INTERNATIONAL JOURNALOF SYSTEMATICBACTERIOLOGY, Oct. 1996, p. 1088-1092 Vol. 46, No. 4 0020-7713/96/$04.00+0 Copyright 0 1996, International Union of Microbiological Societies The Genus Nocardiopsis Represents a Phylogenetically Coherent Taxon and a Distinct Actinomycete Lineage: ProDosal of Nocardiomaceae fam. nov. FRED A. RAINEY,” NAOMI WARD-RAINEY, REINER M. KROPPENSTEDT, AND ERKO STACKEBRANDT DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany The genus Nocardiopsis was shown to be phylogenetically coherent and to represent a distinct lineage within the radiation of the order Actinomycetales. The closest relatives of the genus Nocardiopsis are members of the genera Actinomadura, Thermomonospora,Streptosporangium, and Microtetraspora. The intrageneric structure of the genus Nucardiupsis is shown to consist of a highly related species group containing Nucardiupsis dassonvillei, Nocardiopsis alborubida, and Nocardiopsis antarctica and a second group of less highly related species compris- ing Nocardiopsis alba subsp. alba, Nocardiopsis alba subsp. prasina, and Nocardiopsis listeri. Nocardiopsis lucen- tensis occupies a position intermediate between the two species groups. The results of a 16s ribosomal DNA sequence analysis are generally consistent with the available chemotaxonomic, phenotypic, and DNA-DNA hybridization data. The phylogenetic position and the morpho- and chemotaxonomic properties of Nocardiopsis species support the creation of a family for the genus Nocardiopsis, Nocardiopsaceae fam. nov. Meyer described the genus Nocardiopsis for the species Ac- present. Diagnostic for all members of the genus Nocardiopsis tinomadura dassonvillei in 1976 on the basis of the morpholog- is the combination of 15 to 20% anteiso-C,,:, (14-methylhexa- ical characteristics and cell wall type of this organism (21). The decanoic acid) together with 20 to 25% 10-methyl-C,,,, (tu- genus Nocardiopsis currently comprises seven validly described berculostearic acid; 10-methyl-octadecanoic acid) or its precur- species, Nocardiopsis alborubidus, Nocardiopsis albus, Nocar- sor, oleic acid (7, 12, 22). This combination of fatty acids is diopsis antarcticus, Nocardiopsis dassonvillei,Nocardiopsis halo- unique among bacteria and can be used to differentiate No- phila, Nocardiopsis listen, and Nocardiopsis lucentensis. Nocar- cardiopsis species from all other bacteria. The peptidoglycan diopsis albus includes two subspecies, Nocardiopsis albus subsp. contains meso-diaminopimelic acid, and no diagnostic sugars albus and Nocardiopsis albus subsp. prasina. Arriving at this are present (cell wall chemotype III/C [18]). Other chemotaxo- current taxonomy has involved the exclusion of previously rec- nomic features include a lack of mycolic acids, the presence of ognized Nocardiopsis species and the inclusion of new species muramic acid of the acetyl type, and DNA G+C contents transferred from other genera. Five species, Nocardiopsis co- between 64 and 69 mol% (13). The combination of these emleofusca ,Nocardiopsis Jlava , Nocardiopsis longispora ,Nocar- characteristics can be used to differentiate Nocardiopsis species diopsis mutabilis, and Nocardiopsis synngae, were transferred to from other actinomycetes. the genus Saccharothrix on the basis of their chemotaxonomic Differentiation of Nocardiopsis species is currently based on characteristics (9, 16), and Kroppenstedt et al. (14) have trans- the color of the mycelium and the results of comparative phys- ferred Nocardiopsis afncana to the genus Microtetraspora. iological tests (13). Recently, the presence of novel cell wall The following two species were created by Grund and Krop- teichoic acids has been reported in Nocardiopsis species (24, penstedt (10) on the basis of chemotaxonomic and numerical 25). The authors of these studies suggested that species-spe- taxonomic data: Nocardiopsis alborubidus, for the invalid spe- cific teichoic acids are present in Nocardiopsis dassonvillei,No- cies “Actinomycesalborubidus”; and Nocardiopsis listeri, for the cardiopsis antarcticus, and Nocardiopsis albus subsp. albus, but invalid streptomyce t e “Streptomyceslisten’. ” until this trait has been examined for all Nocardiopsis species, The genus Nocardiopsis is currently defined on the basis of its taxonomic value will remain unknown. chemotaxonomic markers, since Nocardiopsis strains cannot be In recent years the application of rRNA sequence analysis to differentiated from members of the genus Saccharothrix mor- the systematics of the actinomycetes has helped bring some phologically (13, 17). The salient chemotaxonomic features, as order to the taxonomy of this phylum. The combination of new described previously (13), include phospholipid type 111 (19) data from rRNA analyses with previously available phenotypic with the diagnostic phospholipids phosphatidylcholine and phos- information has given us a better understanding of the true phatidylmethylethanolamine.The menaquinone type is type 4c2 relationships among various actinomycete taxa. The recent re- (13), and the main menaquinones contain 10 isoprene subunits view of Embley and Stackebrandt (4) provided a comprehen- in their side chains with variable degrees of saturation. The sive overview of the phylogenetic structure of the actinomyce- fatty acid type is type 3d (12), and the fatty acids include tes. In this review, Nocardiopsis dassonvillei was considered an iso-branched, anteiso-branched, and 10-methyl-branched fatty actinomycete of uncertain phylogenetic affiliation, but was ten- acids. High levels of octadecenoic acid (oleic acid) are also tatively placed between the family Thermornonosporaceae (2) and the family Streptosporangiaceae (8). Although the genus Nocardiopsis is phylogenetically related to these families, it can * Corresponding author. Mailing address: DSMZ-German Collec- be excluded from them by its unique combination of chemot- tion of Microorganisms and Cell Cultures, Mascheroder Weg lb, axonomic markers. D-38124 Braunschweig, Germany. Phone: 49-531-2616101. Fax: 49- In order to clarify the phylogenetic position of the genus 531-2616418. Electronic mail address: [email protected]. Nocardiopsis and to investigate the phylogenetic coherence of 1088 VOL. 46, 1996 NOCARDIOPSACEAE FAM. NOV. 1089 TABLE 1. Strains for which 16s rDNA sequence data contained information for 1,236 unambiguous nucleotide positions present in all were obtained in this study sequences between positions 51 and 1471 (Escherichia coli numbering [l]). When only the Nocardiopsis sequences generated in this study and the sequence of EMBL Strain Microtetraspora glauca were used, a second data set, which contained information accession no. for 1,437 unambiguous nucleotide positions present in these sequences between positions 34 and 1500 (E. coli numbering [l]), was produced. Evolutionary Nocardiopsis alborubidus DSM 40465T ................................... X97882 distances were calculated by the method of Jukes and Cantor (11). Phylogenetic Nocardiopsis albus subsp. albus DSM 43377T........................ X97883 dendrograrns were reconstructed by using treeing algorithms contained in the Nocardiopsis albus subsp. prasina DSM 43845T .................... X97884 PHYLIP package (6). Tree topologies were evaluated by performing bootstrap Nocardiopsis antarcticus DSM 43884= .................................... X97885 analyses (5) of the neighbor-joining data, using 1,000 resamplings. Nocardiopsis dassonvillei DSM 431llT ................................... X97886 Saponification, methylation, extraction, and analysis of fatty acid methyl Nocardiopsis listen DSM 40297r .............................................. X97887 esters. Fatty acid methyl esters were obtained from wet biomass (ca. 40 mg) by Nocardiopsis lucentensis DSM 4404gT..................................... X97888 saponification, methylation, and extraction (15). The fatty acid methyl ester Actinomadura madurae DSM 43067= ..................................... X97889 mixtures were separated by using a model 5898A microbial identification system apparatus (Microbial ID, Newark, Del.). The fatty acid data obtained for the Actinomadura kijaniata DSM 43764T...................................... X97890 strains were compared qualitatively and quantitatively by using Ward’s method Microtetraspora glauca DSM 4331 lT ....................................... X97891 (26) and the Microbial Identification System Library Generation software (Mi- Microtetraspora salmonea DSM 43678T .................................. X97892 crobial ID, Newark, Del.). Themomonospora curvata DSM 43183 ................................ X97893 Nucleotide sequence accession numbers. The 16s rDNA sequences deter- mined in this study have been deposited in the EMBL data library under the accession numbers shown in Table 1. The accession numbers of the sequences of the strains used as representatives of the main actinomycete groups are as fol- lows: Actinoplanes philippinensis DSM 43019T (T = type strain), X93187; Arth- this genus, 16s ribosomal DNA (rDNA) sequence data were robacter globifomis DSM 20124T, M23411; Atopobium rninutum ATCC 33267T, obtained from all available type strains and analyzed. M59059; B$dobacterium bifdum ATCC 29521T, M38018; Cellulomonasjlavigena DSM 20109T, X83799; Daciylosporangium aurantiacum DSM 43157T, X93191; Microbacterium lacticum DSM 20427T, X77441; Microlunatus phosphovorus JCM MATERIALS AND METHODS 9379T, D26169; Micromonospora chalcea DSM 43026T, X92549; Mycobacterium Strains