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Actinosynnemataceae Fam. Nov 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 species, as well as strains of the Center for Agricultural Utilization Research, described species of the related genera Kutzneria, Actinokineospora and Agricultural Research Actinosynnema. These were aligned with published sequences for other Service, US Department of species of these genera, as well as those of the genera'Actinoalloteichus', Agriculture, 1815 N. University Street, Peoria, ,Asiosporangium" Lentzea, Kutzneria, Streptoalloteichus 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 Pseudonocardiaceae. It is proposed that a new family be created within the Braunschweig, Germany class Actinobacteria 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 Amycolatopsis 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
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