8278 International Journal ofSystematic and Evolutionary Microbiology (2000), 50, 331-336 Printed in Great Britain

Phylogenetic analysis of Saccharothrix and related taxa: proposal for Actinosynnemataceae fam. nov.

David P. Labeda' and Reiner M. Kroppenstedt2

Author for correspondence: David P. Labeda. Tel: + I 3096816397. Fax: + 13096816672. e-mail: dlabeda:(rsunca.ncaur.usda.gov

1 Microbial Properties Partial sequences for 165 rONA were determined for strains of the genus Research Unit, National Saccharothrix, including most described , as well as strains of the Center for Agricultural Utilization Research, described species of the related genera , and Agricultural Research . These were aligned with published sequences for other Service, US Department of species of these genera, as well as those of the genera'', Agriculture, 1815 N. University Street, Peoria, ,Asiosporangium" , Kutzneria, and representative IL61604, USA taxa from other actinomycete families. Phylogenetic analysis of the sequence

2 DSMZ-Germany Collection data showed that species of the genera Actinokineospora, Actinosynnema, of Microorganisms and Lentzea and Saccharothrix are members of the same clade, and distinct from Cell Cultures, the . It is proposed that a new family be created within the Braunschweig, Germany class for these genera, to be called the Actinosynnemataceae on the basis of the oldest described genus within this family, Actinosynnema Hasegawa et al. The chemotaxonomic properties of all the genera to be placed within the new family have similar cell wall type (type III), whole-cell sugars (generally galactose although mannose and rhamnose may be present),

phospholipid type (Pit) and menaquinones [MK-9(H 4 ) predominant].

Keywords: Saccharothrix. actinomycetes. Actinosynnemataceae fam. nov.

INTRODUCTION (Stackebrandt et al., 1997), members of the genus Saccharothrix are again loosely grouped with the The genus Saccharothrix was described (Labeda et al., Pseudonocardiaceae. Yassin et al. (1995) noted the 1984) to accommodate actinomycete strains that mor­ close phylogenetic relationship of their new genus phologically resemble the genus Nocardiopsis, but that Lentzea to members of the genera Actinosynnellla. can be chemotaxonomically differentiated from mem­ Kutzneria and Saccharothrix. In order to clarify the bers of this genus based on type III cell wall chemistry phylogenetic position of this genus, as well as to study (Illeso-diaminopimelic acid, lIleso-DAP), rhamnose its phylogenetic continuity, 16S rDNA sequence data and galactose as diagnostic whole-cell sugars, a type were determined for representative type strains of PH phospholipid pattern (phosphatidylethanolamine species of Saccharothrix and allied genera selected as key diagnostic phospholipid), and presence of MK­ from the ARS Culture Collection.

9 (H4) as the predominant menaquinone. The actual phylogenetic position of this genus has not been METHODS certain, although it was suggested by Warwick et al. (1994), on the basis of a phylogenetic study of 16S Strains, cultivation and maintenance. All strains included in rRNA sequences, that this genus should be placed this study are held in the ARS Culture Collection (NRRL), phylogenetically with the Pseudonocardiaceae. In the National Center for Agricultural Utilization Research. str~ins recent proposal for the hierarchic classification of Peoria. IL. USA. The selected for this are shown in actinomycete taxa in the new class Actinobacteria Table 1. Primary storage ofstrains is as lyophilized ampules of mycelial and spore suspensions in sterile beef serum held at 4 dc. Working stock cultures were maintained on slants of ATCC Medium~No. 172 (Cote et al., 1984) and stored at Abbreviation: DAP, diaminopimelic acid. 4°C until needed. Biomass for extraction of DNA was The GenBank accession numbers forthe 165 rONA sequences determined in grown as 7 d streak cultures on ATCC Medium 172 agar this paper are AF114797-AFl1481 5. plates.

01165 331 D. P. Labeda and R. M. Kroppenstedt

Table 1. Strains for which 16S rONA sequence data were determined in this study

Strain GenBank no.

Aclinokineo.ljJora diospyrosa NRRL B-240471' (= IFa 15665'1') AFI14797 AClinokineospora globicalena NRRL B-240481' (= IFa 156641') AFI14798 AClinokineospora inagensis NRRL B-2405QT (= IFa 156631') AFI14799 AClinokineospora riparia NRRL B-1 64321' AFI14802 AClino:>ynnema prelioslllll subsp. prelioslllll NRRL B-1606QT AFI14800 KlIlzneria kofilensis NRRL B-24061 l' (= JCM 3151'1") AFI14801 Saccharolhrix aerocolonigenes NRRL B-32981' AFl14804 Saccharolhrix allslraliensis NRRL 112391' AFl14803 Saccharolhrix coerllleojilsca NRRL B-16115"'" (= ATCC 351081' = DSM 436791') AFl14805 SaccharoillrLy cryophilis NRRL B-162381' AFI14806 Saccharolhrix espanaensis NRRL 157641' AFI14807 Saccharolhrixflava NRRL B-1613j1' (= ATCC 295331' = DSM 438851') AFl14808 Saccharoillrix longispora NRRL B-161161' (= ATCC 351091' = DSM 437491') AFl14809 Saccharolhrix syringae NRRL B-16468 1' (= DSM 438861') AFl14812 Saccharolhrix lexasensis NRRL B-16107 AFl14815 Saccharolhrix lexasensis NRRL B-161341' AFI14814 Saccharolhrix waywayandensis NRRL B-161591' AFI14813 Saccharolhrix species NRRL B-16108 AFI14810 SaccharOlhrix species NRRL B-16133 AFI14811

Chemotaxonomic analysis. The chemotaxonomic profile of CAAGGCCC]. Sequencing reaction mixtures were purified Lenlzea albidocapi/lala was determined using previously as recommended by Applied Biosystems and were electro­ described methods (Grund & KroppenstedL 1989) for phoresed on a 6 % (wIv) polyacrylamide sequencing gel for menaquinones, fatty acids and whole-cell sugars. 9 h using either a 373A or 377 model automated DNA sequence'i- (Applied Biosystems). DNA extraction. Genomic DNA was isolated and purified by a modification ofthe procedure of Rainey el al. (1996). Cells Phylogenetic analysis. The 16S rDNA sequences obtained in scraped from solid growth media (I-2100pfuls) were placed this study were manually aligned \vith actinomycete ref­ in a 1·5 ml microfuge tube with a small amount of 0·1 mm erence sequences obtained from the Ribosomal Database zirconia/silica beads (Biospec Products) and were ground Project (Maidak el al., 1994) and GenBank. Many of the with a conical disposable pestle (Kontes Glass Company). reference sequences obtained from the databases were partial The homogenate was suspended in 400 ~d saline-EDTA sequences and thus the data set used for analysis consisted of buffer (150 mM NaCL 10 mM EDTA: pH 8'0), 1O)l1 1% the sequences generated in this study and actinomycete (wIv) proteinase K and 10)l1 25 % (v.rjv) SDS was added, reference sequences contained information for 1384 nucleo­ mixed and incubated at 55-60°C for 10 min. The resulting tide positions. The programs contained in the PHYLIP preparation \vas extracted sequentially with an equal volume package of Felsenstein (1993) were used to calculate evol­ of phenoL and an equal volume of chloroform. DNA \vas utionary distances by the method of Kimura (1980), and recovered from the aqueous phase by using a Prep-A-Gene linkages by the neighbour-joining method of Saitou & Nei kit (Bio-Rad). The purified DNA was eluted from the (1987). The topographies of the trees resulting from these binding matrix in 30 )ll sterile distilled water. analyses were evaluated by bootstrap analysis of the data with 100 resamplings. The sequences were also subjected to Amplification and direct sequencing of the 165 rRNA gene. likelihood analysis using fastDNAml (Olsen el al., 1994) and The 16S rDNA \vas amplified by the PCR according to the PAUP 3.1 (Swofford, 1993) was used for parsimony analysis. method of Rainey el al. (1996) using the primers 27f Evolutionary trees \vere displayed and printed using version (GAGTTTGATCCTGGCTCAG) and 15251' (AGAAA­ 1.5 of the TREEVIEW program of Page (1996). GGAGGTGATCCAGCC). PCR products were purified and concentrated using a Gene-Clean II kit (Bio 10 I) and Nucleotide sequence accession numbers. The 16S rDNA \vere eluted into 30 JlI sterile distilled water. The sequencing sequences determined in this study have been deposited in reactions were performed with a PRISM Ready-Reaction the GenBank database under the accession numbers shown DyeDeoxy terminator cycle sequencing kit by using Ampli­ in Table 1. The accession numbers for the sequences of the Taq FS (Applied Biosystems) and a Perkin-Elmer Cetus strains used as representatives of the main actinomycete model 2400 thermal cycler according to the protocol and groups are as follows: 'Aclinoalloleichus cyanogriseus' IFO thermal profile recommended by Applied Biosystems. The 14455, AB006178: AClinomadura kijaniala DSM 437641' sequencing primers used \vere: 343r (CTGCTGCCTCCC­ (1' = type strain), X97890; AClinomadura madurae DSM GTA), 357f (TACGGGAGGCAGCAG), 519r [G(T/A)­ 430671', X97889: AClinoplanes phi/ippinensis DSM 430191', ATTACCGCGGC(T/G)GCTG], 536f[CAGC(C/A)GCC­ X93187: AClinosvnnema mirum DSM 438271', X84447: GCGGTAAT(T/A)C], 803f (ATTAGATACCCTGGTA­ medilerranei ATCC 136851' (= DSM 433041' G), 907r (CCGTCAATTCATTTGAGTTT), 1114f (GCA­ = IFO 134151' = IMET 765P), X76957: Amycolalopsis ACGAGCGCAACCC) and 13851' [CGGTGTGT(A/G)- melhanolica NCIB 119461', X54274; Amycolalopsis orienlalis

332 International Journal ofSystematic and Evolutionary Microbiology 50 Actinosynnelllataceae fam. nov.

T T DSM 40040 (= NCIMB 9378 ), X76958 Arthrobacter DISCUSSION T globi(orlllis DSM 20124 (= ATCC 80 OT = NCIMB 1 Intrageneric structure of the genus Saccharothrix 8907 ) M23411: 'Asiosporangillln albidull1 IFO 16102, AB006176: AtopobiulI1 lI1inutum NCFB 275F( = DSM T T T The intrageneric relationship among the species of 20586 = ATCC 33267 = VPI 9428 ), X67148: Bi(ido­ T Saccharothrix in this study based on 16S rDNA bacteriulII bifidulII ATCC 29521 T (= DSM 20456 ), M38018: T sequences, as seen in Fig. L is somewhat surprising Dactylospo,:angiulI1 auramiaculII DSM 43157 , X93191: T KibdelosporangiulII aridum subsp. aridulII ATCC 39323 , since the genus appears phylogenetically hetero­ X53191: Kutzneria viridogrisea DSM 43850T (= ATCC geneous. On the basis ofthe phylogenetic relationships T 2524Yj, X70429: Lentzea albidocapillata DSM 44073 , observed in this study, Saccharothrix sensu stricto X84321: lv!icrobacterium lacticum IFO 14135T (= NCIMB would appear to consist of Saccharothrix australiensis T T T 1 T 8540 = ATCC 8180 = DSM 20427 = NCFB 747 ) NRRL 11239 (the type species), Saccharothrix coeru­ T T D21343: Microlunatus phosphovorus, JCM 9379 , D26169: leojilsca NRRL B-16115 , Saccharothrix espanaensis T T iv!icrolllonospora chalcea DSM 43026 , X92594: Micro­ , Saccharothrix longispora T NRRL 15764 NRRL B­ tetraspora glauca DSM 43311 , X97891: Nonomuria salmo­ T T 16116 , Saccharothrix mutabilis subsp. capreolus DSM nea DSM 43678 • X97892: Mrcobacteriull1 tuberculosis. T T 40225 , Saccharothrix mutabilis subsp. mutabilis DSM NCTC 7416 • X58890: Nocardic; asteroides ATCC 19247"r. T T T 43853 , Saccharothrix syringae NRRL B-16468 and Z36934: Nocardioides albus DSM 43109 (= ATCC 27980), T X53211: Nocardiopsis dassonvillei DSM 4311F, X97886: Saccharothrix texasensis NRRL B-16134 • as well as Propionibacteriull1 ji-eudenreichii subsp. ji-eudenreichii DSM the unnamed Saccharothrix strains NRRL B-16108 T T T 20271 T (= ATCC 6207 = NCTC 10470 = NCIB 5959 ), and NRRL B-16133. The strains of Saccharothrix X53217: alni VKM AC901 T. X76954: texasensis sequenced, NRRL B-16107 and NRRL B­ T T Pseudonocardia halophobica DSM 43089 , Z14111: Pseudo­ 16134 , were previously shown to have 89 % DNA T T nocardia petroleophila ATCC 15777 (= DSM 43193 ), relatedness and not surprisingly had 100 % agreement X80596: SaccharolIIonospora azurea IFO 1465 F, Z38017: in their Saccharothrix waYlvay­ T 16S rDNA sequences. . SaccharolIIonospora caesia' INMI 19125 • X76960: T T andensis NRRL B-16159 appears to be phylo­ Saccharomonospora cyanea IFO 14841 , Z38018: Sacclzaro­ T genetically more closely related to . Asiosporangium monospora glauca DSM 43769 , Z38003: Saccharolllono­ albidum' IFO 16102 and Lent:::ea albidocapillatus DSM .spora viridis Goodfellow' SB-33, Z38005: T T 44073 than the other Saccharothrix species, while gregorii NCIMB 12823 , X76962: SaccharopolY.lpora T T T hirsuta ATCC 27875 (=NCIB 11079 ), M20388: Sac­ Saccharothrix aerocolonigenes NRRL B-3298 , Sac­ T charothrix mutabilis subsp. capreolus DSM 40225 , clzarothrix .tIara NRRL B-16131 T and Saccharothrix X76965: Saccharothrix lIIutabilis subsp. mlltabilis DSM cryophilis NRRL B-16238 T do not seem to belong to T 43853 , X76966: Sporichthya polymorpha DSM 46113, Saccharothrix or any other described actinomycete T X72377: Streptoal!oteichus hindustanus IFO 15115 • genus. The latter strain, however, appears be more T D85497: Streptomyces all1bo(aciens ATCC 23877 , M27245: related to the sporangiate genera' Actinoal!oteichus', Streptomyces griseus KCTC 908QT, X61478: Streptosporan­ Streptoal!oteichus or Kut:::neria. The 16S rDNA se­ giull1longisporum DSM 4318QT, X89944: Streptosporangium quence data do explain anomalies previously observed roseull1 DSM 43021 T, X89947: Thermocrispull1 agreste DSM T T in DNA relatedness studies ofthe genus Saccharothrix 44070 , X79183: Thermocri.\pum lI1unicipale DSM 44069 , T (Labeda & Lechevalier, 1989: Labeda & Lyons, 1989) X79184: Therll1omonspora curvata DSM 43183 , X97893. T T where strains NRRL B-3298 • NRRL B-16159 and NRRL B-16238T exhibited extremely low DNA re­ RESULTS latedness ( < 20 %) to any of the other Saccharothrix Phylogenetic analyses strains. DNA relatedness values below 20 % observed between strains could be interpreted as indicating that Almost complete 16S rDNA sequences were deter­ they belong to different genera, but in the case of the mined for type strains of the genera Actinokineospora, aforementioned Saccharothrix strains. the chemotaxo­ Kut:::neria and Saccharothrix for which these data had nomic and morphological properties observed placed not been previously reported. In addition, similar data them within the description for the genus Saccharo­ were determined for Saccharothrix australiensis T tll/·ix. NRRL 11239 , Saccharothrix coeruleofilsca NRRL B-16115T and Saccharothrix longispOl:a NRRL B­ T 16116 , for which sequence information had been Phylogenetic relationship of the genus Saccharothrix previously reported (Warwick et al., 1994) as well as and related genera with the other actinomycetes Saccharothrix species NRRL B-16108 and NRRL B­ Although it has been suggested in the past (Embley 16133. et at., 1988: Warwick et al., 1994) that the genus The phylogenetic dendrogram shown in Fig. 1 was Saccharothrix should be placed in the family Pseudo­ derived from evolutionary distances by the neighbour­ , the phylogenetic position of the genus joining method. A total of 1384 nucleotides between Saccharothrix and the allied genera Actinokineospora, positions 99 and 1440 (Escherichia coli numbering; Actinosyllnema and Lent:::ea relative to the genera Brosius et al., 1978) were used for the analysis. representative of other actinomycete families was Phylogenetic analyses using maximum-likelihood and consistent and distinct from the Pseudonocardiaceae. parsimony methods generated very similar results and as can be seen in Fig. 1, regardless of phylogenetic are not sho\vn. analysis methods used. The relationship of this clade

International Journal of Systematic and Evolutionary Microbiology 50 333 D. P. Labeda and R. M. Kroppenstedt

Saccharothrix mutabilis subsp. mutabilis DSM 43853T Saccharothrix mutabilis subsp. capreolus DSM 40225T Saccharothrix espanaensis NRRL 15754' Saccharothrix species NRRL 8-16133 Saccharothrix syringae NRRL 8-16468T Saccharothrix coeruleofusca NRRL 8-16115' Saccharothrix australiensis NRRL 11239' Saccharothrix lexasensis NRRL 8-16107

Saccharothrix texasensis NRRL 8-16134 T Saccharothrix species NRRL 8-16108 Saccharothrix longispora NRRL 8-16116' Actinosynnema pretiosum subsp. pretiosum NRRL 8-16060T Actinosynnema mirum DSM 43827' Saccharothrix aerocolonigenes NRRL 8-3298' Sac:ch"rot.hrix nava NRRL 8-16131' Sa,och'arc,thrix waywayandensis NRRL 8-16159' 'AsiOSjJOn3ng·ium albidum 'IFO 16102 '----- Lentzea a/bicapifiata DSM 44073T R?,---- Actinokineospora inagensis NRRL 8-24050T Actinokineosporo riparia NRRL 8-16432T Actinokineospora globicatena NRRL 8-24048' Actinokineosporo diospyrosa NRRL 8-24047 T ---- Kutzneria kofuensis NRRL 8-24061 T ,------Kutzneria viridogrisea DSM 43850' c------Saccharothrix cryophilis NRRL 8-16238' '------Straptoalloteichus hindustanus IFO 15115' 'Actinoalloteichus cyanogriseus'IFO 14455 Saccharomonospora azuraa IFO 14651'

'Saccharomonospora caesia' INMI 19125 T Saccharomonospora cyanea IFO 14641' ,----- Saccharomonospora viridis Goodfellow S8-33 Saccharomonospora g/auca DSM 43769' ,-_-'!.QQ,-_-{======-; Thermocrispum agreste DSM 44070 T - Thermocrispum municipa/e 44069T T L_....!l~_{======~;::: Saccharopo/yspora hirsuta ATCC 27875 Saccharopo/yspora gregorii NCI M8 12823' --'!.QQ--{======- Amyco/atopsis orientans DSM 40040 T r Amycolatopsis mediterranei ATCC 13685' '------Amyco/atopsis methano/ica NCI8 11946 T c----- Pseudonocardia petroleophi/a ATCC 15777 T ~--- PseLfdol~ocaro7a a/niVKM AC901 T '------PseLldoJ~ocard7a ha/ophobica DSM 43089 T '------Kjbde/osporangium aridum subsp. aridum ATCC 39323'

L-~~-c:======-::=;-::;:::~~Mycobacterium tuberculosis NCTC 7416' Nocardia asteroides ATCC 19247' c------Dactylosporangjum aurantiacum DSM 43157 T '------Micromonospora chalcea DSM 43026' '------Actinoplanes philippinensis DSM 43019'

r------Propionibacterium freudenreichii subsp. freudenreichii OSM 2027 T '------Micro/unatus phosphovorus JCM 9379T '------Nocardioides a/bus DSM 43109T Streptosporangium /ongisporum DSM 43180T Streptosporangium roseum DSM 43021' '------Nonomuria sa/monea DSM 43678 T '------Microtetraspora gfauca DSM 43311' r----- Actinomadura madurae DSM 43067' '------Actinomadura kijaniata DSM 43764' '------Thermomonospora cutvala OSM 43183 T '------Nocardiopsis dassonvillei DSM 43111' L .....1.QQ -[=====~S~t~rePtomyces ambofaciens ATCC 23877 T ,- Straptomyces griseus KCTC 9080' '------Sporichthya po/ymorpha DSM 46113 ------Microbacterium /acticum IFO 14135' '------Arthrobacter g/obiformis DSM 20124 T '------Bifidobacterium bilidum ATCC 29521' '------Atopobium minutum NCF8 2751 T 0.1

Fig. 1. For legend see facing page.

334 International Journal ofSystematic and Evolutionary Microbiology 50 Aelinosynnell1a[{[ecae fam. nov.

Table 2. Chemotaxonomic characteristics of members of the proposed family Actinosynnemataceae and related genera

For all genera, the cell wall diamino acid was meso-DAP and the cell wall chemotype was III.

T3xon \Vhole-cell sugar pattern Phospholipid Phospholipid"" Predominant Fany acid lype mcnaquinonc

. Acrinoaf!orcic!Jus"';- PII P\IE !.iIK~9(H1J. \IK-SIH:I. 150. ,:ntcbo MK-9iH,) .·It'ri!z(lk,iJZL'ospora Galactose. mannose. rhamnose PII PE \IK-9(H:l Straight-chain. monoul1satur':I\.'d. i50. antciso Aclinosyl!lIfllio Galac!Osr. m<.!.nnos:: PII PE. PI. PIM. DPG \iIK-9(H:!. \IK-9(H;;1 Straight-chain. monoullsaturattd. :.:nt(;150 . AsiospflrallgilllH'~ Arabinos::. xylose SD SD :"D K!I{:::lIeria~ Trace. galactose. rhamnose PII PE. DPG. PG. PI \IK-9(H:l Str:.:ight-chain. unsaturated. 150. anle15() Lellr::ea Galactose PII PE. DPG_ PG. PI \IK-9(H I) Straight-chain. monouns'llurah:d. iso. anteiso Sacclwro;hrix Ga!

"D. Not determined. "DPG. diphosphatidyl glycerol: PE. phosphatidyl ethanolamine: PG, phosphatidyl glycerol: PI = phosphatidyl inositol: PIM = phosphatidyl inositolmannosides: PME. phosphatidylmethylethanolamine. t Data from !toh ('I al_ (1987). tData from Tamura & Hatano (1998). § Data from Stackebrandt el al. (1994).

to the other actinomycete families studied. including AClinosynnemalaceae. It is likely that these taxa rep­ the Pseudonocardiaceae, is supported by a bootstrap resent a new lineage separate from either family, but value of 77 % and provides good evidence for the this probably can only be clarified by isolating ad­ creation of a new family for these genera. The ditional representatives and sequencing their 165 relationship to the Pseudonocardioaceae vvas consistent rDNA. for all phylogenetic analysis methods used. The levels of 165 rDNA sequence similarity between the taxa in An evaluation of the chemotaxonomic characteristics the proposed new family and the members of other reported in the descriptive literature for all of these actinomycete lineages were 100-95'1 % and 93·4­ genera, seen in Table 2, also supports the creation of a 79·1 %, respectively. A diagnostic nucleotide signature new family, since these genera have cell wall type III pattern of TA (823-975), GC (824-874) was also (meso-DAP), all have galactose in their whole-cell observed for all of the strains proposed for inclusion sugar pattern, most have mannose and/or rhamnose, into the family AClinosynnemataceae. phosphatidylethanolamine is a common constituent of the phospholipid pattern (type PII), and all contain The phylogenetic position of KUlzneria kojilensis, menaquinones with nine isoprenoid units (MK-9). It is Kutzneria viridogrisea, Streptoalloteichus hindusran us, proposed that this family be named the Aetino­ Saccharothrix cryophilis and' Actinoalloteichus cyano­ Svnnemalaceae for the oldest named genus in the griseus' is not clear at this time. These sporangiate taxa family. Aetinosynnema Hasegavva_ Lecllevalier and have the appropriate chemotaxonomic profiles (i.e. Lechevalier 1978 (Hasegawa et al., 1978). A formal meso-DAP, galactose and possibly rhamnose and description of the family follows: mannose as diagnostic sugars, PII phospholipid pat­ tern, and menaquinones with nine isoprenoid units) and exhibit 165 rDNA sequence similarities ranging Description of Actinosynnemataceae fam. nov. from 96·1 to 92·1 % with the taxa in the proposed new Labeda and Kroppenstedt family, but the low bootstrap values observed do not A elinosynnellwtaeeae (Ac.ti.no.syn.ne.mat'ace.ae. support inclusion of these taxa into the Actino­ M.L. n. Aetinosynnema type genus of the family, pI. svnnemataceae and thev also do not have the nucleo­ Aclinosynnemata: L. ending -eeae ending to denote dde signature patter;l observed for the family. a family: M.L. n. Actinosynnemataceae the Aetino­ Saccharothrix cryophilis NRRL B-16238 was recently synnema family). observed to produce sporangia, and thus all of these related genera produce sporangia. Maximum-parsi­ Aerobic, catalase-positive actinomycetes compnsmg mony analysis of the data implied a close relationship the genera Actinokineospora, Actinosynnema, Lentzea of these genera with the Pseudonocardiaceae. while the and Saceharothrix. Vegetative mycelium branches. other algorithms used demonstrated linkage with the diameter approximately 0,5-0,7 ~lm: aerial mycelium

Fig. 1. Phylogenetic dendrogram reconstructed from evolutionary distances (Kimura, 1980) by the neighbour-joining method (Saitou & Nei, 1987), indicating the position of members of the family Actinosynnemataceae compared to the family Pseudonocardiaceae and representatives of the main lineages of the order . Bar, 0-1 nucleotide substitutions per site.

International Journal ofSystematic and Evolutionary Microbiology 50 335 D. P. Labeda and R. M. Kroppenstedt is produced and fragments into chains of smooth­ nov.. and Saccharolhrix II1l11abilis comb. nov. Jill J SYSI surfaced, rod-shaped elements which may be motile. Bacreriol 39.419-423. Synnemata or dome-like bodies mav be also be formed Labeda, D. P. & Lyons, A. J. (1989). Saccharolhrix lexasensis sp. oil the agar surface. Vegetative n~ycelium may also nov. and SaccharOlhrix \vay\vayandensis sp. nov. Jnl J Sysl fragment into coccoidal rod-shaped elements. Gram­ BaclerioI39.355-358. positive. Not acid-fast. Resistant to lysozyme. Soils Labeda, D. P., Testa, R. T., Lechevalier, M. P. & Lechevalier, H. A. and plant materials are typical habitats. The genera (1984). Sacc!lCIrolhrix: a new genus of the AClinoll1ycerales from a coherent phylogenetic unit on the basis of related to Nocardiopsis. Jnl J SYSI Bacleriol 34, 426-431. partial 16S rDNA sequences. Type III cell wall Maidak, B. L, Larsen, N., McCaughey, M. J., Overbeek, R., Olsen, composition (meso-diaminopimelic acid). Whole-cell G. J., Fogel, K., Blandy, J. & Woese, C. R. (1994). The Ribosomal sugars pattern consists of galactose and may also Database Project. Nucleic Acids Res 22, 3485-3487. include rhamnose and mannose. Phospholipid pattern Olsen, G. J., Matsuda, H., Hagstrom, R. & Overbeek, R. (1994). is type PII with a significant amount of phosphatidyl­ fastDNAml: a tool for construction of phylogenetic trees of ethanolamine. The principal menaquinone consists of DNA sequences using maximum likelihood. CABJOS 10, nine isoprenoid units (MK-9), although the degree of 41-48. unsaturation may vary. The G C content of the + Page, R. D. M. (1996). TREEVIEVV: an application to display DNA ranges from 68 to 76 mol %. phylogenetic trees on personal computers. CABJOS 12, 357­ 358. ACKNOWLEDGEMENTS Rainey, F. A., Ward-Rainey, N. L, Kroppenstedt, R. M. & Stacke­ The able technical assistance of E. N. Hoekstra in the brandt, E. (1996). The genus Nocardiopsis represents a phylo­ isolation and purification of DNA, PCR amplification of genetically coherent taxon and a distinct actinomycete lineage: 16S rONA, and sequence determinations for this study is proposal of Nocardiopsaceae fam. nov. Jill J Sysl Bacleriol46. gratefully acknowledged. 1088-IOn. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new REFERENCES method for reconstructing phylogenetic trees. Mol Bioi Evol4, Brosius, 1., Palmer, M. L, Kennedy, J. P. & Noller, H. F. (1978). 406-425. Complete nucleotide sequence of a 16S ribosomal RNA gene Stackebrandt, E., Kroppenstedt, R. M., Jahnke, K.-D., Kemmer­ from Escherichia coli. hoc Narl Acad Sci USA 75. 4801-4808. ling, C. & Gurtler, H. (1994). Transfer of Slreplosporangiull1 Cote, R., Daggett, P.-M., Gantt, M. J., Hay, R., Hay, S.-e. & Pienta, viridogriseull1 (Okuda et al. 1966), Slreplosporangiull1 virido­ P. (1984). ATCC A[edia Handbook. Rockville, MD: American griseulI1 subsp. kojzlense, and SlreplosporangiulI1 albidulI1 (Furu­ Type Culture Collection. mai et a1. 1968) to KUlzneria gen. nov. as Klllzneria viridogrisea KUlzneria kiltzlensis KUlzneria Embley, M. T., Smida, 1. & Stackebrandt, E. (1988). The phylogeny comb. nov.. comb. nov., and of mycolate-Iess wall chemotype IV actinomycetes and de­ albida comb. nov., respectively, and emendation of the genus scription of Pseudonocardiaceae fam. nov. SYSI Appl Aficrobiol SlreplOsporangiwll. Jill J Sysl Bacleriol44. 265-269. 11. 44-52. Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L (1997). Felsenstein, 1. (1993). PHYLIP (phylogenetic inference package) Proposal for a new hierarchic classification system, AClino­ version 3.5. Department ofGenetics, University ofWashington. bacreria classis nov. Jnl J Sysl Bacleriol47. 479-491. Seattle. Swofford, D. L (1993). PAUP: phylogenetic analysis using Grund, E. & Kroppenstedt, R. M. (1989). Transfer of five parsimony, version 3.1. In Narural Hislory Survey. Champaign, Nocardiopsis species to the genus Saccharolhrix Labeda el al. IL: Illinois Natural History Survey. 1984. Sysl Appl AJicrobiol12, 267-274. Tamura, T. & Hatano, K. (1998). Phylogenetic analyses on the Hasegawa, T., Lechevalier, M. P. & Lechevalier, H. A. (1978). A strains belonging to invalidated genera of the order AClino­ new genus of the Acrinoll1ycerales. Aclinosynnell1a gen. nov. Jill lI1ycerales. AClinoll1ycelologica 12, 15-28. J Sysl Bacleriol 28. 304-310. Warwick, S., Bowen, T., McVeigh, H. & Embley, T. M. (1994). A Itoh, T., Kudo, T. & Seino, A. (1987). Chemotaxonomic studies on phylogenetic analysis of the family Pseudonocardiaceae and the new genera of actinomycetes proposed in Chinese papers. genera AClinokineospora and Saccharolhrix with 16S rRNA Aclinoll1ycelologica 1. 43-59. sequences and proposal to combine the genera AlI1ycolara and Kimura, M. (1980). A simple method for estimating evolutionary Pseudonocardia in an emended genus Pseudonocardia. Jill J Sysl rates of base substitutions through comparative studies of BaclerioI44.293-302. nucleotide sequences. J Mol Evo116, 111-120. Yassin, A. F., Rainey, F. A., Brzenzinka, H., Jahnke, K.-D., Wess­ Labeda, D. P. & Lechevalier, M. P. (1989). Amendment of the brodt, H., Budzikiewicz, H., Stackebrandt, E. & Schaal, K. P. genus Saccharolhrix Labeda el al. 1984 and descriptions of (199S). Lelllzea gen. nov., a new genus of the order AClino­ Saccharolhrix espanensis sp. nov., Saccharolhrix cryophilus sp. lI1ycerales. Jill J SYSI Bacreriol45. 357-363.

336 International Journal ofSystematic and Evolutionary Microbiology 50