Halomonas caseinilytica sp. nov., a halophilic bacterium isolated from a saline lake on the Qinghai-Tibet Plateau, China

A halophilic, Gram-negative bacterial strain, designated AJ261^T, which was isolated from a soil sample from a salt lake on the Qinghai–Tibet Plateau, was subjected to a polyphasic taxonomic study. The isolate grew optimally in the presence of 3–5 % NaCl and used various carbohydrates as sole carbon and energy sources. The genomic DNA G+C content was 63.0 mol%. The predominant fatty acids were C_{18 : 1}ω7c, C_{16 : 0} and C_{12 : 0}. A phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate had the highest sequence similarity with respect to type strains of Halomonas elongata (98.2 %), Halomonas eurihalina (98.1 %) and Halomonas halmophila (97.2 %). The DNA–DNA relatedness of strain AJ261^T with respect to H. elongata NBRC 15536^T, H. eurihalina CGMCC 1.2318^T and H. halmophila DSM 5349^T was 42, 25 and 26 %, respectively. Overall, the phenotypic, genotypic and phylogenetic results demonstrate that strain AJ261^T represents a novel species within the genus Halomonas, for which the name Halomonas caseinilytica is proposed. The type strain is AJ261^T (=CGMCC 1.6773^T =JCM 14802^T).

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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AJ261^T is EF527874.

Micrographs of strain AJ261^T and detailed fatty acid compositions of strain AJ261^T and related type strains are available as supplementary material with the online version of this paper.

The family Halomonadaceae of the Gammaproteobacteria includes four genera of halophilic bacteria, Halomonas, Chromohalobacter, Cobetia and Modicisalibacter, and three genera of non-halophilic bacteria, Carnimonas, Halotalea and Zymobacter (Franzmann et al., 1989; Ventosa et al., 1989; Okamoto et al., 1993; Dobson & Franzmann, 1996; Garriga et al., 1998; Arahal et al., 2002, 2007; Ntougias et al., 2007; Ben Ali Gam et al., 2007). The genus Halomonas was proposed by Vreeland et al. (1980), with Halomonas elongata as the type species. As of November 2007, the genus Halomonas contained 48 species (Euzéby, 1997) and is the largest genus in the family Halomonadaceae.

This study describes a novel halophilic bacterium, designated AJ261^T, which belongs to the genus Halomonas. Strain AJ261^T was isolated from a soil sample from the Ayakekum salt lake (3 ° 33' N 8 ° 42' E; 3884 m altitude) on the Qinghai–Tibet Plateau, China. The lake environment has been described previously (Xu et al., 2005). The samples were incubated with halophilic medium (HM) (Ventosa et al., 1982) for approximately 3 days and the liquid was plated by using a 10-fold dilution series.

HM contained the following (l^–1 distilled water): 50.0 g NaCl, 2.0 g KCl, 1.0 g MgSO₄, 0.36 g CaCl₂ . 2H₂O, 0.23 g NaBr, 0.06 g NaHCO₃, trace FeCl₃, 10.0 g yeast extract (Difco), 5.0 g peptone (Difco) and 1.0 g glucose (pH 7.5). After 1 week incubation at 30 °C, a representative colony was picked and maintained on HM. The strain was purified by repeated restreaking; purity was confirmed by the uniformity of cell morphology. Cell morphology and motility were examined by using optical (BX40; Olympus), transmission electron (H-600; Hitachi) and scanning electron microscopy (S260; Cambridge) (see Supplementary Fig. S1, available in IJSEM Online).

The optimal conditions for growth were determined in HM with different salt concentrations (0, 0.5, 1, 3, 5, 7.5, 10, 15, 20, 25 and 30 %, w/v). The pH range for growth was determined by adding MES (pH 5.0–6.0), PIPES (pH 6.5–7.0), Tricine (pH 7.5–8.5) and CHES (pH 9.0–10.0) to the HM at a concentration of 25 mM. The temperature range for growth was determined by using incubation temperatures ranging from 4 to 48 °C. Biochemical and nutritional tests were tested in HM according to the methods of Mata et al. (2002). Antimicrobial-susceptibility tests were performed in liquid HM containing the antimicrobial agent at 50 µg ml^–1. Detailed results are given in the species description.

The 16S rRNA gene was amplified and analysed as described previously (Xu et al., 2007). The almost-complete 16S rRNA gene sequence (1446 bp) of strain AJ261^T was determined. The sequence was compared with closely related sequences of reference organisms from the FASTA network service. Strain AJ261^T showed the highest levels of sequence similarity with respect to type strains of H. elongata (98.2 %), Halomonas eurihalina (98.1 %), Halomonas koreensis (97.3 %) and Halomonas halmophila (97.2 %) and showed less than 97.0 % sequence similarity with respect to other Halomonas species. Sequence data were aligned with CLUSTAL W 1.8 (Thompson et al., 1994). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods (Fitch, 1971) with the MEGA3 program package (Kumar et al., 2004) and using the maximum-likelihood method (Felsenstein, 1981) with the PHYLIP 3.6 program. Evolutionary distances were calculated according to the algorithm of the Kimura two-parameter model (Kimura, 1980) for the neighbour-joining method. Strain AJ261^T always showed the closest phylogenetic affinity to H. elongata, H. eurihalina, H. halmophila and H. almeriensis, with high levels of bootstrap support (Fig. 1).

(54K): Fig. 1. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the phylogenetic relationships of strain AJ261T and related taxa. Bootstrap percentages (based on 1000 replicates) >60 % are shown at branch points. Bar, 0.01 substitutions per nucleotide position.

Fatty acid methyl esters were prepared from lipids extracted from cells grown in HM for 24 h at 30 °C and were analysed by using GC/MS (Kuykendall et al., 1988); data are given in Supplementary Table S1 (available with IJSEM Online). The unsaturated fatty acid content of strain AJ261^T was higher than those of H. elongata NBRC 15536^T and H. eurihalina CGMCC 1.2318^T. The G+C content of genomic DNA was determined by thermal denaturation (T_m) (Marmur & Doty, 1962) using Escherichia coli K-12 DNA as the calibration standard. DNA–DNA hybridizations were performed using the thermal denaturation and renaturation method of De Ley et al. (1970), as modified by Huß et al. (1983), using a Beckman DU 800 spectrophotometer. The hybridization temperature used was 79 °C and the experiments were carried out in triplicate. The DNA–DNA relatedness values between strain AJ261^T and H. elongata NBRC 15536^T, H. eurihalina CGMCC 1.2318^T and H. halmophila DSM 5349^T were 42, 25 and 26 %, respectively. In addition, comparisons of phenotypic properties (Table 1) also indicated differences between strain AJ261^T and recognized Halomonas species, such as cell morphology, colony pigmentation, motility, salt or temperature range for growth, methyl red test results, hydrolysis of substrates and utilization of carbohydrates.

Table 1. Phenotypic characteristics that serve to differentiate strain AJ261T from related Halomonas species Taxa: 1, AJ261T; 2, H. elongata NBRC 15536T; 3, H. eurihalina CGMCC 1.2318T (data in columns 1–3 from this study unless indicated); 4, H. almeriensis (Martínez-Checa et al., 2005); 5, H. halmophila (Dobson et al., 1990; Mata et al., 2002). +, Positive; –, negative; ND, no data available.

On the basis of phenotypic, genotypic and phylogenetic data, therefore, strain AJ261^T represents a novel species of the genus Halomonas, for which the name Halomonas caseinilytica sp. nov. is proposed.

Description of Halomonas caseinilytica sp. nov.
Halomonas caseinilytica (ca.se.i.ni.ly'ti.ca. N.L. n. caseinum casein; Gr. adj. lutikos able to loosen, able to dissolve; N.L. fem. adj. lytica dissolving; N.L. fem. adj. caseinilytica casein-dissolving).

Cells are Gram-negative, motile, short rods or oval in shape, 0.8–2.0x0.4–0.6 µm, and occur singly or in pairs. Colonies on complex agar medium are 1–2 mm in diameter, smooth, circular, elevated and light yellow after 2 days. Moderately halophilic. No growth occurs in the absence of salt. The total salts concentration for growth is 0.5–15 % (w/v), with an optimum at 3–5 %. Grows at pH 5.0–9.0 and 4–48 °C (optimum growth at pH 7.0–8.0 and 30 °C). Does not grow anaerobically in the presence of nitrate, nitrite or fumarate. Oxidase-negative and catalase-positive. Grows on MacConkey agar, but not on cetrimide agar. Produces exopolysaccharide and poly-β-hydroxyalkanoate. Aesculin, casein, gelatin, Tween 20 and tyrosine are hydrolysed. DNA, starch and Tween 80 are not hydrolysed. Negative for gluconate oxidation, indole production, phenylalanine deamination, lecithinase, ornithine decarboxylase and urease. Positive for lysine decarboxylase, ONPG, in the methyl red test and for fermentation of D-glucose. Reduces selenite and nitrate. H₂S is formed from thiosulfate or L-cysteine. Chemo-organotrophic. The following substrates are utilized for growth: L-arabinose, cellobiose, D-fructose, D-galactose, glucose, glycerol, inositol, lactose, maltose, mannitol, D-mannose, raffinose, rhamnose, L-sorbitol, sucrose, trehalose, D-xylose, acetate, citrate, fumarate, gluconate, lactate, malate, propionate, pyruvate, succinate, L-alanine, L-arginine, L-aspartate, L-glutamate, glycine, L-isoleucine, L-histidine, L-ornithine, L-serine and L-valine. The following compounds are not utilized as sole carbon and energy sources: adonitol, L-cysteine, ethanol, formate, malonate, L-methionine, ribose, sorbose and starch. Acid is produced from L-arabinose, D-fructose, D-galactose, glucose, inositol, lactose, maltose, mannitol, D-mannose, rhamnose, L-sorbitol, sucrose and trehalose. Susceptible to chloramphenicol, kanamycin, nalidixic acid, neomycin, nitrofurantoin, polymyxin B, rifampicin and tetracycline, but not to ampicillin, bacitracin, carbenicillin, cefotaxime, erythromycin, penicillin, novobiocin, nystatin or streptomycin. The major fatty acids are C_{18 : 1}ω7c, C_{16 : 0} and C_{12 : 0}/C_{12 : 0} 3-OH. The DNA G+C content of the type strain is 63.0±0.1 mol% (T_m).

The type strain, AJ261^T (=CGMCC 1.6773^T =JCM 14802^T), was isolated from a soil sample collected from a salt lake on the Qinghai–Tibet Plateau, China.