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Nonomuraea kuesteri sp. nov.

¹ Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
² DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, Germany
³ Labor Grün-Wollny, D-35394 Giessen, Germany

Correspondence
Peter Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de

	ABSTRACT

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A Gram-positive, aerobic, non-motile actinomycete, strain GW 14-1925^T, that formed branched substrate and aerial mycelium was studied using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strain GW 14-1925^T was shown to belong to the genus Nonomuraea, being most closely related to Nonomuraea longicatena (97·9 %), Nonomuraea turkmeniaca (98·9 %), Nonomuraea helvata (98·6 %), Nonomuraea polychroma (98·5 %), Nonomuraea salmonaea (98·3 %), Nonomuraea roseoviolacea subsp. roseoviolacea (98·1 %) and Nonomuraea roseoviolacea subsp. carminata (97·7 %). The 16S rRNA gene sequence similarity to other Nonomuraea species was <97·5 %. Chemotaxonomic data [major menaquinones of the MK-9 series with minor amounts of MK-8(H₄); major polar lipids of phospholipid type IV; fatty acids with major amounts of iso- and anteiso- and 10-methyl-branched fatty acids in combination with iso-branched 2-hydroxy fatty acids] supported allocation of the strain to the genus Nonomuraea. The results of DNA–DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW 14-1925^T from closely related species; thus, GW 14-1925^T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea kuesteri sp. nov. is proposed, with GW 14-1925^T (=DSM 44753^T=NRRL B-24325^T) as the type strain.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DSM 44753^T is AJ746362.

	MAIN TEXT

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The genus Nonomuria [sic, corrected by Chiba et al. (1999) to Nonomuraea] was originally proposed by Zhang et al. (1998) as a member of the family Streptosporangiaceae that forms extensively branched substrate and aerial mycelia. On the basis of detailed phylogenetic analysis, the genus presently comprises 18 species and two subspecies, most of which were listed by Gyobu & Miyadoh (2001), Stackebrandt et al. (2001) and Quintana et al. (2003).

During the characterization of organisms from soil, strain GW 14-1925^T was recovered on oatmeal agar (ISP 3 medium; Küster, 1959) at 25 °C; it had slightly yellow-coloured vegetative mycelium with white aerial mycelium. Subcultivation was done on tryptone soy agar (Oxoid) at 25 °C for 24 h. Gram-staining was performed as described by Gerhardt et al. (1994). The cell morphology was observed under a Zeiss light microscope at x1000, using cells that had been grown for 3 days at 25 °C on R2A agar (Oxoid). The 16S rRNA gene was analysed as described previously (Kämpfer et al., 2003). Phylogenetic analysis was performed by using the ARB software package (Strunk et al., 2000) and also the software package MEGA, version 2.1 (Kumar et al., 2001), after multiple alignment of the data by CLUSTAL_X (Thompson et al., 1997).

Distances were obtained (using distance options according to the Kimura two-parameter model; Kumar et al., 2001) and clustering was performed, using the neighbour-joining (Fig. 1) and maximum-parsimony methods, by using bootstrap values based on 1000 replications. The 16S rRNA gene sequence of strain GW 14-1925^T was a continuous stretch of 1496 bp. Sequence similarity calculations after neighbour-joining analysis indicated that the closest relatives of strain GW 14-1925^T were Nonomuraea turkmeniaca (98·9 %), Nonomuraea helvata (98·6 %), Nonomuraea salmonea (98·3 %) and Nonomuraea polychroma (98·5 %). Both the neighbour-joining tree (Fig. 1) and the maximum-parsimony tree (not shown) revealed that strain GW 14-1925^T clustered most closely with Nonomuraea roseoviolacea subsp. roseoviolacea (98·1 %) and Nonomuraea longicatena (97·9 %).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Phylogenetic analysis, based on 16S rRNA gene sequences available from the EMBL database (accession nos are given in parentheses), constructed after multiple alignment of data by CLUSTAL_X (Thompson et al., 1997). Distances were obtained (using distance options according to the Kimura-2 model) and clustering was performed, using the neighbour-joining method, by using the software package MEGA, version 2.1 (Kumar et al., 2001). Bootstrap values based on 1000 replications are listed as percentages at branching points. Bar, 0·005 Knuc.

The quinone system found supports affiliation of strain GW 14-1925^T to the genus Nonomuraea. The principal menaquinones of strain GW 14-1925^T were as follows: MK-9(H₄), 71 %; MK-9(H₆), 15 %; MK-9(H₂), 6 %; and MK-9, 2 %. In addition, MK-8(H₄) was detected (6 %). This is essentially in accordance with the quinone profile reported for members of the genus (Kroppenstedt & Goodfellow, 1991; Stackebrandt et al., 2001; Quintana et al., 2003). The polar lipids of strain GW 14-1925^T are of the PIV type, according to the phospholipid classification of Lechevalier et al. (1977), and include diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, methyl-phosphatidylethanolamine and the diagnostic phospholipid N-acetylglucosamine. Some unidentified glycolipids were also detected. The fatty acids comprised mainly iso- and 10-heptadecanoic-branched fatty acids. Smaller amounts of unbranched saturated and 2-hydroxy fatty acids were detected (fatty acid type 3c of Kroppenstedt, 1985). The detailed fatty acid profile is as follows: 13 : 0 (0·4 %), 14 : 0 iso (0·9 %), 14 : 0 (0·9 %), 15 : 1 iso G (0·2 %), 15 : 0 iso (7·9 %), 15 : 0 anteiso (0·3 %), 15 : 0 (3·8 %), 16 : 1 iso G (8·3 %), 16 : 0 iso (28·9 %), 16 : 1 cis9 (1·8 %), 16 : 0 (1·8 %), 15 : 0 2-OH (0·8 %), 16 : 0 10-methyl (2·8 %), 17 : 0 iso (1·8 %), 17 : 0 anteiso (1·0 %), 17 : 1 cis9 (4·2 %), 16 : 0 iso 2-OH (10·5 %), 17 : 0 (1·6 %), 16 : 0 2-OH (0·5 %), 17 : 0 10-methyl (15·5 %), 18 : 0 iso (0·6 %), 18 : 1 cis9 (0·6 %), 17 : 0 iso 2-OH (0·4 %), 18 : 0 (1·5 %), 17 : 0 2-OH (0·3 %) and 18 : 0 10-methyl (0·9 %). The results of the physiological characterization, performed using methods described previously (Kämpfer, 1990; Kämpfer et al., 1991), are given in Table 1 and in the species description. In addition, degradative tests were performed according to Williams et al. (1983). DNA–DNA hybridizations between strain GW 14-1925^T and the type strains of N. longicatena, N. salmonea and N. turkmeniaca were performed using the method described by Ziemke et al. (1998), except that, for nick translation, 2 µg DNA was labelled during a 3 h incubation at 15 °C. Strain GW 14-1925^T showed relatively low DNA–DNA relatedness to N. roseoviolacea subsp. roseoviolacea DSM 43144^T (15·9 %, mean value of four hybridizations), N. longicatena NRRL 15532^T (16 %, mean value of two hybridizations), N. salmonea DSM 43678^T (32 %, mean value of four hybridizations) and N. turkmeniaca DSM 43926^T (40·5 %, mean value of four hybridizations). It has been shown that Nonomuraea species have high 16S rRNA gene sequence similarities (within the range 97·6–99·4 %) and have low DNA–DNA relatedness values (Fischer et al., 1983; Poschner et al., 1985; Tamura et al., 2000). Stackebrandt et al. (2001) reported 45–48 % as the highest DNA–DNA relatedness values between the type strains of Nonomuraea africana, Nonomuraea dietziae and Nonomuraea recticatena, strains sharing 16S rRNA gene sequence similarities between 98·9 and 99·8 %. For these reasons, it is clear that strain GW 14-1925^T represents a novel species of the genus Nonomuraea, for which we propose the name Nonomuraea kuesteri. The type strain is GW 14-1925^T.

Forms an extensive branched substrate mycelium. Only traces of aerial mycelium are visible on oatmeal agar. Spore chains are spiral; sporangia are not detected. Gram-positive and oxidase-positive; shows oxidative metabolism. Good growth occurs after 3 days incubation on oatmeal agar and nutrient agar (Oxoid) at 25–30 °C. DL-Diaminopimelic acid is the diagnostic amino acid in the peptidoglycan. The main menaquinones of the type strain are MK-9(H₄) (71 %), MK-9(H₆) (15 %), MK-9(H₂) (6 %) and MK-9 (2 %); MK-8(H₄) (6 %) is also present. The polar lipids include diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, methyl-phosphatidylethanolamine and the diagnostic phospholipid N-acetylglucosamine. The fatty acids comprise mainly iso- and 10-heptadecanoic-branched fatty acids. Smaller amounts of unsaturated and 2-hydroxy fatty acids are also detected. Results of carbon-source utilization and degradation tests (including differentiating characteristics) are shown in Table 1. The type strain does not produce acids from the following sugars: glucose, lactose, sucrose, D-mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, L-arabinose, raffinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, methyl D-glucoside, erythritol, melibiose and arabitol. The following carbon sources are utilized (after 7 days incubation): N-acetyl-D-glucosamine, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, D-melibiose, L-rhamnose, sucrose, D-trehalose, D-xylose, adonitol, D-mannitol, fumarate (weak), DL-lactate, malate, 2-oxoglutarate, L-aspartate (weak) and L-proline. The following carbon sources are not utilized: N-acetyl-D-galactosamine, p-arbutin, D-gluconate, D-maltose, D-ribose, salicin, inositol, maltitol, sorbitol, putrescine, acetate, propionate, cis-aconitate, trans-aconitate, adipate, 4-aminobutyrate, azelate, citrate, glutarate, DL-3-hydroxybutyrate, itaconate, mesaconate, pyruvate, suberate, L-alanine, -alanine, L-histidine, L-leucine, L-ornithine, L-phenylalanine, L-serine, L-tryptophan, DL-3-hydroxybenzoate, DL-4-hydroxybenzotae and L-phenylacetate.

The type strain, GW 14-1925^T (=DSM 44753^T=NRRL B-24325^T), was isolated from a soil sample.

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