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Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
Correspondence
S. Shivaji
shivas{at}ccmb.res.in
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Strain HHS 31T and various Exiguobacterium species are compared in a table of fatty acid compositions and a neighbour-joining phylogenetic tree available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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to accommodate Gram-positive, non-spore-forming, facultatively anaerobic, alkaliphilic bacteria isolated from potato-processing effluent (Gee et al., 1980). Since then, nine species have been described: Exiguobacterium aurantiacum (Collins et al., 1983), Exiguobacterium undae and Exiguobacterium antarcticum (Fruhling et al., 2002), Exiguobacterium oxidotolerans (Yumoto et al., 2004), Exiguobacterium aestuarii and Exiguobacterium marinum (Kim et al., 2005), Exiguobacterium mexicanum and Exiguobacterium artemiae (Lopez-Cortes et al., 2006) and Exiguobacterium acetylicum (which was transferred from the genus Brevibacterium; Farrow et al., 1994). In the present study, strain HHS 31T was isolated from the melt water of the Hamta glacier located at a height of 4270 m above sea level in the Himalayan mountain ranges of India. The melt-water sample yielded about 2x104 c.f.u. ml–1 and the pure colonies represented 26 different morphotypes (Shivaji et al., 2005a). Representative strains from each morphotype were then tentatively identified, using their 16S rRNA gene sequences (Shivaji et al., 2005a), as being closely related to Pseudomonas meridiana (GenBank accession no. AJ537602), Pseudomonas antarctica (AJ537601), Pseudomonas veronii (AY267192), Pseudomonas migulae (AY047218), Pseudomonas fluorescens (AY538263), Pseudomonas lini (AY035996), Pseudomonas jessinii (AY391278), Serratia marcescens (AF076038), Janthinobacterium lividum (AY247410), Bacillus subtilis (AY672765), Hafnia alvei (AY572428), E. acetylicum (AY297792) and Pedobacter cryoconitis (AJ585231) (Shivaji et al., 2005a). Two of the strains, HHS 11T and HHS 22T, were identified as representing novel species of Dyadobacter and Pedobacter, namely Dyadobacter hamtensis (Chaturvedi et al., 2005) and Pedobacter himalayensis (Shivaji et al., 2005a). In the present study, strain HHS 31T, a Gram-positive, motile, non-spore-forming, rod-shaped bacterium, was identified, using polyphasic taxonomy, as representing a novel species of the genus Exiguobacterium.
Strain HHS 31T was isolated by plating 1 ml glacial water on nutrient agar plates [0.5 % (w/v) peptone, 0.3 % (w/v) beef extract, 0.5 % (w/v) NaCl and 1.5 % (w/v) agar, pH 7.0] that were then incubated at 22 °C for 3 days. Nutrient agar medium was also used for maintaining strain HHS 31T and for determining growth at various temperatures (5, 10, 15, 22, 28, 37 and 40 °C), at different pH values (pH 4, 6, 7, 8, 10 and 11) and in the presence of various concentrations of NaCl (5, 10, 12 and 15 %) (Shivaji et al., 1989). The buffers used were MOPS (for pH 6.5–7.9) and CAPS (for pH 9.7–11.1). Phenotypic characteristics such as colony morphology, cell morphology, motility, various enzyme activities (as listed in Table 1) and sensitivity to antibiotics at 22 °C were ascertained using standard methods (Lanyi, 1987; Smibert & Krieg, 1994). Gas production was determined according to the method of Hugh & Leifson (1953). For acid production, the medium used was phenol red agar base (pH 7.4) containing proteose peptone (1 %), beef extract (0.1 %), NaCl (0.5 %), phenol red (0.0025 %) and agar (1.8 %). Minimal medium [K2HPO4, 1.05 % (w/v); KH2PO4, 0.45 % (w/v); (NH4)2SO4, 0.1 % (w/v); agar, 1.5 % (w/v)] was used to evaluate the ability of the culture to assimilate various carbon compounds, including amino acids (0.5 %, w/v), when provided as the sole carbon source. Fatty acid methyl esters were prepared according to the method of Sato & Murata (1988) and analysed by GC (Shivaji et al., 2004, 2005b, c). The G+C content of the DNA was determined by the spectrophotometric method (Shivaji et al., 1989). Isoprenoid quinones were extracted according to the method described by Collins et al. (1977), separated by HPLC and identified as described by Reddy et al. (2003). Peptidoglycan was prepared and analysed according to the method described by Komagata & Suzuki (1987). DNA–DNA hybridization was performed by using the membrane filter method (Tourova & Antonov, 1987), as described previously (Shivaji et al., 1992). E. acetylicum DSM 20416T, E. undae DSM 14481T, E. antarcticum DSM 14480T and E. oxidotolerans JCM 12280T were used as reference strains in studies relating to morphology, biochemical characteristics and the identification of fatty acids.
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). The almost-complete rRNA gene sequence (1503 bases) was manually aligned with those of closely related species by using CLUSTAL W (Thompson et al., 1994) (see Supplementary Fig. S1 available in IJSEM Online) and phylogenetic affiliations were inferred using SEQBOOT, DNADIST and neighbour-joining from PHYLIP (Felsenstein, 1993). The Kimura two-parameter method was used for DNA distance calculations (Kimura, 1980), and bootstrap values were generated for 1000 replications of the data. In all cases, the input order of species added to the topology being constructed was randomized with the jumble option, with a random seed of 7 and 10 replications. Majority-rule (50 %) consensus trees were constructed (Page, 1996).
The phenotypic and chemotaxonomic characteristics of strain HHS 31T are presented in the species description and in Table 1
(and in Supplementary Table S1 available in IJSEM Online). Strain HHS 31T, which is Gram-positive, motile, rod-shaped, non-spore-forming, alkaliphilic, possesses MK-7 and MK-8 as the major menaquinones, has the Lys–Gly peptidoglycan type and has a DNA G+C content of 48 mol%, is similar to species of the genus Exiguobacterium (Collins et al., 1983; Farrow et al., 1994; Fruhling et al., 2002; Yumoto et al., 2004; Kim et al., 2005; Lopez-Cortes et al., 2006). Phylogenetic analysis based on the 16S rRNA gene sequence (1480 nt) and performed with the neighbour-joining algorithm further confirms the affiliation of HHS 31T with the genus Exiguobacterium. All of the reported species of Exiguobacterium formed two distinct clusters, as reported previously (Lopez-Cortes et al., 2006; Kim et al., 2005). Strain HHS 31T forms part of one of these clusters, which includes E. acetylicum DSM 20416T, E. oxidotolerans JCM 12280T, E. undae DSM 14481T, E. antarcticum DSM 14480T and E. artemiae DSM 16484T (Supplementary Fig. S1). The coherence of the cluster is obvious from the high bootstrap value (>78 %) between strains in the cluster. The remaining strains, namely E. aurantiacum NCDO 2321T, E. mexicanum DSM 16483T, E. marinum DSM 16307T and E. aestuarii DSM 16306T, form a separate clade.
Using GeneTool 1.0 (http://www.biotools.com), the degrees of affiliation (i.e. 16S rRNA gene sequence similarity) between HHS 31T and reported species of Exiguobacterium were 93.6 % (E. aurantiacum NCDO 2321T; GenBank accession no. X70316), 94 % (E. aestuarii TF-16T; AY594264), 94.2 % (E. marinum TF-80T; AY594266), 94.2 % (E. mexicanum DSM 16483T; AM072764), 96.2 % (E. artemiae DSM 16484T; AM072763), 97.4 % (E. undae DSM 14481T; AJ344151), 97.9 % (E. oxidotolerans T-2-2T; AB105164), 98 % (E. antarcticum DSM 14480T; AJ297437) and 98.9 % (E. acetylicum IFO 12146T; D55730). Thus, strain HHS 31T, which exhibits >97 % 16S rRNA gene sequence similarity with the type strains of E. acetylicum, E. antarcticum, E. oxidotolerans and E. undae, needs to be differentiated from these four species to merit membership of a novel species. At the whole-genome level, as determined by DNA–DNA hybridization when strain HHS 31T was radioactively labelled, the strain showed DNA–DNA hybridization values with E. oxidotolerans JCM 12280T, E. acetylicum DSM 20416T, E. undae DSM 14481T and E. antarcticum DSM 14480T of 50, 63, 67 and 28 %, respectively. However, when E. oxidotolerans JCM 12280T, E. acetylicum DSM 20416T and E. undae DSM 14481T were labelled and used for DNA–DNA hybridization with HHS 31T, the relatedness values were 49, 56 and 60 %, respectively. Furthermore, HHS 31T differs from the closest species, E. oxidotolerans, E. acetylicum, E. undae, E. antarcticum and ‘Exiguobacterium sibiricum’ (Rodrigues et al., 2006), with respect to a number of phenotypic characteristics (Table 1) and also exhibits significant quantitative differences in fatty acid composition (Supplementary Table S1). The predominant fatty acids are iso-C15 : 0 (48.7 %) and iso-C17 : 0 (43 %), which together constituted 
92 % of the total fatty acid content. These two fatty acids were also predominant in the remaining five species of Exiguobacterium (Supplementary Table S1). However, an interesting feature is that the combined levels of iso-C15 : 0 (12–48.7 %) and iso-C17 : 0 (12–43 %) were highest in E. indicum HHS 31T, E. oxidotolerans JCM 12280T and ‘E. sibiricum’ DSM 17290. These three species grow at 2.5 °C, unlike the other species. Thus, these fatty acids (iso-C15 : 0 and iso-C17 : 0) may be required for growth at low temperatures. According to the criteria for species discrimination (Stackebrandt & Goebel, 1994), strain HHS 31T, which exhibits <70 % relatedness at the DNA–DNA level with the type strains of E. oxidotolerans, E. antarcticum, E. acetylicum and E. undae (the most closely related species) and which also differs phenotypically from these four species, represents a novel species of the genus Exiguobacterium, for which the name Exiguobacterium indicum sp. nov. is proposed.
Description of Exiguobacterium indicum sp. nov.
Exiguobacterium indicum (in'di.cum. L. neut. adj. indicum Indian, pertaining to India).
Cells are aerobic, Gram-positive, motile and rod-shaped (2.3 µm long and 0.52 µm wide). Stationary-phase cells are coccobacilli. Colonies (2–4 mm) on nutrient agar are round, shiny, irregular, elevated and orange-coloured after 24 h at 22 °C. No spores are observed. Grows at 5–30 °C and at pH 6–10. The optimum temperature and pH for growth are 25 °C and pH 7.0. Tolerates 5.8 % NaCl and also grows in the absence of salt. Colonies are yellowish orange in colour. The pigment is soluble in chloroform and exhibits multiple absorption maxima at 408, 434, 460 and 495 nm. Positive for catalase, oxidase, 
-galactosidase, phosphatase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, arginine decarboxylase, citrate utilization, malonate utilization, in the Voges–Proskauer test and for reduction of nitrate to nitrite. Negative for urease, lipase, gelatinase, DNase, caseinase, tryptophan deamination, indole production, in the methyl red test, for H2S production and for hydrolysis of aesculin and starch hydrolysis. Utilizes D-glucose, D-galactose, D-rhamnose, L-melibiose, D-cellobiose, sucrose, D-xylose, L-xylose, D-raffinose, glycerol, lactic acid, fumaric acid, N-acetylglucosamine, D-sorbitol, dulcitol, polyethylene glycol, citric acid, sodium acetate, potassium acetate, inulin, dextrin, myo-inositol, glycogen, sodium thioglycolate, methyl 
-D-galactoside, methyl -D-galactoside, arbutin, malic acid, erythritol, sodium gluconate, sodium fumarate, sodium formate, sodium succinate, 
-glucuronic acid, -ketoglutaric acid, valeric acid, 5-ketogluconate, L-glycine, L-ornithine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, L-threonine, L-lysine, L-arginine, L-glutamic acid, L-aspartic acid, glutamine, L-asparagine, L-methionine, L-tyrosine, L-tryptophan, L-proline, L-histidine and L-creatinine, but not L-sorbose, L- or D-arabinose, D-fructose, D-mannose, D-trehalose, D-mannitol, D-ribose, D-lactose, D-adonitol, maltose, D-melezitose, hydroxybutyric acid, dextran, sodium propionate, cellulose, starch, pyruvate, methyl -D-mannoside, methyl -D-glucoside, salicin, amygdalin, xylitol, L-fucose, L-cysteine or L-phenylalanine. Negative for acid production from inulin, D-glucose, sucrose, D-fructose, D-sorbitol, L-melibiose, D-lactose, D-trehalose, maltose, L-rhamnose, D-mannose, L-arabinose, L-xylose, D-ribose, D-cellobiose and adonitol. Resistant to discs containing the following antibiotics (µg): norfloxacin (10), cotrimoxazole (25), clindamycin (25), doxycycline (25), sulfamethoxazole (50), amoxicillin (30), amikacin (30), nalidixic acid (30), nitrofurantoin (300) and colistin (10). Sensitive to the following antibiotics (µg): tobramycin (15), lomefloxacin (30), roxithromycin (30), ciprofloxacin (30), penicillin (10), cefoperazone (75), vancomycin (30), cefuroxime (30), lincomycin (15), cefotaxime (30), cefazolin (30), kanamycin (30), novobiocin (30), chloramphenicol (30), ampicillin (25), tetracycline (30), streptomycin (25), erythromycin (15), bacitracin (10), gentamicin G (30), polymyxin B (50), oleandomycin (15), spectinomycin (100), rifampicin (25) and carbenicillin (100). Major respiratory quinones are MK-7 and MK-8. Peptidoglycan is of the Lys–Gly type. Phosphatidylglycerol and diphosphatidylglycerol are the main phospholipids; minute amounts of phosphatidylethanolamine are also present. The DNA G+C content is 48.0 mol%. The fatty acids present are iso-C13 : 0 (0.2 %), iso-C15 : 0 (48.7 %), iso-C16 : 0 (3.0 %), C16 : 1 (0.3 %), iso-C17 : 0 (43.0 %), anteiso-C17 : 0 (0.3 %) and C18 : 1 (2.0 %).
The type strain, HHS 31T (=LMG 23471T=IAM 15368T), was isolated from melt water from the Hamta glacier located at a height of 4279 m above sea level in the Himalayan mountain ranges of India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230.
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