Nesterenkonia sandarakina sp. nov. and Nesterenkonia lutea sp. nov., novel actinobacteria, and emended description of the genus Nesterenkonia

Two novel actinobacteria isolates, designated YIM 70009^T and YIM 70081^T, were characterized in order to determine their taxonomic position. Cells of strains YIM 70009^T and YIM 70081^T were cocci, although only the latter were motile. The G+C contents of their DNAs were 64·0 and 64·5 mol%, respectively. On the basis of chemotaxonomic characteristics and 16S rRNA gene sequence analysis, the two isolates were classified in the genus Nesterenkonia. DNADNA hybridization and comparison of phenotypic characteristics revealed that strains YIM 70009^T and YIM 70081^T differed from each other and from known species. Therefore, it is proposed that they represent two separate novel species of the genus Nesterenkonia: Nesterenkonia sandarakina sp. nov. (type strain, YIM 70009^T=CCTCC AA 203007^T=DSM 15664^T=KCTC 19011^T) and Nesterenkonia lutea sp. nov. (type strain, YIM 70081^T=CCTCC AA 203010^T=DSM 15666^T=KCTC 19013^T).

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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains YIM 70009^T and YIM 70081^T are AY588277 and AY588278, respectively.

Electron micrographs of strains YIM 70009^T and YIM 70081^T are available as supplementary material in IJSEM Online.

The genus Nesterenkonia was first proposed by Stackebrandt et al. (1995) in the reclassification of Micrococcus halobius Onishi and Kamekura 1972 as Nesterenkonia halobia (Stackebrandt et al., 1995). At present, the genus comprises four valid species: N. halobia (Stackebrandt et al., 1995), Nesterenkonia lacusekhoensis (Collins et al., 2002), and Nesterenkonia halotolerans and Nesterenkonia xinjiangensis (Li et al., 2004). In this report, two novel species of the genus Nesterenkonia that were discovered during our taxonomic study of extremophilic actinomycetes are described.

Strain YIM 70009^T was isolated from a soil sample collected from the eastern desert of Egypt using modified medium A (supplemented with 15 % NaCl, w/v, pH 10·010·5), as described previously (Hozzein et al., 2004). Strain YIM 70081^T was isolated from a saline soil sample from China using a modified glycerol/asparagine agar medium (ISP 5) (Shirling & Gottlieb, 1966) supplemented with 15 % (w/v) MgCl₂.6H₂O. Isolation plates were incubated at 28 °C for 2 weeks. The purified strains were cultivated and maintained on modified TSA medium containing 510 % NaCl (w/v), pH 8·09·0 for strain YIM 70009^T or 510 % MgCl₂.6H₂O (w/v), pH 7·08·0 for strain YIM 70081^T. Biomass for chemical and molecular systematic studies was grown in shaken flasks (about 150 r.p.m.) at 28 °C for 1 week using the same media as above without agar. Morphological properties were examined by light microscopy (Olympus microscope BH-2) and electron microscopy (JEOL JEM-1010).

The G+C contents of isolates YIM 70009^T and YIM 70081^T, determined using the thermal denaturation method of Marmur & Doty (1962), were 64·0 and 64·5 mol%, respectively. 16S rRNA genes were analysed as described previously (Li et al., 2004). Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and MEGA (Molecular Evolutionary Genetics Analysis) version 2.1 (Kumar et al., 2001) after multiple alignment of data by CLUSTAL_X (Thompson et al., 1997). Distances were calculated using distance options according to the Kimura two-parameter model (Kimura, 1980, 1983) and clustering was performed using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was used to evaluate tree topology of the neighbour-joining data by performing 1000 resamplings (Felsenstein, 1985). The neighbour-joining tree (Fig. 1) indicated that strains YIM 70009^T and YIM 70081^T were highly related to each other (99·4 % sequence similarity) and to N. halotolerans YIM 70084^T (99·8 and 99·4 % sequence similarity, respectively), forming a distinct branch with this species. Sequence similarities between strains YIM 70009^T and YIM 70081^T and other members of the genus Nesterenkonia were no more than 97·2 %.

(21K): Fig. 1. Phylogenetic dendrogram, obtained by distance matrix analysis of 16S rRNA gene sequences, showing the position of strains YIM 70009T and YIM 70081T among phylogenetic neighbours. Numbers on branch nodes are bootstrap values (1000 resamplings). The sequence of Streptomyces megasporus DSM 41476T (GenBank/EMBL/DDBJ accession no. Z68100) was used as a root. Bar, 1 % sequence divergence.

Growth at various pH values and salt concentrations was tested as reported by Tang et al. (2003), except that TSA medium was used as the basic medium. Strains were grown on TSA medium containing 0, 1, 3, 5, 10, 15, 20, 25 or 30 % (w/v) total salts (NaCl, KCl or MgSO₄.6H₂O) and at initial pH values of 4·0, 5·0, 6·0, 7·0, 8·0, 9·0, 10·0, 11·0,12·0 and 13·0 for 30 days at 28 °C. Cell morphology and motility, physiological and biochemical test results, and chemotaxonomic properties, determined using previously described methods (Li et al., 2004), are given in detail in Table 1 and the species description.

Table 1. Phenotypic characteristics of strains YIM 70009T, YIM 70081T and N. halotolerans YIM 70084T Strains: 1, YIM 70009T; 2, YIM 70081T; 3, N. halotolerans YIM 70084T. All three strains are Gram-positive, non-spore-forming coccoid cells. All are positive for catalase, milk coagulation, melanin production, growth in cellulose, and lysine decarboxylase, β-glucosidase, β-galactosidase and α-maltosidase activities. All are negative for oxidase, H2S and indole production, decomposition of Tween 20 and Tween 80, casein and starch, and ornithine decarboxylase, arginine dihydrolase and N-acetylglucosaminidase activities. Maltose, sucrose, mannose, mannitol, fructose, salicin and galactose are utilized by the three strains as sole carbon sources for growth; rhamnose, acetamide, inositol, adonitol and sorbitol are not utilized. +, Positive; , negative; W, weak reaction.

DNADNA relatedness tests were performed between YIM 70009^T, YIM 70081^T and N. halotolerans YIM 70084^T using the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992). DNADNA reassociation values of N. halotolerans YIM 70084^T with strains YIM 70009^T and YIM 70081^T were 43·3 and 39·1 %, respectively, and DNADNA hybridization between the two isolates was 45·2 % (repeated twice). DNADNA relatedness data provide decisive evidence that isolates YIM 70009^T and YIM 70081^T, and the related type strain N. halotolerans YIM 70084^T, are members of different genomic species (Wayne et al., 1987). Genomic distinctness was also revealed by differences in a number of phenotypic characteristics (Table 1). It is therefore proposed that strains YIM 70009^T and YIM 70081^T should be classified as representatives of two novel Nesterenkonia species, Nesterenkonia sandarakina sp. nov. and Nesterenkonia lutea sp. nov., respectively.

Emended description of the genus Nesterenkonia Stackebrandt et al. 1995 emend. Collins et al. 2002
The description of the genus Nesterenkonia is as given previously (Stackebrandt et al., 1995; Collins et al., 2002), but with the following amendments. Moderately halophilic or halotolerant. Some species are alkaliphilic or alkalitolerant. Peptidoglycan is of the A4α type, L-lysglyL-Glu, L-lysL-glu or lysglyD-Asp. The G+C content of the DNA is 6472 mol%.

Description of Nesterenkonia sandarakina sp. nov.
Nesterenkonia sandarakina [san.da.ra'ki.na. N.L. fem. adj. sandarakina (from Gr. fem. adj. sandarakinê) of orange colour].

Cells are Gram-positive, non-spore-forming cocci (see electron micrograph available as supplementary material in IJSEM Online). Colony colour on most tested media is orangeyellow. Colonies are circular, opaque and approximately 0·51·0 mm in diameter after 24 h at 28 °C. Growth occurs at 115 % (w/v) NaCl (optimum at 5 %, w/v) and pH 5·012·0 (optimum at 8·09·0). Results of some physiological and biochemical characteristics, and metabolic and enzymic properties are indicated in Table 1. Peptidoglycan type is A4α, LysGlyD-Asp. Cell wall sugars are ribose, xylose and arabinose. Main polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. Predominant menaquinones are MK-7 and MK-8. Cellular fatty acids are C_{16 : 0} (33·11 %), aiC_{15 : 0} (27·63 %), aiC_{17 : 0} (20·68 %), C_{14 : 0} (0·81 %), C_{15 : 0} (0·81 %), C_{17 : 0} (1·46 %), C_{18 : 0} (2·18 %), iC_{16 : 0} (6·33 %), iC_{17 : :0} (0·47 %), A-aiC_{15 : 1} (4·81 %), G-iC_{16 : 1} (0·82 %) and A-aiC_{17 : 1} (0·88 %).

The type strain is YIM 70009^T (=CCTCC AA 203007^T=DSM 15664^T=KCTC 19011^T), isolated from a soil sample collected from the eastern desert of Egypt. The DNA G+C content of the type strain is 64·0 mol%.

Description of Nesterenkonia lutea sp. nov.
Nesterenkonia lutea (lu'te.a. L. fem. adj. lutea goldyellow).

Cells are Gram-positive, non-spore-forming, motile cocci with flagella (see electron micrograph available as supplementary material in IJSEM Online). Colony colour on most tested media is light yellow to primrose yellow. Colonies are circular, opaque, somewhat convex and approximately 0·51·0 mm in diameter after 24 h at 28 °C. Growth occurs at 020 % (w/v) MgCl₂.6H₂O (optimum at 510 %, w/v) and pH 6·510·0 (optimum at 7·08·0). Results of some physiological and biochemical characteristics, and metabolic and enzymic properties are indicated in Table 1. Peptidoglycan type is A4α, LysGlyD-Asp. Cell wall sugars are ribose, xylose, arabinose and glucose. Predominant menaquinones are MK-8 and MK-7. Main polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. Cellular fatty acids are aiC_{15 : 0} (10·27 %), iC_{16 : 0} (31·40 %), aiC_{17 : 0} (15·27 %), A-aiC_{15 : 1} (15·12 %), G-iC_{16 : 1} (11·09 %), G-iC_{15 : 1} (0·84 %), C_{14 : 0} (0·34 %), 2-OH C_{14 : 0} (0·24 %), C_{16 : 0} (5·75 %), iC_{14 : 0} (0·99 %), iC_{15 : 0} (0·57 %), iC_{17 : 0} (0·96 %), C_{17 : 1}ω6c (0·36 %), A-aiC_{17 : 1} (3·21 %), iC_{18 : 0} (1·55 %), C_{18 : 1}ω7c (1·64 %) and summed feature 3 (C_{16 : 1}ω7c and/or 2-OH i-C_{15 : 0}; 0·40 %).

The type strain is YIM 70081^T (=CCTCC AA 203010^T=DSM 15666^T=KCTC 19013^T), isolated from a saline soil sample from the Xinjiang Province, China. The DNA G+C content of the type strain is 64·5 mol%.