Polyphasic Taxonomy of Thermophilic Actinomycetes Thesis submitted in accordance with the requirement of the University of Newcastleupon Tyne, U. K. for the Degreeof Doctor of Philosophy by Bongcheol Kim (B. Sc., Seoul National University, Seoul, Korea) Department of Agricultural and Environmental Science, University of Newcastle upon Tyne, U. K. February1999 NEWCASTLE-UNIVERSITY-LIBRARY -------------------------- 098 14280 5 --------- l-ý'ýý7 ýý ý__I Q-1çovJ ýý ýý ýýý ý ý'ý ý"ýý -4/ t-ýt? Dedicated to my loving father and mother Acknowledgements I would like to express my sincere gratitude to my supervisor Professor Michael Goodfellow for his guidance, patience and advice during the preparation of the thesis and, most of all, for his care and concern about my life in Newcastle. I am also grateful to Professor David E. Minnikin for his sound advice on the chemotaxonomic work and to Dr. Alan C. Ward for his time and help with the computing. I am also indebted to Professor Yung Chi] Hah for introducing me to microbial systematics and for his interest in my work. Special thanks go to Dr. Nevzat Sahin whose project provided the basis for much of my research work. I am also very grateful to Drs. Seung Bum Kim and Jongsik Chun for their invaluableadvice on my project. I am grateful to Professor Marian Mordarski and Dr. Jolanta Zakrzewska- Czerwinska for arranging my stay in Wroclaw, Poland; to Maria Guzik and Bashia Lis for their wonderful assistance with the DNA: DNA hybridisation experiments and to Dr. Stefan Rowinski for his friendship and his `passionate' tours of Poland. I am also indebted to Professor Reiner M. Kroppenstedt for providing cultures and to Professor Erko Stackebrandt for kindly comparing my 16S rRNA sequences with the Streptomycete 16S rRNA sequence databaseheld at DSMZ. I would also like to thank my friends, Amira M. Al-Tai, Noelle Brennan, Ann Brown, Rose Brown, Russell Davenport, Elayne V. Ferguson, Kamil Isik, Seong-Gyun Kang, Sam-Bong Kim, Jeongjin Lee, Luis Maldonado, Gilson P. Manfio, Hye-Sook Mheen, Sun-Woo Nam, Gail Payne, Ehsan Rashidian, Jae-Young Rho, Langkah Sembiring, Fiona Stainsby and Ampaitip Sukhoom who shared the `tough' life in the laboratory. I gratefully acknowledge receipt of an Overseas Research Student Award. Finally, to my wife, SuKyung, for her incredible and unconditional moral support and love. Publications Part of the work describedin this thesisis alsoincluded in the following publications: Kim, B., Sahin, N., Minnikin, D. E., Zakrzewska-Czerwifiska, J., Mordarski, M. & Goodfellow, M. (1999). Classification of thermophilic streptomycetesincluding the descriptionof Streptomycesthe rmoalcalitolerans. International Journal of Systematic Bacteriology 49,7-17. Kim, B, Al-Tai, A. M., Kim, S. B., Somasundaram, P. & Goodfellow, M. (1999) Streptomyces thermocoprophilus sp. nov., a cellulase-free endo-xylanase producing streptomycete. International Journal of Systematic Bacteriology (submitted). Kim, B., Sahin, N., Zakrzewska-Czerwinska, J., Mordarski, M. & Goodfellow, M. (1999).Amycolatopsis eurythermus sp. nov., a thermophilic memberof the genus Amycolatopsis. International Journal of Systematic Bacteriology (submitted). Poster presentation Kim, B., Sahin, N. & Goodfellow, M. (1997). Taxonomic studies on novel thermophilic streptomycetes. 10th International Symposium on Biology of Actinomycetes. Beijing, China. Abstract Molecular systematicmethods were applied in a series of studies designed to resolve the taxonomic relationships of thermophilic actinomycetes known to be difficult to classify using standard taxonomic procedures.The test strains included representativesof clusters defined in an extensivenumerical phenetic survey of thermophilic streptomycetesand twelve marker strains. The resultantgenotypic data together with the results of correspondingphenotypic studies were used to highlight novel taxa and to improve the circumscription of validly describedspecies. The most comprehensive study was undertaken to clarify relationships within and between representative alkalitolerant, thermophilic and neutrophilic, thermophilic streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA: DNA relatedness studies, supported the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and Streptomyces thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of the priority, Streptomyces thermonitrificans is a later subjective synonym of Streptomyces thermovulgaris. Similarly, eight out of eleven representative alkalitolerant, thermophilic isolates and three out of sixteen representative neutrophilic, thermophilic isolates had a combination of properties consistent with their classification as Streptomyces thermovulgaris. One of the remaining alkalitolerant, thermophilic isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic, thermophilic isolate, Streptomyces strain NAR85, was identified as Streptomyces thermodiastaticus. Four other neutrophilic thermophilic isolates assigned to a numerical phenetic cluster and a thermophilic isolates from poultry faeces were also considered to warrant species status; the names Streptomyces eurythermophilus sp. nov. and Streptomyces thermocoprophilus sp. nov. are proposed to accommodate these strains. It was also concluded that additional comparative taxonomic studies are required to clarify the relationships between additional thermophilic streptomycete strains included in the present investigation. A corresponding polyphasic approach was used to clarify the taxonomy of six thermophilic isolates provisionally assigned to either the genera Amycolatopsis or Excellospora. Two of the isolates, strain NT202 and NT303, had properties consistent with their classification in the genus Amycolatopsis. However, the genotypic and phenotypic data also showed that these strains formed a is new centre of taxonomic variation for which the name Amycolatopsis eurythermus sp. nov. taxonomic proposed. Similarly, the four remaining strains formed two new centre of variation TA114 be designated within the genus Excellospora. It is proposed that isolates TA113 and Excellospora alcalithermophilus sp. nov. Similarly, the name Excellospora thermoalcalitolerans sp. description is also given for the genus nov. is proposed for strains TA86 and TA111. An emended Excellospora. Contents Acknowledgements Publications Abstract Chapter I. Current Trends in Bacterial Systematics 1. Polyphasic taxonomy 1 2. Suprageneric classification: Nucleic acid sequencing 5 (a) Background 5 (b) 16S rDNA sequence analysis 9 (c) Phylogentictrees 11 (d) Supragenericclassification of actinomycetes 17 3. Classificationat genusand specieslevels 25 (a) Chemotaxonomy 25 (b) Numerical taxonomy 37 (c) Molecular systematics 38 4. Classification at and below the species level 44 (a) Nucleic acid fingerprinting 44 (b) Chemical fingerprinting 51 Chapter II. Classification of Novel Thermophilic Amycolatopsis and Excellospora Strains Introduction 1. Thermophiles 54 2. Thermophilic actinomycetes 60 3. Presentstudy 92 Materials and Methods 97 .1. Strains and cultivation 97 2. Acquisition of phenotypicdata 3. Chemotaxonomy 100 (a) Analysis of menaquinones 100 (b) Analysis of polar lipids 101 (c) DNA basecomposition 103 4. Sequencingand analysisof 16SrDNA 103 (a) Small scalepreparation of genomicDNA 104 (b) PCR amplification of 16SrDNA 109 (c) Dye-DeoxyTMterminator Taq cycle sequencingof 16SrDNA 113 (d) Analysis of 16S rRNA sequence data 114 5. DNA: DNA hybridisation 121 (a) Large scale isolation of DNA 121 (b) Loading DNA on nitrocellulose filters 124 (c) Preparation of labelled reference chromosomal DNA 125 (d) Pre-hybridisation 126 (e) Hybridisation 126 (f) Scintillation counting 127 Results and Discussion 1. Classification of thermophilic Amycolatopsis strains 130 2. Classificationof alkalitolerant,thermophilic excellosporae 144 Chapter HE Systematics of Thermophilic Streptomycetes Introduction 1. Circumscription of the genus Streptomyces 167 2. Thermophilic streptomycetes 171 3. Streptomycetesystematics: the early years 173 4. Application of modern taxonomic methods 176 (a) Numerical taxonomy 176 186 (b) Molecular systematics (c) Chemotaxonomy 201 207 5. Polyphasic taxonomy Materials and Methods 210 1. Strainsand cultivation 210 2. Morphology andpigmentation 217 3. Degradationand nutritional tests 4. Menaquinone and polar lipid analyses 217 5. DNA base composition 217 6. Sequencing and analysis of 16S rRNA 217 7. DNA: DNA relatedness studies 220 8. Molecular fingerprinting: Ribotyping 221 Results 1. Phylogenetic analyses based on 16S rDNA sequences 229 (a) Reference strains 229 (b) Thermophilic isolates 234 2. DNA: DNA relatednessstudy 243 3. Ribotype patterns 245 4. Chemotaxonomic markers 248 5. Phenotypicproperties 248 Discussion 261 References 292 Appendix 1. World Wide Web sites 348 Appendix 2. Secondary structuresof bacterial 16S ribosomal RNA 352 Appendix 3. Formulae used for calculating oligonucleotide primer concentrations 356 Appendix 4. Culture media and reagents 358 CHAPTER 1 BACTERIALSYSTEMATICS 1 ChapterI: Current Trends in Bacterial Systematics 1. Polyphasic taxonomy Bacterial classification and identification are data