cl 18 S. althioticus 20408T + + S. amakusaensis 19350T + + cl 14 + S. ambofaciens 20409T + M27245 + S. aminophilus 19319T cl 5 cl 17 ( c) cl 8 cl 36 S. anandii 8600T + cl 8 S. anthocyanicus 20411T cl 4 cl 22 S. antibioticus 20412T + cl 13 S. antibioticus 19334 cl 15 - S. antimycoticus 20413T + cl 16 S. anulatus 19301T + cl 18 (a) (e) cl 2 cl 23 S. arabicus 20414T cl 23 (b) cl 8 S. ardus Streptoverticillium 6 20415T + (j) cl 17 S. arenae 20416T + AJ399485 cl 1 S. argenteolus 5967T + AB045872 cl 23 S. armeniacus 20418T + + S. asterosporus 20419T cl 8 cl 40 S. atratus 20420T + cl 23 S. atroaurantiacus 20421T + cl 30 S. atrovirens 20422T + + S. atroolivaceus 19306T + + AJ781320 cl 2 cl 23 S. aurantiacus 19358T cl 1 + ( c) AJ781383 cl 5 cl 24 S. aurantiogriseus 19298T + + cl 14 + S. aureocirculatus 21794T + cl 22 S. aureofaciens 5968T cl 28 Y15504 cl 29 S. aureorectus 19908T cl 8 cl 40 S. aureoversilis Streptoverticillium 6 20425T + (j) cl 48 S. aureoverticillatus 20426T + + S. avellaneus 20427T cl 28 cl 29 S. avidinii 20428T + cl 22 K. azatica 20429T + U93312 cl 30 S. azureus 20430T + AJ399470 cl 1 S. baarnensis 20431T + cl 23 S. bacillaris 8585T + cl 50 S. badius 19353T + + cl 2 cl 23 S. bambergiensis 19299T + + cl 14 + S. bellus 19401T + + AJ399476 cl 17 cl 1 S. bikiniensis 19367T + + X79851 cl 1 cl 23 S. biverticillatus Streptoverticillium 6 20433T + (j) AJ781381 cl 13 S. blastmyceticus Streptoverticillium 6 20434T + (j) cl 17 S. bluensis 5969T + X79324 cl 7 S. bobili 20436T + cl 22 S. bottropensis 20437T + D63868 + S. brasiliensis Elytrosporangium 1 20438T + + S. bungoensis 20439T + cl 22 S. cacaoi asoensis 20440T + + S. cacaoi cacaoi 19320T cl 5 cl 17 ( c) cl 8 cl 36 S. caelestis 20441T + + S. caelicus' 19391 + - cl 14 - S. caeruleus 19399T cl 19 cl 14 ( c) cl 9 cl 47 S. californicus 19309T + + cl 2 cl 23 S. calvus 20442T cl 8 cl 40 S. canarius 20443T + cl 22 S. canus 19329T + + cl 9 + S. canescens 20445T cl 22 (k) Z76684 cl 8 S. caniferus 20446T + cl 23 S. capoamus 20447T - AB045877 + S. capillispiralis 19909T + + S. carpaticus 20448T + + S. carpinensis Elytrosporangium 1 19913T + + S. catenulae 20449T + AJ621613 cl 23 S. cavourensis cavourensis 20450T + cl 22 S. cavourensis washingtonensis 20451T + cl 23 S. cellostaticus 20452T + cl 55 S. celluloflavus 21796T + cl 42 S. cellulosae 19315T + + cl 17 cl 4 S. champavatii 20454T + cl 8 S. chartreusis 20455T + AJ399468 + S. chattanoogensis 19339T + + AJ621611 cl 12 cl 23 S. chibaensis 20456T cl 26 (a) cl 52 S. chrestomyceticus 20457T + AJ621609 cl 23 S. chromofuscus 19317T + + cl 10 cl 9 S. chryseus 20458T + cl 23 S. chrysomallus chrysomallus 20459T cl 18 (a) cl 23 S. chrysomallus fumigatus 21793T + + S. cinereorectus 20461T cl 30 (b) cl 28 S. cinereoruber cinereoruber 20462T + cl 23 S. cinereoruber fructofermentans 20463T + cl 20 S. cinereospinus 20464T + cl 22 S. cinereus Microellobosporia 4 21310T + + S. cinerochromogenes 20466T + cl 57 S. cinnabarinus 20467T + AJ399487 + S. cinnamonensis 20468T + cl 22 S. cinnamoneus albosporus Streptoverticillium 6 20469T + cl 17 S. cinnamoneus cinnamoneus Streptoverticillium 6 8602T + (j) cl 17 S. cinnamoneus lanosus Streptoverticillium 6 20471T + cl 17 S. cinnamoneus sparsus Streptoverticillium 6 20472T + cl 17 S. cirratus 20473T + cl 22 S. ciscaucasicus 20474T cl 5 AY508512 cl 19 S. citreofluorescens 20475T cl 18 (a) cl 23 S. clavifer 20476T cl 5 cl 19 S. clavuligerus 20477T + AB045869 + K. cochleata 20478T cl 30 (b) U93316 cl 28 S. coelescens 20479T + (k) cl 22 S. coelicoflavus 20480T + cl 23 S. coelicolor 8571T + (d) (k) Z76678 cl 8 S. coeruleofuscus 20482T + AJ399473 cl 1 S. coeruleoprunus 20483T + cl 33 S. coeruleorubidus 20484T + (g) cl 1 S. coerulescens 8590T + cl 1 S. collinus 20486T + + S. colombiensis 20487T cl 13 (b) (I) - S. corchorusii 20488T cl 26 (a) cl 52 S. cremeus 20489T + cl 22 S. crystallinus 20490T + cl 34 S. curacoi 20491T + cl 55 S. cuspidosporus 20492T + cl 56 S. cyaneofuscatus 20493T + cl 23 S. cyaneogriseus' 19381T + - cl 4 - S. cyaneus 20494T + (g) + S. cyanoalbus 19343T + + cl 14 + K. cystargineus 20495T + U93318 + S. daghestanicus 20496T cl 11 cl 37 S. diastaticus ardesiacus 20497T + + S. diastaticus diastaticus 19322T + + cl 9 S. diastatochromogenes 20498T + D63867 cl 25 S. distallicus Streptoverticillium 6 20499T cl 13 (b) (j) cl 13 S. djakartensis 21795T + cl 4 S. durhamensis 20501T + cl 22 S. echinatus 5972T + AJ399465 + S. ederensis 20504T + cl 19 S. ehimensis Streptoverticillium 6 20505T + (j) cl 12 S. endus 19393T cl 8 cl 29 (f) AJ391821 cl 8 cl 16 S. enissocaesilis 20506T cl 6 cl 58 S. erumpens 20507T + AJ621603 cl 23 S. erythraeus 20508T + + S. erythrogriseus 19406T + cl 25 AJ781328 cl 17 cl 1 S. eurocidicus Streptoverticillium 6 20509T + (j) cl 13 S. eurythermus 20510T + D63870 cl 8 S. exfoliatus 19307T + + cl 1 cl 23 S. felleus Streptoverticillium 6 20511T cl 22 (k) Z76681 cl 8 S. fervens melrosporus Streptoverticillium 6 19897T + (j) cl 38 S. filamentosus 20512T cl 24 (b) cl 23 S. filipinensis 19333T + + cl 9 cl 23 S. fimbriatus 20513T + + S. fimicarius 21044T + cl 23 S. finlayi 19373T + + cl 2 cl 23 S. flaveolus 19328T + + cl 17 cl 59 S. flaveus Microellobosporia 4 19323T + - - S. flavidofuscus 20515T + - S. flavidovirens 19387T + + + S. flaviscleroticus Chainia 3 19886T cl 3 (a) cl 46 S. flavofungini 21799T + + S. flavofuscus 19900T cl 19 cl 23 S. flavogriseus 19887T + cl 53 S. flavotricini 19880T + cl 22 S. flavovariabilis 19905T + AJ781334 + S. flavovirens 20516T cl 9 (a) cl 53 S. flavoviridis 19881T + cl 35 S. flocculus 19889T + cl 18 S. floridae 19899T + cl 23 S. fluorescens 8579T cl 18 (a) cl 23 S. fradiae 19371T + cl 27 (b) cl 9 cl 45 S. fragilis 19874T + + S. fulvorobeus 19901T + AJ781331 cl 23 S. fulvissimus 19310T + + (h) cl 14 cl 43 S. fumanus 19882T + AJ399463 cl 12 S. fumigatiscleroticus Chainia 3 19911T + + S. "fungicidicus" 19386 + + - S. "fungicidicus" 21792T + + S. galbus 19879T + X79852 cl 22 S. galilaeus 21790T + AB045878 cl 22 S. gancidicus 19898T + cl 4 S. gardneri 19876T + cl 44 S. gelaticus 19376T + + + S. geysiriensis 19893T + cl 39 S. ghanaensis 19894T + + S. gibsonii 19912T cl 20 AJ781753 cl 18 S. glaucosporus 19907T + cl 44 S. glaucescens 19330T + + cl 57 S. glaucus 19902T + AJ781332 + S. globisporus caucasicus 19895T + cl 8 S. globisporus globisporus 8578T + cl 23 S. globosus 19896T + AJ781330 cl 31 S. glomeratus 19903T + AJ781754 cl 9 S. gobitricini 19910T + AJ781335 cl 14 S. goshikiensis 19884T + cl 22 S. gougerotii 19888T + (k) Z76687 cl 9 S. graminearus 19904T cl 10 AJ781333 cl 4 S. graminofaciens 19892T + AJ781329 + S. griseinus 19875T + - S. griseoaurantiacus 21045T + + S. griseobrunneus 19877T + cl 50 S. griseocarneus Streptoverticillium 6 19383T + + (e) (j) X99943 cl 17 S. griseochromogenes 19891T + AJ310923 cl 22 S. griseoflavus 19344T + + AJ781322 cl 1 S. griseofuscus 19885T + cl 6 S. griseoincarnatus 19316T + cl 25 AJ781321 cl 17 cl 1 S. griseoloalbus 21308T + cl 22 S. griseoluteus 19356T + + cl 41 S. griseolosporeus 19962T - cl 22 S. griseolus 19878T + cl 53 S. griseomycini 19883T cl 10 cl 4 S. griseoplanus 19923T + cl 28 S. griseorubens 19931T + cl 1 S. griseoruber 19325T + + cl 15 + S. griseorubiginosus 19941T + AJ781339 cl 19 S. griseosporeus 19947T + + S. griseostramineus 19932T cl 10 cl 4 S. griseoverticillatus Streptoverticillium 6 19944T + (j) cl 17 S. griseoviridis 19321T + cl 11 (h) cl 37 S. griseus alpha 19953T cl 12 (a) cl 23 S. griseus cretosus 19946T cl 12 (a) cl 23 S. griseus griseus 19302T + + cl 2 cl 23 S. griseus solvifaciens 19952T + cl 8 S. hachijoensis Streptoverticillium 6 19928T + (j) cl 13 S. halstedii 19303 - cl 6 - S. hawaiiensis 5975T + AJ399466 cl 1 S. heliomycini 19960T + AJ781343 cl 5 S. helvaticus 19940T + cl 23 S. herbaricolor 19929T + cl 30 S. hiroshimensis Streptoverticillium 6 19924T + (j) cl 12 S. hirsutus 19927T + cl 49 S. humidus 19936T + cl 22 S. humiferus Actinopycnidium 2 20519T + (k) AF503491 cl 22 S. hydrogenans 19948T + - S. hygroscopicus "ascomyceticus" 19398 + - cl 8 - S. hygroscopicus angustmyceticus 19958T + cl 23 S. hygroscopicus decoyicus 19954T + cl 23 S. hygroscopicus glebosus 19950T + cl 23 S. hygroscopicus hygroscopicus 19335T cl 8 cl 29 (f) AJ391820 cl 8 cl 16 S. hygroscopicus ossamyceticus 19951T + cl 26 S. iakyrus 19942T + AJ399489 cl 1 S. indiaensis 19961T + AJ781344 cl 4 S. indigoferus 19930T + cl 30 S. intermedius 19304T + + Z76686 cl 14 cl 8 S. inusitatus 19955T + AJ781340 cl 13 S. ipomoeae 20520T + AY207593 cl 26 S. janthinus 8591T + AJ399478 cl 1 S. kanamyceticus 19351T + + cl 2 cl 23 S. kashmirensis Streptoverticillium 6 19937T + (j) AJ781337 cl 12 S. kasugaensis 19949T + cl 42 S. katrae 19945T + cl 22 K. kifunense 19957T + AB022874 cl 29 S. kishiwadensis Streptoverticillium 6 19939T + (j) AJ781338 cl 11 S. kunmingensis Chainia 3 20521T + cl 22 S. kurssanovii 19933T + cl 21 S. labedae 19956T cl 25 cl 1 S. lanatus 19380T + + AJ399469 cl 9 cl 22 S. lateritius 19372T + + AJ781326 cl 7 cl 22 S. laurentii 19959T + AJ781342 cl 22 S. lavendofoliae 19935T + AJ781336 cl 14 S. lavendulae grasserius 19938T - - S. lavendulae lavendulae 19925T - (e) (I) - S. lavenduligriseus Streptoverticillium 6 19943T + cl 59 S. lavendulocolor 19934T + cl 12 S. levis 20090T + cl 1 S. libani libani 20077T + cl 23 S. libani rufus 20087T + AJ781351 cl 23 S. lienomycini 20091T + AJ781353 cl 22 S. lilacinus Streptoverticillium 6 20059T + (j) AJ781346 cl 12 S. limosus 8570T cl 22 (k) Z76679 cl 8 S. lincolnensis 20068T + cl 13 S. lipmanii 20047T cl 12 (a) cl 23 S. litmocidini 20052T + cl 22 S. lomondensis 20088T + AJ781352 cl 4 S. longisporoflavus 19347T + + cl 6 cl 19 S. longispororuber 20082T + cl 1 S. longisporus 20053T + AJ399475 cl 22 S. longwoodensis 20096T + AJ781356 cl 22 S. lucensis 20065T + + S. luridus 19365T + + - S. lusitanus 20078T + - S. luteofluorescens 20522T + + S. luteogriseus 20073T + AJ399490 cl 1 S. luteosporeus Streptoverticillium 6 20085T + (j) + S. luteoverticillatus Streptoverticillium 6 20045T + (j) cl 12 S. lydicus 19331T + + Y15507 cl 12 cl 23 S. macrosporus 20089T + + S. "malachticus" 19400 - cl 16 - S. malachitofuscus 20067T + + AJ781347 cl 4 S. malaysiensis 20099T + AF117304 cl 15 S. mashuensis Streptoverticillium 6 8603T + (j) X79323 cl 11 S. massasporeus 19362T + + AJ781323 cl 17 cl 1 S. matensis 20055T + cl 1 S. mauvecolor 20100T + AJ781358 cl 34 S. mediolani 20093T cl 16 AJ781354 cl 23 S. megasporus 20092T + - S. melanogenes 20056T + cl 13 S. melanosporofaciens 20066T + AJ271887 cl 16 S. michiganensis 20042T + cl 14 S. microflavus 19327T + cl 12 (a) cl 2 cl 23 S. minutiscleroticus Chainia 3 20062T cl 3 (a) cl 46 S. mirabilis 20076T + AF112180 cl 20 S. misakiensis 19369T + + AJ781325 cl 29 S. misionensis 20063T cl 15 cl 54 S. mobaraensis Streptoverticillium 6 20086T + (j) cl 56 S. morookaensis Streptoverticillium 6 20074T + (j) AJ781349 + S. murinus 10475T + cl 59 S. mutabilis 20054T + + S. mutomycini 20098T + AJ781357 cl 23 S. naganishii 21042T + + S. narbonensis 20043T + cl 44 S. nashvillensis 20064T + cl 23 S. netropsis Streptoverticillium 6 5979T + (j) cl 13 S. neyagawaensis 20080T + AJ399493 cl 26 S. niger Chainia 3 20101T + + S. nigrescens 19332T + + cl 12 cl 23 S. nigrifaciens 20048T cl 9 (a) cl 53 S. nitrosporeus 20044T + + S. niveoruber 19379T + + cl 4 + S. niveus 19395T cl 19 cl 14 ( c) cl 9 cl 47 S. noboritoensis 19337T + + cl 6 cl 34 S. nodosus 19430T + + cl 11 cl 4 S. nogalater 19338T + + AB045886 cl 14 cl 4 S. nojiriensis 20094T + AJ781355 cl 22 S. noursei 5982T + + S. novaecaesareae 20069T + cl 22 S. ochraceiscleroticus Chainia 3 19349T + + cl 8 + S. odorifer 8572T + (k) Z76682 cl 8 S. olivaceiscleroticus Chainia 3 20081T + AJ781350 + S. olivaceoviridis 19324T + + cl 53 S. olivaceus 19394T + + cl 16 cl 13 S. olivaceus 19374 - cl 2 - S. olivochromogenes 20071T + AY094370 cl 20 S. olivoreticuli cellulophilus Streptoverticillium 6 20097T + (j) cl 13 S. olivoreticuli olivoreticuli Streptoverticillium 6 20050T + (j) AJ781345 cl 12 S. olivoverticillatus Streptoverticillium 6 20058T + (j) cl 13 S. olivoviridis 20057T + cl 23 S. omiyaensis 20075T + cl 23 S. orinoci Streptoverticillium 6 20079T + (j) cl 15 S. pactum 19357T + + + S. pallidus 19396T + + - K. paracochleata 20095T + U93328 cl 28 S. paradoxus Actinosporangium 5 20523T + AJ276570 cl 1 S. parvisporogenes Streptoverticillium 6 20072T + (j) AJ781348 cl 12 S. parvus 20524T + cl 2 cl 23 S. parvulus 21789T + + cl 17 cl 6 S. peucetius 20084T + AB045887 cl 21 S. phaeochromogenes 19348T + + cl 6 - S. phaeofaciens 20070T + + S. phaeopurpureus 20051T cl 2 (b) cl 51 S. phaeoviridis 20061T cl 2 (b) cl 51 S. phosalacineus 20102T + AJ781359 cl 27 S. pilosus 20049T + cl 35 S. platensis 20046T + AB045882 cl 23 S. plicatus 20288T + cl 39 S. pluricolorescens 8576T + - S. polychromogenes 20287T + cl 22 S. poonensis Chainia 3 19326T + + cl 14 + S. praecox 20290T + cl 23 S. prasinus 20259T + cl 14 cl 49 S. prasinopilosus 19345T + + cl 14 cl 49 S. prasinosporus 19346T + + cl 10 cl 54 S. prunicolor 19311T + + + S. psammoticus 20525T cl 28 cl 29 S. pseudoechinosporeus Microellobosporia 4 21052T + + S. pseudogriseolus 20252T - cl 4 S. pseudovenezuelae 20276T + AJ399481 cl 19 S. pulveraceus 20322T + AJ781377 cl 23 S. puniceus 20258T + cl 22 S. purpeofuscus 20283T + AJ781364 cl 30 S. purpurascens 20526T + (g) AB045888 cl 1 S. purpureus 19368T + + AJ781324 cl 29 S. purpurogeneiscleroticus Chainia 3 20331T + + S. racemochromogenes 20273T + cl 22 S. rameus 20326T + AJ781379 cl 2 S. ramulosus 19354T + + cl 12 cl 23 S. rangoonensis 20295T cl 20 AJ781366 cl 18 S. recifensis 20261T + cl 41 S. rectiverticillatus Streptoverticillium 6 20292T + (j) cl 48 S. rectiviolaceus 20310T + - S. regensis 20300T + cl 52 S. resistomycificus 21797T + AJ310926 + S. rimosus paromomycinus 20308T + cl 23 S. rimosus rimosus 19352T + + AB045883 cl 13 cl 10 S. rimosus 19390 - cl 13 - S. rishiriensis 20297T + + S. rochei 19313T + + cl 17 cl 39 S. roseiscleroticus Chainia 3 20284T cl 1 cl 38 S. roseodiastaticus 20327T cl 21 (b) cl 2 S. roseoflavus 20535T cl 27 (b) cl 45 S. roseofulvus 20263T + cl 22 S. roseolilacinus 20264T + cl 12 S. roseolus 20265T + cl 22 S. roseosporus 20262T cl 24 (b) cl 23 S. roseoverticillatus Streptoverticillium 6 20255T + (j) AJ781361 cl 13 S. roseoviolaceus 8594T + AJ399484 cl 1 S. roseoviridis 20266T + cl 44 S. ruber Chainia 3 20285T cl 1 cl 38 S. rubiginosohelvolus 20267T + cl 23 S. rubiginosus 20268T + cl 4 S. rubrogriseus 20318T + AJ781373 cl 22 S. rutgersensis castelarensis 20304T + cl 16 S. rutgersensis rutgersensis 8568T + (k) Z76688 cl 9 S. salmonis Streptoverticillium 6 20306T + (j) cl 13 S. sampsonii 8574T cl 22 (k) D63871 cl 8 S. sannanensis 20329T cl 4 cl 22 S. sapporonensis Streptoverticillium 6 20324T + (j) AJ781378 cl 17 S. scabiei 20323T + D63862 cl 25 S. sclerotialus Chainia 3 20528T + cl 2 S. septatus Streptoverticillium 6 8604T + (j) + K. setae 20529T + AB022868 cl 27 S. setonii 20291T cl 19 D63872 cl 23 S. showdoensis 20298T + cl 23 S. sindenensis 21041T + cl 23 S. sioyaensis 20531T cl 6 cl 58 S. somaliensis 21049T - AJ007403 cl 33 S. sparsogenes 19378T + - - S. spectabilis 5986T t2 + + S. spheroides 19392T cl 19 + ( c) cl 9 cl 47 S. spinoverrucosus 20321T + + S. spiralis Elytrosporangium 1 20332T + + S. spiroverticillatus 20254T + cl 22 S. sporocinereus 20311T + AJ781368 cl 16 S. sporoclivatus 20312T + AJ781369 cl 16 S. spororaveus 20313T + AJ781370 cl 22 S. sporoverrucosus 20314T + cl 22 S. subrutilus 20294T + X80825 cl 22 S. sulfonofaciens 20325T + + S. sulphureus 19355T + + - S. syringium Streptoverticillium 6 20320T + (j) AJ781375 cl 13 S. tanashiensis 20274T + AJ781362 cl 22 S. tauricus 20301T + AB045879 cl 24 S. tendae 19314T + + D63873 + cl 6 S. termitum 20289T + cl 22 S. thermoalcalitolerans 19858T + + S. thermocarboxydovorans 19860T + U94489 cl 4 S. thermocoprophilus 19857T + AJ007402 + S. thermodiastaticus 20302T + Z68101 cl 4 S. thermogriseus 20532T cl 7 cl 32 S. thermolineatus 20309T + + S. thermoviolaceus apingens 20307T + Z68101 cl 3 S. thermoviolaceus thermoviolaceus 19359T + + Z68095 cl 11 cl 3 S. thermovulgaris 19342T + cl 7 Z68094 cl 10 cl 32 S. thioluteus Streptoverticillium 6 21037T + (j) AJ781360 cl 12 S. torulosus 20305T + AJ781367 cl 26 S. toxytricini 20269T + cl 31 S. tricolor 20328T cl 21 (b) AJ781380 cl 2 S. tubercidicus 19361T + + AJ621612 cl 12 cl 23 S. tuirus 20299T + AF503493 cl 1 S. umbrinus 20280T cl 15 cl 54 S. variabilis 20270T cl 25 cl 1 S. variegatus 20315T + AJ781371 + S. varsoviensis 20083T cl 12 + ( c) cl 13 S. vastus 21043T + + S. venezulae 19308T + + AB045890 cl 7 cl 22 S. vinaceus 20533T cl 23 (b) cl 8 S. vinaceusdrappus 20296T + cl 39 S. violaceochromogenes 20271T + cl 1 S. violaceolatus 20293T + (k) AJ781365 cl 22 S. violaceorectus 20281T + cl 23 S. violaceoruber 20256T + (d) (k) AF503492 cl 22 S. violaceorubidus 20319T + AJ781374 cl 8 S. violaceus 20257T + AJ781755 cl 1 S. violaceusniger 19336T + + (f) AJ391822 cl 9 cl 15 S. violarus 20275T + AJ399477 cl 1 S. violascens 20272T + cl 23 S. violatus 19397T cl 12 + ( c) AJ399480 cl 14 cl 1 S. violens Chainia 3 20303T + + S. virens 20316T cl 8 cl 40 S. virginiae 20534T + D85123 cl 22 S. virginiae ATCC 13161 + + + S. virginiae 21028 + D85117 + S. virginiae 21048 + + S. virginiae 21537 + + S. virginiae MAFF 116014 + + S. virginiae 899 variant PHIBRO V899-1 + - S. virginiae 899 variant PHIBRO V899-2 + - S. virginiae 899 variant PHIBRO V899-3 + - S. virginiae 899 variant PHIBRO V899-4 + - S. virginiae 899 variant PHIBRO V899-5 + - S. virginiae 899 variant PHIBRO V899-6 + - S. virginiae 899 variant PHIBRO 844 + - S. virginiae 899 variant PHIBRO R4237 + - S. virginiae 899 variant PHIBRO V1403 + - S. virginiae 899 variant PHIBRO 285184 + - S. virginiae 899 variant PHIBRO 286164 + - S. virginiae 899 variant PHIBRO 286763 + - S. virginiae 899 variant PHIBRO 287524 + - S. virginiae 899 variant PHIBRO 180059 + - S. virginiae 899 variant PHIBRO 277277 + - S. virginiae 899 variant PHIBRO 236379 + - S. virginiae 899 variant PHIBRO PD170 + - + S. virginiae 899 variant PHIBRO PD282 + - S. virginiae 899 variant PHIBRO PD321 + - S. "pristinaespiralis" ATCC 25486 + + S. " mitakaensis" ATCC 15297 + + S. viridiviolaceus 20282T + + - S. viridobrunneus 20317T + AJ781372 cl 22 S. viridochromogenes 20260T + cl 54 S. viridodiastaticus 20279T + + S. viridiflavus Streptoverticillium 6 20277T + (j) AJ781363 cl 13 S. viridosporus 20278T + + S. vitaminophilus Actinosporangium 5 21051T + + S. wedmorensis 21050T + cl 23 S. werraensis 21047T + cl 4 S. willmorei 21046T cl 12 (a) cl 23 S. xanthochromogenes 19366T + + cl 2 + S. xanthocidicus 19370T + + cl 3 cl 29 S. xantholiticus 19402T + + cl 4 cl 21 S. xanthophaeus 21039T + cl 22 S. yerevanensis Microellobosporia 4 21053T + + S. yokosukanensis 21040T + cl 22 S. zaomyceticus 19853T + cl 23 List of strains not analysed (based on list valid names DSMZ version 07/2004)

S. albidochromogenes, S. asiaticus, S. aureus, S. avermectinius, S. avermitilis, S. baldaccii, S. beijiangensis, S. candidus, S. cangkringensis, S. caniferus, S. caviscabies, S. cellulolyticus, S. costaricanus, S. drozdowiczii, S. echinoruber, S. europascabiei, S. fervens, S. flavopersicus, S. glaucosporus, S. globisporus subsp. globisporus, S. glomeroaurantiacus, S. halstedii, S. hebeiens S. indonesiensis, S. javensis, S. kentuckensis, S. laceyi, S. ladakanum, S. luridiscabiei, S. luteireticuli, S. malachitospinus, S. mediocidicus, S. mexicanus, S. monomycini, S. niveiscabiei, S. olivomycini, S. puniscabiei, S. reticuliscabiei, S. rhizosphaericus, S. sanglieri, S. scabrisporus, S. sclerotialus, S. seoulensis, S. speibonae, S. spleomycini, S. spitsbergiensis, S. stelliscabiei, S. stramineus, S. thermoautrophicus, S. thermocarboxydus, S. thermonitrificans, S. thermospinosisporus, S. turgidiscabies, S. viridiviolaceus, S. yatensis, S. yeochonensis, S. yogyakartensis, S. yunnanensis.

ε: LMG number unless given otherwise *: Lanoot et al. (2002) **: Lanoot et al. (2004), in press $: Lanoot et al. (submitted) #: not published α: Lanoot et al. (submitted) β: Lanoot et al. (in press) +': analysed and holding separate position or similar pattern to another strain -' or blank: not analysed cl: cluster

1: Goodfellow et al. (1986d). 2: Goodfellow et al. (1986a). 3: Goodfellow et al. (1986b). 4: Goodfellow et al. (1986d). 5: Goodfellow et al. (1986a). 6: Witt & Stackebrandt, 1990 (a): Lanoot et al. (in press) (b): Lanoot et al. (2004) ( c): Lanoot et al. (2002) (d): Monson et al. (1969) (e): Mordarski et al. (1986) (f): Labeda & Lyons, 1991a (g):Labeda & Lyons, 1991b (h): Labeda (1998) (I): Labeda, 1993 (j): Hatano et al. (2003) (k): Kim (2004) ANNEX III. List of Streptomyces isolates used in this study

R- Original Soil sample Number number from province Bioactivity (&) Closest neighbours

Anti Anti Anti Anti G-- Anti G+- Anti F. Anti Anti Producer E. coli B. subtilis yeast bacteria bacteria oxysporum R. solani C. albicans of

5974 PR4.00008 Yunnan 5975 PR4.00027 Yunnan anthracycline S. niveoruber, S. badius** 5976 PR4.00017 Yunnan anthracycline 6120 PR4.00029 Yunnan anthracycline S. niveoruber, S. badius** 6121 PR4.00036 Yunnan anthracycline 6122 PR4.00011 Yunnan anthracycline 6123 PR4.00030 Yunnan anthracycline 6124 PR4.00028 Yunnan anthracycline 6125 PR4.00007 Yunnan aclacinomycin S. diastatochromogenes** 6126 PR4.00002 Yunnan anthracycline S. niveoruber, S. badius** 6127 PR4.00019 Yunnan anthracycline S. niveoruber, S. badius** 6128 PR4.00001 Yunnan anthracycline S. niveoruber, S. badius** 6129 PR4.00031 Yunnan anthracycline S. niveoruber, S. badius** 6130 PR4.00012 Yunnan antitumor glucoside 6131 PR4.00010 Yunnan aclacinomycin 6132 PR4.00022 Yunnan anthracycline S. diastatochromogenes** 6133 PR4.00013 Yunnan aclacinomycin S. diastatochromogenes** 6134 PR4.00317 Yunnan cyclopeptide antibiotics 6147 PR4.00020 Yunnan anthracycline S. niveoruber, S. badius** 6148 PR4.00005 Yunnan anthracycline S. diastatochromogenes** 6149 PR4.00026 Yunnan anthracycline S. niveoruber, S. badius** 6150 PR4.00006 Yunnan aclacinomycin S. diastatochromogenes** 6151 PR4.00025 Yunnan anthracycline 6152 PR4.00018 Yunnan anthracycline 6153 PR4.00034 Yunnan inhibitor of β-lactamase S. anulatus** 6154 PR4.00024 Yunnan anthracycline S. niveoruber, S. badius** 6155 PR4.00032 Yunnan inhibitor of β-lactamase 6156 PR4.00016 Yunnan nontypical anthracycline S. cellulosae** 6183 PR4.00065 Yunnan inhibitor of β-lactamase S. venezulae, S. hygroscopicus, S. intermedius, S. fulvissimus** 6184 PR4.00066 Yunnan inhibitor of β-lactamase S. anulatus** 6185 PR4.00056 Yunnan 6186 PR4.00054 Yunnan inhibitor of β-lactamase 6187 PR4.00048 Yunnan inhibitor of β-lactamase 6188 PR4.00050 Yunnan inhibitor of β-lactamase 6189 PR4.00052 Yunnan inhibitor of β-lactamase S. lydicus** 6190 PR4.00042 Yunnan inhibitor of β-lactamase 6191 PR4.00049 Yunnan inhibitor of β-lactamase S. lydicus** 6192 PR4.00055 Yunnan inhibitor of β-lactamase 6193 PR4.00041 Yunnan inhibitor of β-lactamase 6194 PR4.00062 Yunnan inhibitor of β-lactamase 6195 PR4.00014 Yunnan anthracycline S. niveoruber, S. badius** 6242 PR4.00075 Yunnan inhibitor of β-lactamase 6243 PR4.00073 Yunnan inhibitor of β-lactamase 6244 PR4.00241 Yunnan inhibitor of chitin synthetase 6245 PR4.00236 Yunnan inhibitor of chitin synthetase 6246 PR4.00321 Yunnan cyclopeptide antibiotics 6247 PR4.00211 Yunnan inhibitor of chitin synthetase 6248 PR4.00238 Yunnan inhibitor of chitin synthetase 6249 PR4.00078 Yunnan inhibitor of β-lactamase 6250 PR4.00072 Yunnan inhibitor of β-lactamase 6251 PR4.00299 Yunnan alkaloids 6252 PR4.00076 Yunnan inhibitor of β-lactamase 6253 PR4.00308 Yunnan alkaloids 6296 PR4.00309 Yunnan alkaloids 6297 PR4.00315 Yunnan cyclopeptide antibiotics 6298 PR4.00310 Yunnan alkaloids 6299 PR4.00298 Yunnan alkaloids 6300 PR4.00322 Yunnan cyclopeptide antibiotics 6301 PR4.00074 Yunnan inhibitor of β-lactamase S. olivaceus** 6302 PR4.00307 Yunnan alkaloids 6303 PR4.00160 Yunnan inhibitor of chitin synthetase 6304 PR4.00080 Yunnan inhibitor of β-lactamase 6305 PR4.00318 Yunnan cyclopeptide antibiotics 6306 PR4.00157 Yunnan inhibitor of chitin synthetase S. lydicus** 6307 PR4.00156 Yunnan inhibitor of chitin synthetase 6308 PR4.00210 Yunnan inhibitor of chitin synthetase 6309 PR4.00239 Yunnan inhibitor of chitin synthetase 6310 PR4.00311 Yunnan alkaloids S. venezulae, S. hygroscopicus, S. intermedius, S. fulvissimus** 6311 PR4.00320 Yunnan cyclopeptide antibiotics 6312 PR4.00077 Yunnan inhibitor of β-lactamase 6347 PR4.00316 Yunnan cyclopeptide antibiotics 6348 PR4.00319 Yunnan cyclopeptide antibiotics S. olivaceus** 6349 PR4.00314 Yunnan cyclopeptide antibiotics S. olivaceus** 6350 PR4.00301 Yunnan alkaloids 6351 PR4.00312 Yunnan alkaloids S. venezulae, S. hygroscopicus, S. intermedius, S. fulvissimus** 6352 PR4.00313 Yunnan cyclopeptide antibiotics 6353 PR4.00300 Yunnan alkaloids 6354 PR4.00112 Yunnan inhibitor of chitin synthetase 6355 PR4.00306 Yunnan alkaloids 6356 PR4.00079 Yunnan inhibitor of β-lactamase 6357 PR4.00070 Yunnan inhibitor of β-lactamase 6358 PR4.00037 Yunnan inhibitor of β-lactamase 6359 PR4.00045 Yunnan inhibitor of β-lactamase 6861 PR4.00051 Yunnan inhibitor of β-lactamase 6862 PR4.00067 Yunnan inhibitor of β-lactamase 6863 PR4.00063 Yunnan inhibitor of β-lactamase S. cellulosae** 6864 PR4.00038 Yunnan inhibitor of β-lactamase 6865 PR4.00044 Yunnan inhibitor of β-lactamase 6866 PR4.00069 Yunnan inhibitor of β-lactamase 6867 PR4.00040 Yunnan inhibitor of β-lactamase 6868 PR4.00058 Yunnan inhibitor of β-lactamase 6869 PR4.00061 Yunnan inhibitor of β-lactamase 6870 PR4.00059 Yunnan inhibitor of β-lactamase S. olivaceus** 6871 PR4.00064 Yunnan inhibitor of β-lactamase 6872 PR4.00068 Yunnan inhibitor of β-lactamase 6873 PR4.00039 Yunnan inhibitor of β-lactamase 6874 PR4.00047 Yunnan inhibitor of β-lactamase 6875 PR4.00046 Yunnan inhibitor of β-lactamase 10424 FSSM 20 Morocco 10425 FSSM 14 Morocco 10426 FSSM 25 Morocco S. microflavus** 10427 FSSM 27 Morocco S. rubiginosohelvolus£ 10428 FSSM 23 Morocco S. microflavus** 10429 FSSM 3 Morocco S. olivaceus** 10430 FSSM 5 Morocco S. odorifer£ 10431 FSSM 21 Morocco 10432 FSSM 13 Morocco 10433 FSSM 26 Morocco S. rubiginosohelvolus£ 10434 FSSM 2 Morocco 10435 FSSM 28 Morocco 10436 FSSM 6 Morocco S. odorifer£ 10437 FSSM 1 Morocco 10782 NI 659 Hainan - Guangxi + + 10783 NI 251 Hainan - Guangxi + + amylase 10784 NI 81 Hainan - Guangxi + 10785 NI 07 Hainan - Guangxi + amylase 10786 NI 364 Hainan - Guangxi + + amylase 10787 NI 111 Hainan - Guangxi + + 10788 NI 178 Hainan - Guangxi + + amylase 10789 NI 168 Hainan - Guangxi + + 10790 NI 134 Hainan - Guangxi + 10791 NI 503 Hainan - Guangxi amylase 10792 NI 82 Hainan - Guangxi + + 10793 NI 433 Hainan - Guangxi + + 10794 NI 430 Hainan - Guangxi + + amylase 10795 NI 98 Hainan - Guangxi + + 10796 NI 344 Hainan - Guangxi + amylase 10797 NI 351 Hainan - Guangxi + + amylase 10977 NI 12 Hainan - Guangxi + + 10978 NI 11 Hainan - Guangxi + + 10979 NI 428 Hainan - Guangxi + amylase 10980 NI 518 Hainan - Guangxi + + 10981 NI 76 Hainan - Guangxi + amylase 10982 NI 345 Hainan - Guangxi + + S. phaeochromogenes** 10983 NI 83 Hainan - Guangxi + + + 10984 NI 244 Hainan - Guangxi + amylase 10985 NI 213 Hainan - Guangxi + + 10986 NI 346 Hainan - Guangxi 10987 NI 70 Hainan - Guangxi + amylase S. exfoliatus** 10988 NI 324 Hainan - Guangxi + amylase 10989 NI 448 Hainan - Guangxi + 10990 NI 94 Hainan - Guangxi + + amylase 10991 NI 584 Hainan - Guangxi + + 10992 NI 405 Hainan - Guangxi + 10993 NI 87 Hainan - Guangxi + 10994 NI 559 Hainan - Guangxi + + 10995 NI 399-1 Hainan - Guangxi + + amylase 11210 NI 392 Hainan - Guangxi + 11211 NI 286 Hainan - Guangxi + + 11212 NI 154 Hainan - Guangxi + + 11213 NI 02 Hainan - Guangxi + amylase 11214 NI 410 Hainan - Guangxi + amylase S. phaeochromogenes** 11215 NI 10 Hainan - Guangxi + 11216 NI 46 Hainan - Guangxi + + 11217 NI 132 Hainan - Guangxi + 11218 NI 292-A Hainan - Guangxi 11219 NI 331 Hainan - Guangxi + S. phaeochromogenes** 11220 NI 73 Hainan - Guangxi + + 11221 NI 101 Hainan - Guangxi S. phaeochromogenes** 11709 NI 376 Hainan - Guangxi + amylase 14324 A25 Jiangxi S. ambofaciens, S. coerulescens, S. caelestis, S. nogalater, S. intermedius* 14325 A36 S. venezulae* 17451 fxj 13 Guangxi + related to > 10 strains*** 17452 fxj 2 Guangxi + + + + amylase S. yunnanensis, S. albulus, S. albogriseus* 17453 fxj 5 Yunnan + + + S. fimicarius£ 17454 fxj 1 Guangxi + amylase S. fimicarius£ 17455 fxj 15 Guangxi + S. canarius*** 17456 fxj 17 Yunnan + + amylase S. albollongus£, S. psammoticus£ 17457 fxj 35 Guangxi + related to > 10 strains*** 17458 fxj 33 Beijing + + S. albollongus£, S. psammoticus£ 17459 fxj 43 Guangxi + amylase S. flavogriseus£ 17460 fxj 21 Guangxi + S. cyaneus, S. olivochromogenes*** 17461 fxj 48 Beijing + no RFLP results 17462 fxj 27 Beijing + related to >10 strains*** 17463 fxj 47 Beijing + related to >10 strains*** 17464 fxj 22 Guangxi + + amylase S. eurythermus, S. nogalater* 17465 fxj 12 Guangxi + Actinomadura glucoflavus, A. citrea* 17466 fxj 25 Guangxi + amylase related to >10 strains*** 17467 fxj 28 Guangxi + + amylase related to >10 strains*** 17468 fxj 36 Beijing + + amylase S. fimicarius£ 17469 fxj 38 Yunnan + + amylase related to >10 strains*** 17470 fxj 8 Guangxi S. aureoverticillatus£ 17471 fxj 50 Beijing + + S. albollongus£, S. psammoticus£ 17472 fxj 4 Guangxi + + amylase S. peuceticus, S. lavendulae* 17473 fxj 39 Guangxi + + S. capoamus, S. hygroscopicus, S. fimbriatus* 17474 fxj 41 Beijing + amylase related to >10 strains*** 17475 fxj 19 Guangxi + + amylase S. fimicarius£ 17476 fxj 3 Guangxi + S. fimicarius£ 17477 fxj 29 Guangxi + S. albollongus£, S. psammoticus£ 17478 fxj 20 Yunnan + + S. viridodiastaticus, S. albogriseolus, S. flavovariabilis, S. flavidovirens*** 17479 fxj 24 Beijing + + S. eurythermus£ 17502 fxj 44 Guangxi + + related to >10 strains*** 17696 fxj 6 S. albollongus£ 17697 fxj 7 Yunnan S. coerulescens£ 17698 fxj 9 Guangxi + + amylase related to >10 strains*** 17699 fxj 10 Yunnan + + S. viridocyaneus* 17700 fxj 11 Yunnan + + amylase related to >10 strains*** S. rimosus, S. malaysiensis, S. sparsogenes, S. yeochonensis* 17701 fxj 14 Guangxi + + S. thermoviolaceus£ 17702 fxj 16 Yunnan + amylase S. canarius*** 17703 fxj 18 Guangxi + + S. mirabilis, S. cinereoruber s. fructofermentans*** 17704 fxj 23 Beijing + + + S. kasugaensis, S. platensis, S. griseocarneum# 17705 fxj 26 Beijing + amylase S. canarius*** 17706 fxj 30 Guangxi + amylase S. cyaneus, S. olivochromogenes*** 17707 fxj 31 Guangxi ++ related to >10 strains*** 17708 fxj 32 Beijing + S. eurythermus£ 17709 fxj 34 Guangxi + amylase no RFLP results 17710 fxj 37 Yunnan + S. fimicarius£ 17711 fxj 40 Beijing + amylase S. flavogriseus£ 17712 fxj 42 Beijing + + S. flavogriseus£ 17713 fxj 45 Beijing + + related to >10 strains*** 17714 fxj 46 Beijing + S. griseoaurantiacus, S. nogalater* 17715 fxj 49 Beijing + amylase related to > 10 strains*** 21497 III-63102 $ + + 12 12 S. werraensis£ 21498 III-62119 $ + + 14 S. werraensis£ 21499 III-62208 $ 21500 III-62220 $ + + 21501 III-62223 $ 12 21502 III-53117 $ 21503 III-63108 $ + + S. werraensis£ 21504 III-61208 $ 21505 III-62113 $ + 12 S. werraensis£ 21506 III-63223 $ + + S. werraensis£ 21507 III-62226 $ + 12 S. werraensis£ 21508 III-62209 $ + + S. werraensis£ 21509 III-62210 $ + S. werraensis£ 21510 III-62118 $ + S. werraensis£ 21511 III-62229 $ + 13 S. werraensis£ 21512 III-7C204 $ + + 12 21513 III-63101 $ + S. werraensis£ 21514 III-7C222 $ + 21515 III-63227 $ S. werraensis£ 21516 III-62110 $ 21517 III-71211 $ 21518 III-53108 $ + + 14 21519 III-61107 $ 21520 III-62206 $ + + S. werraensis£ 21521 III-62222 $ + + 12 12 S. werraensis£ 21522 III-62116 $ S. werraensis£ 21523 III-62215 $ S. werraensis£ 21524 III-63214 $ 21525 III-62123 $ + S. werraensis£ 21526 III-71203 $ 13 14 21527 III-63228 $ + 10 21528 III-62214 $ + 12 S. werraensis£ 21529 III-62102 $ 21530 III-7C220 $ + 21532 III-62219 $ + 11 S. werraensis£ 21533 III-61214 $ 21536 III-7C210 $ 14 21537 III-62213 $ + + 14 S. werraensis£ 21590 III-61206 $ + + S. werraensis£ 21591 III-63222 $ 13 21592 III-7C203 $ 21593 III-7C221 $ + 21594 III-71201 $ 12 S. microflavus£ 21773 II1.4 $ + 21774 II1.6 $ 11 H 21775 II1.7 $ 21776 II1.9 $ 10 S. hygroscopicus£ 21777 II1.10 $ 12 21778 II1.11 $ 11 21779 II1.12 $ 11 21780 II1.13 $ 13 S. hygroscopicus£ 21781 II1.14 $ 16 11 21782 II1.15 $ 11 S. werraensis£ 21783 II1.18 $ 12 12 12 21784 II1.19 $ 11 S. werraensis£ 21785 II1.22 $ 10 S. werraensis£ 21786 II1.23 $ 15 10 21787 II1.24 $ 15 12 13 21788 II1.27 $ 15 12 21789 II1.30 $ 14 11 14 21790 II1.32 $ 12 + 21791 II1.33 $ 16 21792 II1.37 $ 21793 II1.38 $ 10 11 21794 II1.39 $ 11 + 21795 II1.42 $ 11 11 21796 II1.46 $ 12 S. coerulescens£ 21797 II1.5 $ + 13 S. hygroscopicus£ 21798 II1.54 $ 12 21799 II1.55 $ 13 21800 II1.56 $ 11 12 + 21801 II1.58 $ 12 21802 II1.65 $ 21803 II1.83 $ 13 21804 II1.84 $ 11 21805 II1.88 $ 12 11 + 21806 II1.89 $ + 21807 II1.91 $ 12 13 21808 II1.96 $ + 21809 II1.100 $ 11 F 21810 II1.101 $ F 21811 II1.102 $ 11 S. werraensis£ 21812 II1.60 $ 11 S. hygroscopicus£ 21814 II1.70 $ 13 S. ederensis£ 21815 II1.78 $ 21816 II1.93 $ 9 10 F 21817 II1.97 $ 11 + 21818 II1.25 $ 11 S. purpurascens£ 21819 II1.29 $ 11 13 21871 II1.40 $ 12 + 21872 II1.43 $ 9 12 F 21873 II1.44 $ 13 + S. indigoferus£ 21874 II1.47 $ 12 10 11 21875 II1.59 $ F 21821 II1.64 $ 11 11 21916 II1.66 $ 12 11 21822 II1.69 $ 13 21917 II1.71 $ 12 S. coerulescens£ 21876 II1.74 $ 12 11 21877 II1.87 $ + 21918 II1.90 $ + 21823 II1.92 $ 12 21878 II1.95 $ 9 + 21825 II1.98 $ + 21820 II1.35 $ 10 11 11 22180 II2.105 $ 11 21942 II2.109 $ + 22063 II2.113 $ 21994 II2.114 $ 21943 II2.115 $ 21944 II2.116 $ 12 S. griseoplanus£ 21945 II2.119 $ + 11 S. griseoplanus£ 21946 II2.120 $ + 11 21947 II2.122 $ 11 22133 II2.123 $ + + 21948 II2.124 $ 11 11 21949 II2.125 $ 12 21950 II2.126 $ 11 S. biverticillatus£ 21951 II2.129 $ + 21953 II2.142 $ 11 21954 II2.146 $ 11 21955 II2.147 $ + 21985 II2.148 $ 11 21956 II2.151 $ 12 22181 II2.152 $ 21957 II2.153 $ 11 21958 II2.157 $ + 21959 II2.158 $ + 21995 II2.162 $ 12 21960 II2.163 $ + S. luridus£ 21961 II2.167 $ 21996 II2.168 $ + 21962 II2.169 $ 21986 II2.172 $ 21963 II2.173 $ 21964 II2.174 $ H 21997 II2.175 $ 21952 II2.139 $ 21965 II2.177 $ 21966 II2.178 $ 21967 II2.179 $ 11 21968 II2.180 $ 21988 II2.181 $ 21969 II2.182 $ H 21989 II2.183 $ 21999 II2.184 $ 21998 II2.185 $ + 21970 II2.186 $ 12 11 21971 II2.187 $ + H 21972 II2.189 $ + H 21973 II2.190 $ + H 21974 II2.191 $ 10 11 22000 II2.192 $ 12 12 S. roseiscleroticus£ 21990 II2.193 $ 12 S. microflavus£ 22077 II2.194 $ 10 21975 II2.196 $ 21976 II2.197 $ 12 21977 II2.198 $ 12 H 21991 II2.199 $ 14 21992 II2.200 $ 21978 II2.201 $ + + H 21979 II2.202 $ 21980 II2.203 $ + 22001 II2.204 $ 11 S. lavendofoliae£ 22002 II2.205 $ + S. coerulescens£ 21981 II2.207 $ 10 + + H 21982 II2.208 $ + H 21993 II2.209 $ + H 22134 III1.1 $ 22078 III1.3 $ S. werraensis£ 22064 III1.5 $ + 22182 III1.6 $ 22183 III1.7 $ + 22184 III1.8 $ 22065 III1.9 $ 22259 III1.10 $ + S. ederensis£ 22209 III1.11 $ + 22135 III1.12 $ 22210 III1.13 $ + 22066 III1.15 $ 22136 III1.16 $ 22067 III1.24 $ + 22068 III1.25 $ + S. nojiriensis£ 22137 III1.26 $ + S. avellaneus£ 22121 III1.27 $ S. nojiriensis£ 22185 III1.29 $ 22186 III1.30 $ + + 25 S. ederensis£ 22122 III1.31 $ 22079 III1.32 $ + + 22123 III1.33 $ + 22080 III1.34 $ 11 + 22069 III1.35 $ 12 22138 III1.36 $ 22139 III1.38 $ 13 + 22140 III1.39 $ 22070 III1.40 $ + 22124 III1.41 $ + 22125 III1.42 $ 12 + 22071 III1.43 $ S. werraensis£ 22081 III1.44 $ + 22082 III1.45 $ S. coerulescens£ 22187 III1.46 $ + S. coerulescens£ 22188 III1.47 $ 15 S. coerulescens£ 22189 III1.49 $ 22083 III1.50 $ 13 22190 III1.52 $ 13 S. coerulescens£ 22191 III1.53 $ + 22072 III1.55 $ 12 S. werraensis£ 22084 III1.56 $ + 25 22085 III1.57 $ 11 22 S. microflavus£ 22086 III1.58 $ 11 30 S. coerulescens£ 22087 III1.59 $ + + 22192 III1.60 $ + + 22141 III1.62 $ 22126 III1.63 $ 11 22088 III1.64 $ S. coerulescens£ 22127 III1.65 $ 12 S. coerulescens£ 22089 III1.66 $ + 12 S. microflavus£ 76 22142 III1.67 $ + 22090 III1.68 $ + 22091 III1.69 $ + S. werraensis£ 22128 III1.73 $ S. laurentii£ 22092 III1.76 $ 22145 III1.77 $ 22073 III1.78 $ + 22193 III1.79 $ 22146 III1.81 $ 22074 III1.82 $ 22194 III1.84 $ S. coerulescens£ 22075 III1.86 $ S. werraensis£ 22129 III1.89 $ + 22260 III2.92 $ H 22261 III2.93 $ + 22327 III2.94 $ + 22211 III2.95 $ 22212 III2.97 $ 22213 III2.99 $ 22262 III2.100 $ + 22198 III2.101 $ 11 22199 III2.109 $ + 22214 III2.113 $ S. coerulescens£ 22263 III2.115 $ 22215 III2.122 $ 22328 III2.125 $ H 22216 III2.127 $ 22329 III2.128 $ H 22264 III2.129 $ S. coerulescens£ 22217 III2.130 $ 22200 III2.136 $ + 22265 III2.138 $ + S. coerulescens£ 22218 III2.139 $ 22219 III2.140 $ S. coerulescens£ 22201 III2.141 $ 22202 III2.142 $ + S. coerulescens£ 22220 III2.143 $ + 22221 III2.144 $ + 22222 III2.145 $ S. coerulescens£ 22203 III2.146 $ + S. coerulescens£ 22223 III2.147 $ 22266 III2.149 $ + S. coerulescens£ 22267 III2.150 $ 22330 III2.151 $ 22204 III2.153 $ 22268 III2.156 $ H 22269 III2.157 $ 22270 III2.158 $ 22271 III2.163 $ 22272 III2.164 $ 19 22331 III2.165 $ 22273 III2.166 $ H 22224 III2.167 $ + 22225 III2.168 $ 22274 III2.169 $ H 22205 III2.170 $ 22206 III2.171 $ 22275 III2.172 $ 22276 III2.173 $ + 22207 III2.174 $ + + 22226 III2.176 $ H 22227 III2.177 $ 22332 III2.182 $ 22228 III2.184 $ 22229 III2.185 $ 22333 III2.186 $ 22334 IV.1 $ 14 11 13 22358 IV.2 $ 15 + 12 22335 IV.3 $ 11 + 12 22336 IV.4 $ 11 21 S. microflavus£ 22337 IV.5 $ 13 S. coerulescens£ 22338 IV.7 $ 11 23022 IV.8 $ 12 22549 IV.9 $ 13 12 22359 IV.10 $ 23023 IV.11 $ 13 22339 IV.12 $ 11 15 S. coerulescens£ 22340 IV.13 $ + S. coerulescens£ 22550 IV.16 $ H 22551 IV.17 $ H 22341 IV.18 $ + S. luridus£ 22361 IV.19 $ + 22362 IV.24 $ + 22342 IV.25 $ 22552 IV.26 $ H 22343 IV.33 $ + 22363 IV.34 $ 13 22344 IV.36 $ 22345 IV.38 $ + 12 22532 IV.39 $ + 22364 IV.40 $ + + 22365 IV.41 $ + H 22553 IV.43 $ H 22366 IV.44 $ + 22367 IV.45 $ H 23045 IV.47 $ 22368 IV.49 $ 22346 IV.50 $ H 22347 IV.51 $ 22348 IV.52 $ + H 22369 IV.53 $ 22370 IV.54 $ 22533 IV.55 $ 12 S. prasinosporus£ 22349 IV.56 $ + H 22350 IV.57 $ + + S. prasinosporus£ 23046 IV.58 $ + + S. prasinosporus£ 22351 IV.59 $ + 22534 IV.60 $ 22352 IV.63 $ H 22535 IV.64 $ S. michiganensis£ 22605 IV.70 $ H 22371 IV.71 $ H 22353 IV.73 $ 22372 IV.74 $ 22536 IV.76 $ 22354 IV.78 $ H 22373 IV.79 $ 22537 IV.81 $ H 22355 IV.82 $ + + 22374 IV.84 $ 22606 IV.88 $ 23227 V.41202 $ 23047 V.41203 $ 13 22607 V.42204 $ 22842 V.43201 $ 13 23048 V.43401 $ 22843 V.42313 $ 22844 V.42314 $ + 22845 V.43301 $ 12 22846 V.4C201 $ 20 22847 V.33308 $ 23025 V.33313 $ 16 20 16 12 23049 V.3C302 $ 22848 V.33304 $ + + 22849 V.33307 $ 23026 V.12201 $ 10 22850 V.4320-1 $ + 22851 V.52202 $ 22608 V.52105a $ 13 23027 V.52117 $ + 23050 V.52113 $ 22852 V.52101 $ + + 22853 V.52104 $ 12 16 22854 V.52105b $ 13 22609 V.52206 $ 22610 V.53101 $ 23228 V.53108 $ + + 14 22611 V.53102 $ 23028 V.52114 $ 13 22612 V.53104 $ 12 23051 V.5C101 $ 22613 V.4302.1 $ 22857 V.61201 $ 20 17 14 22614 V.62216 $ 22615 V.61103 $ + + 13 S. werraensis£ 22616 V.62217 $ S. werraensis£ 22855 V.63218 $ 23052 V.62116 $ 13 S. werraensis£ 22617 V.62121 $ S. werraensis£ 22618 V.62109 $ 12 S. werraensis£ 22619 V.63207 $ 22620 V.3.187 $ + 22621 V.3.188 $ 15 22622 V.3.191 $ + 23053 V.3.192 $ S. werraensis£ 22623 V.3.194 $ + 22624 V.3.196 $ 22625 V.3.201 $ + 22626 V.3.203 $ + 22856 V.3.205 $ + 22627 V.3.206 $ + 23029 V.4.92 $ 12 23054 V.4.91 $ H 23030 V.4.93 $ + + H 23031 V.4.100 $ + S. goshikiensis£

all strains isolated by CCCM / IMCAS except R-10424 to R-10437 isolated by FFSM

$: Yunnan / Jiangxi province or Beijing city *: as revealed by 16S rRNA gene sequence analysis and subsequent BLAST with EMBL database (CCCM / IMCAS) **: as revealed through protein profiling against 109 Streptomyces reference strains (BCCM / LMG) ***: as revealed through 16S-ITS RFLP fingerprinting (BCCM / LMG) H: similar BOX-PCR pattern to S. hygroscopicus F: similar BOX-PCR pattern to S. afghaniensis &: analysis performed by CCCM / IMCAS; number denotes diameter in millimeters of inhibition zone £: as revealed through BOX-PCR fingerprinting (BCCM / LMG) screened in protein profiling blank: not tested

ANNEX IV.

Dendrogram of BOX-PCR patterns of reference strains

Pea rson corr el ati on (Op t:0. 55%) [ 5.7%- 84.6 %] S. flavovir idis LMG 19881 T rep-PCR rep-PCR S. flavovariabilis LMG 19905 T S. ol ivaceoviridis LMG 19324 T S. Sc.ana anulriatusus LMLMG 1920430431 TT S . Sc.h cibitareoensf luois rescens LMLMG 2020447565 TT

0 S . gr iseus solvifaciens LMG 19 9 5 2 T S. corchorusii LMG 20488 T

50 10 S. fluor esc ens LMG 8579 T S. cyanoalbus LMG 19343 T S . pa r v ul us LMG 21789 T S. p urp ur og en ei scle ro ti cus LMG 20331 T S. chrysomallus subsp. chrysomallus LMG 20459 T S. carpaticus LMG 20448 T S. rimosus subsp. rimosus LMG 19352 T S. h ydr og en an s LMG 19948 T S. praecox LMG 20290 T S. atrovirens LMG 20422 T S. fumanus LMG 19882 T S . chrys omallus f umigatus LMG 21793 T S. lavendulae lavendulae LMG 19925 T S. Ste. ndcirraaetus LMLMG 2019347143 TT S . pr a s i n us LMG 20259 T S. roseol us LMG 20265 T S. S.ol ifvlaorveti diocfulsi ccuesllulophilus LMLMG 2020051975 TT S. bambergiensis LMG 19299 T S. S.vio olacrinoeocci hromogenes LMLMG 2020207719 TT S. viridSo. chineromreoorgeenect uss LMG 2L0M260G 2 T0461 T S. collinus LMG 20486 T S. ederensi s LMG 20504 T S. vitaS.m icnopochhileluastus LMG 2L1M051G 2 T0478 T S. luteogriseus LMG 20073 T S. coelicolor LMG 8571 T S. gris eocarneus LMG 19383 T S. h ygr osc op icu s gl eb osu s LMG 19950 T S. kurssanovii LMG 19933 T S . ar a b i c us LMG 20414 T S. S.vio l elacv iseorubidus LMLMG 2020309190 TT S. vinaSc. elusibani subsp. rufus LMG 2L0M533G 2 T0087 T S. Sg.r issaelmofuonscisus LMLMG 2019830856 TT S. diasSt.a otichrusa scueibssp.cle droiastictausti cus LMG 1L9M322G 1 T9349 T S . fulvissim us LMG 19310 T S. a ure ov er ti cil lat u s LMG 20426 T S. gougS.er l yodtiici us LMG 1L9M888G 1 T9331 T S. griseoruber LMG 19325 T S. rutgersensis subsp. rutgersensi s LMG 8568 T S. albogriseolus LMG 20395 T S. c oe rul eo pru nu s LMG 20483 T S . um b ri n us LMG 20280 T S . l o ng i sp o r of l a vu s LMG 19347 T S. Sm. igsrioisneoenausisranti ac us LMLMG 2120004635 TT S. caneS.s hcyengrsosc opicus subsp. hygroscopiLMcusG 2L0M445G 1 T9335 T S. Sh.i roslivutuacseus LMLMG 1919939274 TT S . felleS.u sendus LMG 2L0M511G 1 T9393 T S. tuirus LMG 20299 T S. limosus LMG 8570 T S. flaveol us LMG 19328 T S. libani subsp. libani LMG 20077 T S. arenae LMG 20416 T S. sampsonii LMG 8574 T S. v iri div io lac eus LMG 20282 T S. malachitofuscus LMG 20067 T S. nigrescens LMG 19332 T S. odorifer LMG 8572 T S. Sb.o ottlrivoorpeetnsiciusli s ubsp. oi lvore ti cul i LMLMG 2020405370 TT S. gl obi sporus caucasicus LMG 19895 T S. longisporus LMG 20053 T S. S.la nthatuermsogr iseus LMLMG 2019353802 TT S . al bi d of l a vu s LMG 21791 T S. durhamensis LMG 20501 T S. c avo ur en sis wash in gt o ne nsi s LMG 20451 T S. thermovulgari s LMG 19342 T S. champavatii LMG 20454 T S. filipinensis LMG 19333 T S. viridoS. brauurneneocusirculatus LMG 2L0M317G 2 T1794 T S. c lav ifer LMG 20476 T S. chartreusis LMG 20455 T S. meSga. swpeorrrusae nsi s LMG 2L0M092G 2 T1047 T S. Sd.i acsisctaaticucusa sisucubssp. ardesiacus LMLMG 2020447974 TT S. lavendofoliae LMG 19935 T S. graminofaciens LMG 19892 T S . Scu. rreageconsi is LMLMG 2020430910 TT S . therm oalcali tolerans LMG 19858 T S. Sip. ogmrisoeeoaefl av us LMLMG 1920534204 TT S. psaSm. malbotoicfluavsus LMG 2L0M525G 2 T1038 T S . no v a ec a es a r ea e LMG 20069 T S. aurS.eof macuireinsus LMG 5L9M68G T 10475 T S. pilosus LMG 20049 T S. violaceol atus LMG 20293 T S. avelS.la cneuansferus LMG 2L0M427G 2 T0446 T S. Sc.o grelesisecinuenss LMLMG 1920487795 TT S. therm olineatus LMG 20309 T S. v iolens LMG 20303 T S. Ss.ann c innanabenarsiinsus LMLMG 2020346297 TT S. roseofulvus LMG 20263 T S . San. rti hmocosyuasn iscuubss p. rimo su s LMLMG 1920435112 TT S . muSta.b silipso rov erru co sus LMG 2L0M054G 2 T0314 T S. violaceoruber LMG 20256 T S . libani subsp. libani LMG 20077 T S. fumanus LMG 19882 T S. goshikiensis LMG 19884 T S. humiferus LMG 20519 T S . ni gr e s ce n s LMG 19332 T S. t e nd ae LMG 19314 T S. neyagawaensis LMG 20080 T S. litmocidini LMG 20052 T S . oc h r ac e isc l e r o tic u s LMG 19349 T S . Sro. soeliov lorilaetcicnulsi c ell ul ophi lus LMLMG 2020209647 TT S. lydiSc.u sspir overticillatus LMG 1L9M331G 2 T0254 T S. Sin. daliaboenfascisiens LMLMG 2019939614 TT S. hygroscopicus glebosus LMG 19950 T S. acidiscabies LMG 19856 T S. griseochromogenes LMG 19891 T S . libani subsp. ruf us LMG 20087 T S. niv eoruber LMG 19379 T S. torul osus LMG 20305 T S. hawaiiensis LMG 5975 T S. coeruleoprunus LMG 20483 T S. S.cin perruenoricubeolorr fructofermentans LMLMG 1920431631 TT S . pl atSe.n hesi slvaticus LMG 2L0M046G 1 T9940 T S. Sk.a rismhmosiruesn spiasro momycinus LMLMG 2019930378 TT S . ca vSo.u chr e nrysises wuassh in g t on e n si s LMG 2L0M451G 2 T0458 T S. Ss.c vaiboileaic eo chr omo ge ne s LMLMG 2020327231 TT S. aureocirculatus LMG 21794 T S. s ap po ron en sis LMG 20324 T S. lienomycini LMG 20091 T S. c oll inus LMG 20486 T S. hygroscopicus subsp. hygroscopicus LMG 19335 T S. phaeoviridis LMG 20061 T S. enduS.s erythraeus LMG 1L9M393G 2 T0508 T S. l uteogr iseus LMG 20073 T S. phaeopurpureus LMG 20051 T S. purSp.ur fogrageni liseiscleroticus LMG 2L0M331G 1 T9874 T S. Sg.a kulilarsesusanovi i LMLMG 1921793903 TT S. hydrS.o gceynanstarsgi neus LMG 1L9M948G 2 T0495 T S. S.m av sioslasacpoeorreubusidus LMLMG 2019331629 TT S . l a ve n d ul ae l a ve n d ul a e LMG 19925 T S. puniceus LMG 20258 T S. Sg.r igsreisoeruobifugiscunossus LMLMG 1919988415 TT S. cinereorectus LMG 20461 T S. phaeofaciens LMG 20070 T S. cocS.hl efalotruidsae LMG 2L0M478G 1 T9899 T S. fulvi ssimus LMG 19310 T S. humidus LMG 19936 T S . l o ng i sp o r us LMG 20053 T S. virginiae LM G 2 15 37 S. Sro. cgrhiseieoruber LMLMG 1919332135 TT S . al boSf.l amviussakiensi s LMG 2L1M038G 1 T9369 T S . S.dj akumabr rteinnussis LMLMG 2021728950 TT S. murinus LMG 10475 T S. clavuligerus LMG 20477 T S. c ali forn icus LMG 19309 T S. misionensis LMG 20063 T S . pr u n i c ol o r LMG 19311 T S. coerulescens LMG 8590 T S . ri mosus paromomyci nus LMG 20308 T S. hirs ut us LMG 19927 T S. pol ychr omogenes LMG 20287 T S. racemochromogenes LMG 20273 T "S. f ungi cidicus" LMG 21792 T S. resi stomyc ificus LMG 21797 T S . S.m itruairbuiliss LMLMG 2020029769 TT S. longwoodensis LMG 20096 T S. Sk.unm a reni naegensis LMLMG 2020541216 TT S. cineSr.o mchorbaomraogeennesiss LMG 2L0M466G 2 T0086 T S. Sp.s meuadlaoecchhnitofiusspcouresus LMLMG 2021006527 TT S. spinSov. gerrrisuecoolusutesu s LMG 2L0M321G 1 T9356 T S. lusitanus LMG 20078 T S. minutiscleroticus LMG 20062 T S. bottropensis LMG 20437 T S. reci fens is LMG 20261 T S . al bi re t i cu l i LMG 20393 T S. flaviscleroticus LMG 19886 T S. l anatus LMG 19380 T S. p lic at us LMG 20288 T S. daghestani cus LMG 20496 T S. caelestis LMG 20441 T S. Sg.r idsueorhvamiridiens sis LMLMG 2019350211 TT S. rosSeo. lpsuseudovenezuelae LMG 2L0M265G 2 T0276 T S. Sa.nan f il ipdiinei ns is LMLMG 198603303 T T S. varS.ieg aabtui kso ens is LMG 2L0M315G 2 T0386 T S . ch r es t om yc e tic u s LMG 20457 T S. morookaensis LMG 20074 T S. c ha rtr eu sis LMG 20455 T S. cuspi dosporus LMG 20492 T S. noboritoensis LMG 19337 T S. diastaticus subsp. ardesi acus LMG 20497 T S . bl as t my ce ti cu s LMG 20434 T S. cremeus LMG 20489 T S. katrSa.e me la no ge ne s LMG 1L9M945G 2 T0056 T S . S.pe cuucretaci cousi LMLMG 2020049841 TT S. parvisporogenes LMG 20072 T S. flavofungini LMG 21799 T S. Sli.nc i polonemoensaei s LMLMG 2020052680 TT S. albaduncus LMG 20392 T S. will mor ei LMG 21046 T S. antibioti cus LMG 20412 T S . griseoloal bus LMG 21308 T S. novaecaesareae LMG 20069 T S. aurantiacus LMG 19358 T S. g ris eu s s. c ret os us LMG 19946 T S. roseolilacinus LMG 20264 T S . al bo v i r i d i s LMG 20403 T S. viridosporus LMG 20278 T S . l a ve n d ul ae gr a s se r i u s LMG 19938 T S. l ipm an ii LMG 20047 T S. Sg.han indiaeaensnsisis LMLMG 1919896941 TT S. mi crofl av us LMG 19327 T S . S.gr ahmawi naeai iernussi s LMLMG 591997504 T T S. griseus s. alpha LMG 19953 T S. S.gr ivsieoolamc eyocilniatus LMLMG 2019829833 TT S. griseostramineus LMG 19932 T S. c oeles cens LMG 20479 T S. re ct iv ert ic ill at us LMG 20292 T S . l a ve n d ul oc o l o r LMG 19934 T S. c inereus LMG 21310 T S . Sni. grsai fnna cani enens sis LMLMG 2020032489 TT S. ma sh ue nsi s LM G 8 60 3 T S. Sfl.av anotvhiroencyasni cus LMLMG 2020541161 TT S. flavogriseus LMG 19887 T S. hygr osc opicus decoy icus LMG 19954 T S. humi ferus LMG 20519 T S. rectiverticillatus LMG 20292 T S. v iolac eoruber LMG 20256 T S. me di ol an i LMG 20093 T S. cinereus LMG 21310 T S. litmocidini LMG 20052 T S. alb ov inaceus LMG 20402 T S. spiralis LMG 20332 T S . Sri.s ghririsieeonscishromogenes LMLMG 1920289971 TT S. ru bi gi no soh el vo lu s LMG 20267 T S. Ss.app c ineorroenenorubseisr fr uct o fe rment an s LMLMG 2020346243 TT S. pluricolorescens DS M 40 01 9T S. Se.r ykathshraemusirensis LMLMG 1920593087 TT S. globisporus globisporus LM G 8 57 8 T S. cavourensis cavourensis LMG 20450 T S. s cabiei LMG 20323 T S. s etonii LMG 20291 T S . bl ue n s i s LMG 5969 T S . S.br al iesnilieomnsyicsini LMLMG 2020409381 TT S. flavofuscus LMG 19900 T S. Sk.ifu phnaeenovsisi ridis LMLMG 2019906571 TT S. b aa rne ns is LMG 20431 T S. Sv.a phstuaesopurpureus LMLMG 2021005431 TT S. galbus LMG 19879 T S. fimicarius LMG 21044 T S. gal ilaeus LMG 21790 T S . fumi gat is cler otic us LMG 19911 T S. parv us LMG 20524 T S . Sgr. ims eaossapl aspn uosreus LMLMG 1919936232 TT S. l aurentii LMG 19959 T S . Sto. xgry trisiecoinriubiginosus LMLMG 1920294691 TT S. x anthol iticus LMG 19402 T S. S.ro sroeicshceleri oticus LMLMG 1920231843 TT S. ruber LMG 20285 T S. flavovir idis LMG 19881 T S. djakartensis LMG 21795 T S . colom bi ensis LMG 20487 T S. flavovariabi lis LMG 19905 T S. S.di sgtal lbliucus s LMLMG 1920487999 TT S. anulatus LMG 19301 T S. Sc.aer fuumligeuastiscleroticus LMLMG 1919391991 TT S. Sgniveruisseoplanus LMLMG 199939523 TT S. biverticillatus LMG 20433 T S. lilacinus LMG 20059 T Salbulus LMG 20404 T S. djakartensis LMG 21795 T S. rubr ogriseus LMG 20318 T S. galbus LMG 19879 T S. mauv ec olor LMG 20100 T S. fumigatiscleroticus LMG 19911 T S. af ghaniensi s LMG 20390 T S. g ris eo pla nu s LMG 19923 T S. purpur asc ens LMG 20526 T S. toxytrici ni LMG 20269 T S. v iolar us LMG 20275 T S. kifunensis LMG 19957 T S. janthinus LMG 8591 T S. ro se isc ler ot icu s LMG 20284 T S. albolongus LMG 20396 T S. ruber LMG 20285 T S. violatus LMG 19397 T S. kunmingensis LMG 20521 T S. v iolac eus LMG 20257 T S. phaeofaciens LMG 20070 T S. roseov iol ac eus LMG 8594 T S. humi dus LMG 19936 T S. biki niens is LMG 19367 T S. nogalater LMG 19338 T S. azur eus LMG 20430 T S. c ell ul osa e LMG 19315 T S. fervens melrosporus LMG 19897 T S. c anus LMG 19329 T S. thermocarboxydovorans LMG 19860 T S. l ucens is LMG 20065 T S. olivoviridis LMG 20057 T S. pseudoechnisporeus LMG 21052 T S. at rooliv ac eus LMG 19306 T S. mutomyc ini LMG 20098 T S. nitrosporeus LMG 20044 T S. albus pathocidicus LMG 20406 T S. v irens LMG 20316 T S. ki shi wadens is LMG 19939 T S. c alv us LMG 20442 T S. c ell ul of la vu s LMG 21796 T S. ast erosporus LMG 20419 T S. luteosporeus LMG 20085 T S. aureor ect us LMG 19908 T S. aburaviensis LMG 19305 T S. naganishii LMG 21042 T S. c ine re osp in us LMG 20464 T S. hygr oscopicus angustmyceti cus LMG 19958 T S. kasugaensis LMG 19949 T S. kanamyceticus LMG 19351 T S. o liv ov ert i cill at u s LMG 20058 T S. macrosporus LMG 20089 T S. v iri dofl av us LMG 20277 T S. diastatochromogenes LMG 20498 T S. p urp eo f usc us LMG 20283 T S. sporocinereus LMG 20311 T S. o liv ac eo vir idi s LMG 19324 T S. paracochl eatus LMG 20095 T S. c anari us LMG 20443 T S. matensis LMG 20055 T S. c hib ae ns is LMG 20456 T S. rectiviolaceus LMG 20310 T S. c orchor usi i LMG 20488 T S. glaucus LMG 19902 T S. p arv ul us LMG 21789 T S. bungoensis LMG 20439 T S. eury thermus LMG 20510 T S. yokosukanensis LMG 21040 T S. lavenduligriseus LMG 19943 T S. glaucusporus LMG 19907 T S. paradoxus LMG 20523 T S. sulfonofaciens LMG 20325 T S. s clerotialus LMG 20528 T S. crystal linus LMG 20490 T S. albov ertic ill atus LMG 20401 T S. albospinus LMG 20398 T S. septatus LMG 8604 T S. graminearus LMG 19904 T S. nodosus LMG 19340 T S. griseomycini LMG 19883 T S. poonensis LMG 19326 T S. gris eost rami neus LMG 19932 T S. badius LMG 19353 T S. l av enduloc olor LMG 19934 T S. s ind en en si s LMG 21041 T S. nigrifaciens LMG 20048 T S. ehi mensis LMG 20505 T S. flavovirens LMG 20516 T S. h ac hij oe ns is LMG 19928 T S. flavogriseus LMG 19887 T S. phaeochromogenes LMG 19348 T S. rubi gi nosus LMG 20268 T S. achromogenes achromogenes LMG 20387 T S. glomeratus LMG 19903 T S. achromogenes rubradiris LMG 20388 T S. l usi tanus LMG 20078 T S. v irginiae LMG 20534 T S. albireti culi LMG 20393 T S. v irginiae LMG 21048 S. daghestanicus LMG 20496 T S. t h ermo co pro ph ilu s LMG 19857 T S. gris eoviri di s LMG 19321 T S. gardneri LMG 19876 T S. c uspidospor us LMG 20492 T S. gris eus LMG 19302 T S. c remeus LMG 20489 T S. ramulos us LMG 19354 T S. peuceticus LMG 20084 T S. bac ill ari s LMG 8585 T S. l inc ol nensi s LMG 20068 T S. n arb on en si s LMG 20043 T S. yerevanensi s LMG 21053 T S. fradi ae LMG 19371 T S. anandii LMG 8600 T S. roseoflavus LMG 20535 T S. c hrest omyc et ic us LMG 20457 T S. phosalacineus LMG 20102 T S. v iri dosporus LMG 20278 T S. s ub rut il us LMG 20294 T S. ghanaensis LMG 19894 T S. s howdoensi s LMG 20298 T S. c hatt anoogensis LMG 19339 T S. coelicoflavus LMG 20480 T S. rutgersensi s castelarensi s LMG 20304 T S. echinatus LMG 5972 T S. spectabilis LMG 5986t2 S. i akyr us LMG 19942 T S. malaysiensis LMG 20099 T S. c inn amon eu s lan os us LMG 20471 T S. v iolac eorec tus LMG 20281 T S. c inn amon eu s sub sp . a lbo sp oru s LMG 20469 T S. c inereoruber cinereor uber LMG 20462 T S. c inn amon eu s spa rsu s LMG 20472 T S. flavi dovir eus LMG 19387 T S. c inn amon eu s cin na mon eu s LMG 8602 T S. finlayi LMG 19373 T S. e rumpe ns LMG 20507 T S. tanashi ensis LMG 20274 T S. armeniacus LMG 20418 T S. atroaurantiacus LMG 20421 T S. gris eosporeus LMG 19947 T S. argent eol us LMG 5967 T S. i ndigoferus LMG 19930 T S. hygr oscopicus ossamyceticus LMG 19951 T S. o liv oc hro mog en es LMG 20071 T S. v iri dodiast at ic us LMG 20279 T S. netropsis LMG 5979 T S. pras inosporus LMG 19346 T S. c oe rul eo fu scu s LMG 20482 T S. oliv ac eis clerot i cus LMG 20081 T S. hiroshimensis LMG 19924 T S. inusitatus LMG 19955 T S. a ure ov er sili s LMG 20425 T S. mi chiganens is LMG 20042 T S. amak usaensis LMG 19350 T S. xanthochromogenes LMG 19366 T S. at rat us LMG 20420 T S. noj iri ensis LMG 20094 T S. sioyaensis LMG 20531 T S. c oe rul esc en s LMG 8590 T S. eni ssocaesil is LMG 20506 T S. racemochromogenes LMG 20273 T S. gel aticus LMG 19376 T S. mi rabil is LMG 20076 T S. f ulvorobeus LMG 19901 T S. prasinopilosus LMG 19345 T S. gris eobrunneus LMG 19877 T S. ru br og rise us LMG 20318 T S. gris eolus LMG 19878 T S. ma uv ec olo r LMG 20100 T S. glaucescens LMG 19330 T S. a f gh an ien si s LMG 20390 T S. gris eoinc arnat us LMG 19316 T S. erythrogriseus LMG 19406 T S. ru br og rise us LMG 20318 T S. distallicus LMG 20499 T S. mauv ec olo r LMG 20100 T S. c aerul eus LMG 19399 T S. a f gh an ien si s LMG 20390 T S. niveus LMG 19395 T S. purpur asc ens LMG 20526 T S. biverticill atus LMG 20433 T S. v iolar us LMG 20275 T S. l ilacinus LMG 20059 T S. janthinus LMG 8 591 T S. albulus LMG 20404 T S. albolongus LMG 20396 T S. noursei LMG 5 982 T S. violatus LMG 19397 T S. roseov ert ic ill at us LMG 20255 T S. v iolac eus LMG 20257 T S. kat rae LMG 19945 T S. roseov iol ac eus LMG 8 594 T S. bluensis LMG 5 969 T S. biki niens is LMG 19367 T S. parvisporogenes LMG 20072 T S. azur eus LMG 20430 T S. c anesc ens LMG 20445 T S. fervens melrosporus LMG 19897 T S. f elleus LMG 20511 T S. thermocarboxydovorans LMG 19860 T S. l imosus LMG 8 570 T S. olivoviridis LMG 20057 T S. s ampsonii LMG 8 574 T S. a t rooliv ac eus LMG 19306 T S. nitrosporeus LMG 20044 T S. odorifer LMG 8 572 T S. v irens LMG 20316 T S. globisporus caucasicus LMG 19895 T S. c alv us LMG 20442 T S. albidoflavus LMG 21791 T S. asterosporus LMG 20419 T S. c hampavati i LMG 20454 T S. aureor ectus LMG 19908 T S. v it ami no philu s LMG 21051 T S. naganishii LMG 21042 T S. coelicolor LMG 8 571 T S. h ygr osc opicu s angust myceti cus LMG 19958 T S. arabic us LMG 20414 T S. kanamyceticus LMG 19351 T S. v inaceus LMG 20533 T S. macrosporus LMG 20089 T S. diastaticus subsp. diastaticus LMG 19322 T S. diastatochromogenes LMG 20498 T S. gougerotii LMG 19888 T S. s poroci nereus LMG 20311 T S. rutgersensis subsp. rutgersensis LMG 8 568 T S. paracochl eatus LMG 20095 T S. longisporoflavus LMG 19347 T S. mat ensis LMG 20055 T S. pras inus LMG 20259 T S. rectiviolac eus LMG 20310 T S. b amb er giens is LMG 19299 T S. glaucus LMG 19902 T S. v iri do chromo ge nes LMG 20260 T S. bungoensis LMG 20439 T S. v iri do brunn eu s LMG 20317 T S. yokosukanensis LMG 21040 T S. megasporus LMG 20092 T S. glaucusporus LMG 19907 T S. lavendofoliae LMG 19935 T S. sulfonofaciens LMG 20325 T S. t h ermoal cal it ol era ns LMG 19858 T S. c rystal linus LMG 20490 T S. euroci dic us LMG 20509 T S. albospinus LMG 20398 T S. v ars ovi ensis LMG 20083 T S. graminear us LMG 19904 T S. a mbo f aci ens LMG 20409 T S. gris eomycini LMG 19883 T S. griseus sol vifaciens LMG 19952 T S. g ris eo st rami ne us LMG 19932 T S. lavendulocolor LMG 19934 T S. c yan oa lb us LMG 19343 T S. n igrif ac iens LMG 20048 T S. atrovirens LMG 20422 T S. flavovirens LMG 20516 T S. chrysomallus fumigatus LMG 21793 T S. flavogriseus LMG 19887 T S. c arpat ic us LMG 20448 T S. ru bi gi no sus LMG 20268 T S. s pheroides LMG 19392 T S. glomeratus LMG 19903 T S. acrimycini LMG 21798 T S. l usi tanus LMG 20078 T S. t h ermovi olace us su bs p. ther mov iol ac . LMG 19359 T S. albireti culi LMG 20393 T S. t h ermovi olace us su bs p. apinge ns LMG 20307 T S. daghestanicus LMG 20496 T S. wedmor ensis LMG 21050 T S. gris eoviri di s LMG 19321 T S. ra ngoone nsis LMG 20295 T S. c uspidospor us LMG 20492 T S. almqui st ii LMG 21307 T S. c remeus LMG 20489 T S. a lb us su bs p. al bu s LMG 20405 T S. peuceticus LMG 20084 T S. gibsonii LMG 19912 T S. l inc ol ne nsi s LMG 20068 T S. flocculus LMG 19889 T S. yerevanensis LMG 21053 T S. l omo nd ensis LMG 20088 T S. anandii LMG 8 600 T S. i nt er medius LMG 19304 T S. c hre st omyc et ic us LMG 20457 T S. c yan eu s LMG 20494 T S. v iri dosporus LMG 20278 T S. s yringium LMG 20320 T S. ghanaensis LMG 19894 T S. albosporeus labilomyceticus LMG 20400 T S. c ha tt anoogensis LMG 19339 T S. gobitricini LMG 19910 T S. rutgersensis castelarensis LMG 20304 T S. a ve lla ne us LMG 20427 T S. s pectabili s LMG 5 986t 2 S. aureofaciens LMG 5 968 T S. malaysiensis LMG 20099 T S. psammoticus LMG 20525 T S. v iolac eorec tu s LMG 20281 T S. t h ermoli neat us LMG 20309 T S. c inereoruber cinereor uber LMG 20462 T S. flavidovireus LMG 19387 T S. roseof ul vus LMG 20263 T S. finlayi LMG 19373 T S. mut abili s LMG 20054 T S. t anashi ensis LMG 20274 T S. v ari egat us LMG 20315 T S. gris eosporeus LMG 19947 T S. aculeolatus LMG 19906 T S. a rge nt e ol us LMG 5 967 T S. longwoodensis LMG 20096 T S. hygroscopicus ossamyceticus LMG 19951 T S. cinerochromogenes LMG 20466 T S. v iri dodiast at ic us LMG 20279 T S. s pinov err ucosus LMG 20321 T S. pras inosporus LMG 19346 T S. c lav ul igerus LMG 20477 T S. o liv ac eis clerot i cus LMG 20081 T S. c avour ensis cavourensis LMG 20450 T S. inusitatus LMG 19955 T S. mi nu t iscl ero t icu s LMG 20062 T S. a mak usaensis LMG 19350 T S. flaviscleroticus LMG 19886 T S. atratus LMG 20420 T S. c aeles ti s LMG 20441 T S. s ioyaensi s LMG 20531 T S. morookaensis LMG 20074 T S. eni ssocaesil is LMG 20506 T S. blastmyceti cus LMG 20434 T S. gel aticus LMG 19376 T S. albaduncus LMG 20392 T S. f ulvorobeus LMG 19901 T S. griseoloalbus LMG 21308 T S. griseobrunneus LMG 19877 T S. nashvillensis LMG 20064 T S. gris eolus LMG 19878 T S. alboviridis LMG 20403 T S. glaucescens LMG 19330 T S. lavendulae grasserius LMG 19938 T S. gris eoinc arnatus LMG 19316 T S. erythrogriseus LMG 19406 T

ANNEX V. Dendrogram of 16S-ITS RFLP patterns of reference strains

Dice ( Opt: 0.32%) (T ol 0. 9%-0. 9%) ( H>0.0 % S>0. 0%) [6 .0%-8 8.6%] .S. filamentosus L MG 20 51 2 T RFLP RFLP S. violescens L MG 20 27 2 T S. nashv illensis L MG 20 06 4 T S. mediolani L MG 20 09 3 T

0 S. albov inaceus L MG 20 40 2 T 60 80 10 S. spi rov ert ic ill at us L MG 20254 T S. rubiginosohelvolus L MG 20 26 7 T S. violaceoruber L MG 20256 T S. cavourensis washingtonensis L MG 20 45 1 T .S. an th oc yanicus L MG 20411 T S. pu lve rac eu s L MG 20 32 2 T S. crem eus L MG 20489 T S. kanamyceticus L MG 19 35 1 T S. cavourensis cavourensis L MG 20450 T S. atratus L MG 20 42 0 T S. polychromogenes L MG 20287 T S. fl ori dae L MG 19 89 9 T .S. hyg ros cop icu s gl eb os us L MG 19 95 0 T S. race mo chro mo ge ne s L MG 20273 T S. mutomycini L MG 20 09 8 T S. violaceolatus L MG 20293 T S. libani subsp. rufus L MG 20 08 7 T S. cirratus L MG 20473 T S. fi nlayi L MG 19 37 3 T S. humidus L MG 19936 T S. gri seu s su bsp. g ris eus L MG 19 30 2 T S. rubrogriseus L MG 20318 T S. fi lipinensis L MG 19 33 3 T S. galbus L MG 19879 T S. californicus L MG 19 30 9 T S. spo ror ave us L MG 20313 T S. fi micarius L MG 21 04 4 T S. virginiae PHIBRO 287524 S. baarnensis L MG 20 43 1 T S. cinnamonensis L MG 20468 T S. fl av of us cus L MG 19 90 0 T S. virginiae L MG 20534 T S. sindenensis L MG 21 04 1 T S. go shi kiensi s L MG 19884 T .S. badius L MG 19 35 3 T S. virginiae L MG 21048 S. setonii L MG 20 29 1 T .S. virginiae L MG 21028 S. parvus L MG 20 52 4 T S. subruti lus L MG 20294 T .S. fulv orobeus L MG 19 90 1 T S. roseofulvus L MG 20263 T S. rimosus par omomycinus L MG 20 30 8 T S. viridobrunneus L MG 20317 T S. platensis L MG 20 04 6 T S. chrestomyceticus L MG 20 45 7 T S. humif erus L MG 20519 T S. coelescens L MG 20479 T S. microflavus L MG 19 32 7 T S. an ula t us L MG 19 30 1 T S. rose ol us L MG 20265 T .S. gri seu s s. alp ha L MG 19 95 3 T S. laurentii L MG 19959 T S. fl uo res cen s LMG 8579 T S. fl av ot ric ini L MG 19880 T S. chry soma ll us su bs p. chr ysomall us L MG 20 45 9 T S. avidinii L MG 20428 T S. lipmanii L MG 20 04 7 T S. xan t hophae us L MG 21039 T .S. citreofluorescens L MG 20 47 5 T .S. sannanensis L MG 20329 T .S. albov iri dis L MG 20 40 3 T S. virginiae PHIBRO 844 S. argenteol us LMG 5967 T S. virginiae PHIBRO PD 170 S. griseus cretosus L MG 19 94 6 T S. virginiae PHIBRO ATCC 13161 S. wil lmorei L MG 21 04 6 T S. bu ngoensi s L MG 20439 T S. cyan eof usc at us L MG 20 49 3 T S. novaecaesareae L MG 20069 T S. atrooli vaceus L MG 19 30 6 T .S. gri seo ch romo ge ne s L MG 19891 T S. oli vovir idi s L MG 20 05 7 T S. bo bil i L MG 20436 T S. tu be rci dic us L MG 19 36 1 T S. kunmingensi s L MG 20521 T S. hygros copicus angustmyceticus L MG 19 95 8 T .S. galil aeus L MG 21790 T S. hygros copicus decoyicus L MG 19 95 4 T S. pu nic eu s L MG 20258 T S. glo bi sporu s gl ob isp oru s LMG 8578 T S. cha t ta no og en sis L MG 19 33 9 T S. gri seo lo al bus L MG 21308 T S. helvaticus L MG 19 94 0 T S. litmocidini L MG 20052 T S. ramulosus L MG 19 35 4 T .S. venez ulae L MG 19308 T S. catenulae L MG 20 44 9 T S. no jiri en sis L MG 20094 T S. lydi cus L MG 19 33 1 T .S. tanashiensi s L MG 20274 T S. coelicoflavus L MG 20 48 0 T S. canari us L MG 20443 T S. chry seus L MG 20 45 8 T .S. katrae L MG 19945 T S. canferus L MG 20 44 6 T S. spo rov err ucosus L MG 20314 T S. eru mpens L MG 20 50 7 T S. cinere os pinus L MG 20464 T .S. libani subsp. libani L MG 20 07 7 T S. du rhamen sis L MG 20501 T S. nigrescens L MG 19 33 2 T S. cel lostaticus L MG 20452 T S. praecox L MG 20 29 0 T .S. curacoi DSM 40107T S. alb os poreu s labi lomycet ic us L MG 20 40 0 T S. clavuligerus L MG 20477 T S. nitrosporeus L MG 20 04 4 T S. termit um L MG 20289 T .S. capoamus L MG 20 50 3 T S. ach romog en es achr omogene s L MG 20387 T S. caerul eus L MG 19 39 9 T S. yokosukanensis L MG 21040 T S. niv eus L MG 19 39 5 T S. longwoodensis L MG 20096 T S. spheroides L MG 19 39 2 T S. chi baensis L MG 20 45 6 T S. gra minof ac iens L MG 19892 T S. corc horus ii L MG 20 48 8 T S. longi sporus L MG 20053 T S. regensis L MG 20 30 0 T S. lienomyci ni L MG 20091 T S. gri seolus L MG 19 87 8 T S. lanatus L MG 19380 T S. fl av og rise us L MG 19 88 7 T S. gri seolosporeus L MG 19962 T .S. nigrifaciens L MG 20 04 8 T S. aureocirculatus L MG 21794 T S. fl av ovi ren s L MG 20 51 6 T S. lateritius L MG 19372 T S. oli vaceovi ridis L MG 19 32 4 T S. wedmorensis L MG 21050 T S. bacill ar is LMG 8585 T S. zaomyceticus L MG 19853 T S. gri seo bru nn eu s L MG 19 87 7 T S. showdoensis L MG 20298 T S. aureoversil is L MG 20 42 5 T S. omi yae nsi s L MG 20075 T S. recti ver ti cil latus L MG 20 29 2 T S. rose os porus L MG 20262 T S. pru ni col or L MG 19 31 1 T S. exfoliatus L MG 19307 T .S. yerevanensi s L MG 21 05 3 T .S. biki ni ensis L MG 19367 T S. cinereus L MG 21 31 0 T S. violaceorectus L MG 20281 T S. melanogenes L MG 20 05 6 T S. cinereoruber cinereoruber L MG 20462 T .S. netrops is LMG 5979 T .S. filamentosus L MG 20512 T S. hachi joensis L MG 19 92 8 T S. violescens L MG 20272 T S. olivoverticillatus L MG 20 05 8 T .S. biverti cill atus L MG 20 43 3 T S. na shv ill en sis L MG 20064 T Slincolnensis LMG 20068 T S. ha chi joen sis LMG 19 92 8 T S. olivoverticillatus L MG 20 05 8 T S. flavovariabilis LMG 19905 T .S. biverticillatus L MG 20 43 3 T S. viri dodi ast ati cus LMG 20279 T .S. lincol nens is L MG 20 06 8 T S. albogris eolus LMG 20395 T S. syringi um L MG 20 32 0 T .S. rimosus subsp. rimosus LMG 19352 T .S. roseov ert i cil lat us L MG 20 25 5 T S. albofaciens LMG 20394 T S. olivoreticuli cellulophilus L MG 20 09 7 T S. set ae LMG 20529 T S. anti bi ot icus L MG 20 41 2 T S. phosalacineus LMG 20102 T S. sal monis L MG 20 30 6 T S. toxytricini LMG 20269 T S. dis tall icus L MG 20 49 9 T S. globosus LMG 19896 T S. viri do f lav us L MG 20 27 7 T S. prasinopilosus LMG 19345 T S. oli vaceu s L MG 19 39 4 T S. hir sut us LMG 19927 T S. inusitatus L MG 19 95 5 T S. prasinus LMG 20259 T .S. var sov iensi s L MG 20 08 3 T S. roseov iri di s LMG 20266 T S. eu roci di cus L MG 20 50 9 T S. gardneri LMG 19876 T S. albireticuli L MG 20 39 3 T S. narbonensis LMG 20043 T S. ardus L MG 20 41 5 T S. glauc uspor us LMG 19907 T S. cinnamoneus lanosus L MG 20 47 1 T .S. violaceus LMG 20257 T S. cinnamoneus sparsus L MG 20 47 2 T .S. mass aspor eus LMG 19362 T .S. cinnamoneus subsp. albosporus L MG 20 46 9 T .S. roseov iolaceus LMG 8594 T .S. griseoverticillatus L MG 19 94 4 T S. luteogriseus LMG 20073 T S. sapporonensi s L MG 20 32 4 T S. paradoxus LMG 20523 T S. cinnamoneus cinnamoneus L MG 86 02 T .S. bellus LMG 19401 T S. alboverticillatus L MG 20 40 1 T S. af ghaniensi s LMG 20390 T S. blastmyceticus L MG 20 43 4 T S. gri seorubens LMG 19931 T S. gri seocarneus L MG 19 38 3 T S. coerul eofuscus LMG 20482 T S. no bo rit oe ns is L MG 19 33 7 T S. arenae LMG 20416 T S. mauvecolor L MG 20 10 0 T S. jant hi nus LMG 8591 T S. crystallinus L MG 20 49 0 T S. hawaiiensis LMG 5975 T S. gri seoruber L MG 19 32 5 T S. violarus LMG 20275 T S. xant hochromogenes L MG 19 36 6 T .S. iaky rus LMG 19942 T S. fl av of ungi ni L MG 21 79 9 T .S. purpuras cens LMG 20526 T S. fulvissimus L MG 19 31 0 T .S. coerul escens LMG 8590 T S. alboflavus L MG 21 03 8 T S. violaceochromogenes LMG 20271 T S. michiganensis L MG 20 04 2 T .S. azureus LMG 20430 T S. go bit ri cin i L MG 19 91 0 T S. mat ensi s LMG 20055 T S. lav endof oli ae L MG 19 93 5 T .S. violatus DSM 40209T S. chartreusis L MG 20 45 5 T S. coeruleorubidus LMG 20484 T S. lucensis L MG 20 06 5 T S. lev is LMG 20090 T S. gri seo au ran t iacus L MG 21 04 5 T .S. tuirus DSM 40505T S. sulfonofaciens L MG 20 32 5 T S. gri seof lav us LMG 19344 T S. olivochromogenes L MG 20 07 1 T S. gri seoincar natus LMG 19316 T S. mirabi lis L MG 20 07 6 T .S. longispororuber LMG 20082 T S. cinereoruber fructofermentans L MG 20 46 3 T S. var iabil is LMG 20270 T S. resi stomycificus L MG 21 79 7 T S. erythrogriseus LMG 19406 T S. ph ae ovi ridis L MG 20 06 1 T S. labedae LMG 19956 T S. phaeopurpureus L MG 20 05 1 T .S. gri seosporeus LMG 19947 T S. cyaneus L MG 20 49 4 T S. albus pat hoci di cus LMG 20406 T S. alboniger JCM 4309T .S. bluensi s LMG 20435 T S. pseudovenezuelae L MG 20 27 6 T S. althioticus LMG 20408 T S. longi sporofl av us L MG 19 34 7 T .S. collinus LMG 20486 T S. griseorubiginosus L MG 19 94 1 T S. poonensis LMG 19326 T S. ed ere nsi s L MG 20 50 4 T S. fl av eolus LMG 19328 T S. ciscaucas icus L MG 20 47 4 T S. lavenduligriseus LMG 19943 T .S. clavifer L MG 20 47 6 T S. muri nus LMG 10475 T S. bo tt ro pe nsi s L MG 20 43 7 T S. umbrinus LMG 20280 T S. niv eo rub er L MG 19 37 9 T S. misionensis LMG 20063 T S. canus L MG 19 32 9 T S. viridochromogenes LMG 20260 T S. violens L MG 20 30 3 T S. prasinos porus LMG 19346 T S. mashuens is L MG 86 03 T S. mut abil is LMG 20054 T S. kishiwadensis L MG 19 93 9 T S. rochei LMG 19313 T S. albospinus L MG 20 39 8 T S. geysiri ensis LMG 19893 T S. mobaraensis L MG 20 08 6 T S. pli cat us LMG 20288 T S. cuspidosporus L MG 20 49 2 T S. vinaceusdrappus LMG 20296 T S. diastaticus subsp. ardesiacus L MG 20 49 7 T S. gri seoluteus LMG 19356 T .S. enissocaesilis L MG 20 50 6 T S. recifensis LMG 20261 T .S. sioyaensi s L MG 20 53 1 T .S. tri color LMG 20328 T S. albosporeus s. albosporeus L MG 19 40 3 T .S. roseodiast at ic us LMG 20327 T S. auranti ac us L MG 19 35 8 T S. rameus LMG 20326 T S. tauri cus L MG 20 30 1 T S. sclerotialus LMG 20528 T S. heliomyci L MG 19 96 0 T .S. canes cens LMG 8567 T .S. acri myci ni L MG 21 79 8 T .S. felleus LMG 8569 T S. albaduncus L MG 20 39 2 T S. arabi cus LMG 20414 T S. albul us L MG 20 40 4 T .S. anandii LMG 20410 T S. macr osporus L MG 20 08 9 T S. vinac eus LMG 20533 T S. gelaticus L MG 19 37 6 T S. globisporus caucasicus LMG 19895 T S. ba mbergi en sis L MG 19 29 9 T S. odori fer LMG 8572 T S. caelest is L MG 20 44 1 T S. limosus LMG 8570 T S. fi mbri at us L MG 20 51 3 T .S. coelicolor LMG 8571 T S. echinat us L MG 59 72 T S. sampsonii LMG 8574 T S. thermoli neat us L MG 20 30 9 T S. albidoflavus LMG 21791 T S. no ursei L MG 59 82 T S. gri seus solvi faciens LMG 19952 T S. fl av idovi reus L MG 19 38 7 T S. violaceorubidus LMG 20319 T S. flavovariabilis L MG 19 90 5 T S. champavat ii LMG 20454 T S. viri dodi ast ati cus L MG 20 27 9 T S. eurythermus LMG 20510 T S. albogriseolus L MG 20 39 5 T S. intermedius LMG 19304 T S. tendae LMG 19314 T S. kasugaensi s LMG 19949 T S. eurythermus LMG 20510 T S. cel lul oflavus LMG 21796 T S. intermedius LMG 19304 T S. olivaceiscleroticus LMG 20081 T S. tendae LMG 19314 T S. rishiriensis LMG 20297 T S. gri seof usc us LMG 19885 T .S. parvulus LMG 21789 T S. carpati cus LMG 20448 T S. achromogenes rubradi ris LMG 20388 T .S. thermovi ol aceus s ubsp. apingens LMG 20307 T S. rutgersensis subsp. rutgersensis LMG 8568 T S. thermovi ol aceus s ubsp. t hermoviolaceusLMG 19359 T S. gougeroti i LMG 19888 T S. morookaensi s LMG 20074 T S. glomerat us LMG 19903 T S. brasiliensis LMG 20438 T S. diast at i cus subs p. di ast ati cus LMG 19322 T .S. ochraceiscleroticus LMG 19349 T S. chromofuscus LMG 19317 T S. glauc esc ens LMG 19330 T S. parvisporogenes LMG 20072 T S. cinerochromogenes LMG 20466 T S. alanosinicus LMG 20391 T S. coerul eoprunus LMG 20483 T S. olivoreticuli subsp. oilvoreticuli LMG 20050 T S. somali ensis LMG 21049 T S. abikoensis LMG 20386 T S. fradi ae LMG 19371 T S. ehimensis LMG 20505 T .S. roseofl avus LMG 20535 T S. minutiscleroticus LMG 20062 T .S. lut eov ert ic ill at us LMG 20045 T S. fl av iscl erot icus LMG 19886 T S. lil aci nus LMG 20059 T S. capillispiralis LMG 19909 T S. lavendulocolor LMG 19934 T S. fragilis LMG 19874 T S. hir oshimensis LMG 19924 T S. thermocopr ophil us LMG 19857 T S. kashmir ensis LMG 19937 T S. spi ral is LMG 20332 T .S. spectabilis LMG 5986 T t1 S. roseol il aci nus LMG 20264 T S. carpinensis LMG 19913 T S. fumanus LMG 19882 T S. auranti ogris eus LMG 19298 T S. thioluteus LMG 21037 T S. cal vus LMG 20442 T S. rangoonensis LMG 20295 T S. ast erospor us LMG 20419 T S. almquist ii LMG 21307 T S. virens LMG 20316 T S. aureorec tus LMG 19908 T S. gibsonii LMG 19912 T S. aureoverticillatus LMG 20426 T S. albus s ubsp. al bus LMG 20405 T S. mal achit of uscus LMG 20067 T S. fl occ ulus LMG 19889 T S. djakartensis LMG 21795 T S. amakus aensi s LMG 19350 T S. cel lul os ae LMG 19315 T S. acidiscabies LMG 19856 T S. thermodias tat icus LMG 20302 T .S. wer raens is LMG 21047 T S. spinoverrucosus LMG 20321 T .S. pseudogriseolus LMG 20252 T S. diastatochromogenes LMG 20498 T S. rubiginosus LMG 20268 T S. scabiei LMG 20323 T S. ganci di cus LMG 19898 T S. purpurogeneiscleroticus LMG 20331 T S. lomondensis LMG 20088 T S. cacaoi s. cacaoi LMG 19320 T S. indiaensis LMG 19961 T S. aminophilus LMG 19319 T S. gri seost ramineus LMG 19932 T S. thermocarboxydov orans LMG 19860 T S. luteosporeus LMG 20085 T .S. nogalat er LMG 19338 T S. aburaviens is LMG 19305 T S. graminearus LMG 19904 T S. kifunensis LMG 19957 T S. nodosus LMG 19340 T S. chrysomallus fumigatus LMG 21793 T S. gri seomyci ni LMG 19883 T S. atroaurantiacus LMG 20421 T .S. thermovulgari s LMG 19342 T S. thermogriseus LMG 20532 T S. azaticus LMG 20429 T S. pil osus LMG 20049 T S. purpeofuscus LMG 20283 T S. fl av ovi ridis LMG 19881 T S. indigoferus LMG 19930 T S. glauc us LMG 19902 T S. herbaricolor LMG 19929 T .S. fumigatiscleroticus DSM 43154T S. cinereorectus LMG 20461 T S. atrovirens LMG 20422 T .S. thermoalcalit olerans LMG 19858 T S. coc hleat us LMG 20478 T S. pact um LMG 19357 T S. paracochleat us LMG 20095 T S. viri dosporus LMG 20278 T .S. gri seopl anus LMG 19923 T S. ghanaensi s LMG 19894 T .S. psammoticus LMG 20525 T S. violaceusniger LMG 19336 T S. avellaneus LMG 20427 T .S. ori noci LMG 20079 T S. aureofaciens LMG 5968 T S. malaysiensis LMG 20099 T S. cinnabarinus LMG 20467 T .S. xanthocidicus LMG 19370 T S. cacaoi asoensis LMG 20440 T S. purpureus LMG 19368 T S. niger LMG 20101 T S. misakiensis LMG 19369 T S. naganis hi i LMG 21042 T S. albolongus LMG 20396 T S. melanosporofaciens LMG 20066 T S. cystar gineus LMG 20495 T S. rutgersensis castel arensi s LMG 20304 T S. sporoc liv at us LMG 20312 T S. pseudoechnisporeus LMG 21052 T S. anti mycoti cus LMG 20413 T S. ambofaciens LMG 20409 T S. hygroscopicus subsp. hygroscopicus LMG 19335 T S. sept at us LMG 8604 T S. endus LMG 19393 T .S. vitaminophil us LMG 21051 T S. sporoc inereus LMG 20311 T .S. hygros copicus os samycet ic us LMG 19951 T S. ruber LMG 20285 T S. fervens melrosporus LMG 19897 T S. neyagawaensi s LMG 20080 T S. roseiscleroticus LMG 20284 T S. torulosus LMG 20305 T .S. daghest anic us LMG 20496 T S. ipomoeae LMG 20520 T S. gri seovi ridis LMG 19321 T .S. vas tus LMG 21043 T 'S. cyaneogriseus' LMG 19381 T S. phaeof ac iens LMG 20070 T S. xant holit ic us LMG 19402 T S. peucet icus LMG 20084 T .S. aculeolatus LMG 19906 T S. kurssanovii LMG 19933 T S. eryt hraeus LMG 20508 T .S. var iegat us LMG 20315 T S. armeniacus LMG 20418 T S. kasugaensi s LMG 19949 T S. cel lul of lavus LMG 21796 T S. oli vacei scl eroti cus LMG 20081 T Sihii i LMG 20297T

Curriculum vitae

1. Personalia

Benjamin LANOOT Born on 2 September 1974, Geraardsbergen

2. Education

Universiteit Gent, licentiate in the Biotechnology Thesis: “Identificatie van pathogene en niet pathogene bacteriën aanwezig op rijstzaad”

3. Professional employment

1998-present

Universiteit Gent, PhD student. Involved in the following research projects:

• OSTC project (ref BL/02/C10): Identification and classification of actinomycetes, specifically bioactive Streptomyces strains isolated from Chinese soils (1998- 2002)

• OSTC project (ref BL/02/C10): Molecular assessment of diversity of bioactive Streptomyces isolates from China and use of bioinformatic tools for enhanced data exchange and processing (2002-2004)

4. Publications (peer-reviewed)

• Lanoot B, Vancanneyt M, Cleenwerck I, Wang L, Li W, Liu Z & Swings J. (2002). The search for synonyms among streptomycetes by using SDS-PAGE of whole-cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violaceus. Int J Syst Evol Microbiol 52(3), 823-829.

• Lanoot B, Vancanneyt M, Dawyndt P, Cnockaert MC, Zhang J, Huang Y, Liu Z & Swings J. (2004). BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol 27(1), 84- 92.

• Lanoot B, Vancanneyt M, Van Schoor A, Liu Z & Swings J. (2005a). Reclassification of Streptomyces nigrifaciens as a later synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as later synonyms of Streptomyces anulatus; Streptomyces chibaensis as later synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as later synonym of S. minutiscleroticus; Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as later synonyms of Streptomyces microflavus. Int J Syst Evol Microbiol, in press.

• Lanoot B, Vancanneyt M, Hoste B, Vandemeulebroecke K, Cnockaert MC, Dawyndt P, Liu Z & Swings J. Phylogenetic grouping of streptomycetes using 16S- ITS RFLP fingerprinting. Submitted to Research in Microbiology.

• Lanoot B, Vancanneyt M , Hoste B , Cnockaert MC, Piecq M*, Gosselé F*, Swings J. (2005b). Phenotypic and genotypic characterisation of variants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae. System Appl Microbiol, in press.

* in collaboration with

• Li W, Lanoot B, Zhang Y, Vancanneyt M, Swings J & Liu Z. (2002). Streptomyces scopiformis sp. nov., a novel streptomycete with fastigiate spore chains. Int J Syst Evol Microbiol 52(5), 1629-1633.

• Huang Y, Li W, Wang L, Lanoot B, Vancanneyt M, Rodriguez Z, Liu Z & Swings J. (2004). Streptomyces glauciniger sp. nov., a novel mesophilic streptomycete isolated from soil in south China. Int J Syst Evol Microbiol 54, 2085- 2089.

• He L, Wang L, Li W, Liu Z, Lanoot B, Vancanneyt M, Swings J & Huang Y. Streptomyces jietaisiensis sp. nov., isolated from soil in North China. Submitted to Int J Syst Evol Microbiol.

• Cottyn B, Regalado E, Lanoot B, De Cleene M, Mew TW & Swings J. (2001). Bacterial populations associated with rice seed in the tropical environment. Phytopathology 91, 282-292.

5. Oral presentations

• Genotyping as a means towards a more transparent Streptomyces taxonomy (2003). 13th Int Symposium on the Biology of actinomycetes, 1-5 December, Melbourne, Australië.

• Improved classification of streptomycetes for an enhanced search for novel bioactive compounds (2004). Belgo-Chinese workshop on bioactive compounds from actinomycetes, Universiteit Gent, 17 September 2004, Gent, België.

6. Poster presentations

• Lanoot B, Vancanneyt M, Cleenwerck I, Wang L, Li W, Liu Z & Swings J. (2000). Searching for synonymous species among streptomycetes using a polyphasic approach. 3rd symposium of the Belgian Society for Microbiology (BSM), 8-9 June, Brussels, Belgium

• Lanoot B, Vancanneyt M, Cleenwerck I, Wang L, Li W, Liu Z & Swings J. (2001). Searching for synonymous species among streptomycetes using a polyphasic approach. 12th Int Symposium on the Biology of actinomycetes, 5-9 August, Vancouver, Canada.

• Lanoot B, Vancanneyt M, Swings J., Liu Z, Li W & Zhang J. (2002). Evaluation of the molecular techniques RFLP of 16S, RFLP of 16S-ITS, tDNA-PCR and rep- PCR within Streptomyces systematics. 10th International Congress of Bacteriology and Applied Microbiology, 27 July-1 August, Paris, France.

• Lanoot B, Vancanneyt M, Li W, Zhang J, Liu Z & Swings J. (2003). Evaluation of the molecular techniques RFLP of 16S, RFLP of 16S-ITS, tDNA-PCR and rep- PCR within Streptomyces systematics. Doctoraatssymposium, Faculty of Sciences, Universiteit Gent, 30 April, ICC Gent, Belgium.

• Lanoot B, Vancanneyt M, Dawyndt P, Cnockaert MC, Huang Y, Liu Z & Swings J. (2003). Applicability of BOX-PCR fingerprinting to differentiate Streptomyces species. 1st FEMS Congress of European Microbiologists, 29 June-3 July, Ljubljana, Slovenia.

• Lanoot B, Vancanneyt M, Gosselé F, Piecq M, Cnockaert MC & Swings J. (2003). Intra-species genotypic relationships among bioactive Streptomyces virginiae strains and mutants of the virginiamycin producing strain 899. 13th Int Symposium on the Biology of actinomycetes, 1-5 December, Melbourne, Australië.

• Huang Y, Li W, Wang L, Lanoot B, Vancanneyt M, Rodriguez Z, Liu Z, Swings J & Goodfellow M. (2003). A new Streptomyces species from soil in South China. 13th Int Symposium on the Biology of actinomycetes, 1-5 December, Melbourne, Australië.

• Lanoot B, Vancanneyt M, Cnockaert MC, Liu Z & Swings J (2004). Genotyping as a means towards a more transparent Streptomyces taxonomy. 10th International Congress of Culture Collections, 10-15 October, Tsukuba, Japan.