Introduction

Micro-organisms requiring salt for growth, the halophiles, are found among all the domains of life, the Archaea, Eucarya, and Bacteria (Oren 2002). The different branches of the phylum Proteobacteria have various halophilic representatives with close relatives that are non-halophilic (Oren 2002). Among the bacterial families that form part of the class Gammaproteobacteria, the family Halomonadaceae is mainly represented by halophilic and halotolerant species belonging to different genera. The genus Halomonas was first established by Vreeland et al. (1980) and the members are mainly represented by halophilic and halotolerant species. The aim of this study was to determine the exact taxonomic position of the halophilic strains YIM 93003T and YIM 94343T using phenotypic, phylogenetic and genomic characteristics, from which they were finally determined as two new species belonging to the genus Halomonas.

Materials and methods

Strains and culture conditions

The samples used in this study were shore sediments obtained from Qijiaojing Lake, which is a salt lake in Xinjiang province, north-west China (43°23′01″ N 91°36′11″ E). Two strains, YIM 93003T and YIM 94343T were isolated by using the dilution-plating technique on Glucose–Tryptone–Yeast (GTY) medium (contained 10% NaCl) described by Tang et al. (2010) and incubated at 37°C for 1 week. Colonies were picked and repeatedly re-streaked onto tryptic soy agar (TSA, BD) medium containing 5% NaCl, until purity was confirmed. Strains YIM 93003T and YIM 94343T were maintained on TSA slants containing 5% NaCl at 4°C and in 10% (v/v) glycerol suspensions at −80°C. Strain YIM 93003T was deposited in the Collection Center of Typical Cultures, China (CCTCC) as strain CCTCC AB 208133T and in the Korean Collection for Type Cultures (KCTC) as strain KCTC 22228T. Strain YIM 94343T was deposited in the CCTCC as strain CCTCC AB 2010382T and in the KCTC as strain KCTC 23356T. Biomass for chemical and molecular studies was obtained by cultivation in shake flasks (about 200 r.p.m) using tryptic soy broth (TSB, BD) containing 5% NaCl at 37°C for about 3 days.

Phenotypic characterization

Gram staining was carried out using the standard Gram reaction combined with the KOH lysis test (Cerny 1978). Colony morphology was observed on TSA medium containing 5% NaCl (pH 7.5) after incubation at 37°C for 6, 12, 24, 48, and 72 h and cellular morphology was examined using scanning electron microscopy (QUANTA200; FEI). Cell motility was confirmed by the presence of turbidity throughout a tube containing semisolid medium (Leifson 1960). Accumulation of Poly-β-hydroxyalkanoates (PHB) was determined by the Sudan Black staining method (Smibert and Krieg 1994) under a light microscope. Extracellular polysaccharide production was observed using the phenol/sulfuric acid method with glucose as a standard (Zhou et al. 2007). Growth was tested at various temperatures (4, 10, 15, 20, 28, 37, 40, 45, 50, and 55°C) on TSA medium containing 5% NaCl, pH 7.5 for 3–10 days. The pH range for growth was investigated between pH 4.0 and 10.0 (in increments of 1 pH unit) with the buffer system described by Xu et al. (2005). Liquid cultures were grown in tubes at 37°C for 3–14 days, using TSB medium containing 5% NaCl, pH 7.5 as the basal medium. The salt concentrations ranged from 0 to 30% (w/v) at intervals of 1% were tested by using TSA medium without NaCl as the basal medium. Catalase activity was determined by assessing bubble production after the addition of a drop of 3% H2O2. Oxidase activity was determined by assessing the oxidation of tetramethyl-p-phenylenediamine. Hydrolysis of casein, gelatin, starch, Tweens (20, 40, 60, 80) and urease activity were determined as described by Cowan and Steel (1965). Methyl red and Voges–Proskauer tests, hydrolysis of aesculin and ONPG, indole and H2S production from l-cysteine, nitrate and nitrite reduction, oxidation/fermentation of d-glucose, respiration in fumarate, nitrate and nitrite, DNAse activity were tested as recommended by Smibert and Krieg (1994). The utilization of different compounds as sole carbon or nitrogen and energy sources was tested as described by Carrasco et al. (2006). Antibiotic susceptibility was determined by the method of Williams (1967). Other enzymatic activities were assayed by using API ZYM strips (bioMérieux) according to the manufacturer’s instructions. Acid production from carbohydrates was determined by using the API 50 CHB system (bioMérieux). Halomonas anticariensis DSM 16096T, obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), was used as a reference strain for physiological and biochemical characteristics tests. All the tests in this study were repeated three times.

Chemotaxonomy and determination of G+C content of DNA

Chemotaxonomic properties, including quinones and fatty acids were analyzed. For fatty acids analysis, cells of strains YIM 93003T and YIM 94343T were cultured on tryptic soy broth (TSB; BD) containing 5% NaCl, pH 7.5 at 37°C shaking for 48 h (about 200 r.p.m), cellular fatty acids were extracted, methylated and analysed by using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. The fatty acid methyl esters were analyzed by using the Microbial Identification software package (Sherlock Version 4.0; MIDI database: TSBA40). Isoprenoid quinones were extracted and purified as described by Komagata & Suzuki (1987). The purified ubiquinones were dissolved in acetone and separated by reversed-phase HPLC. To determine the G+C contents of strains YIM 93003T and YIM 94343T, genomic DNAs were prepared according to the method of Marmur (1961). The G+C contents of the DNAs were determined by reversed-phase HPLC (Mesbah et al. 1989) with DNA from H. anticariensis DSM 16096T as a control.

16S rRNA gene sequencing, phylogenetic analysis and DNA–DNA hybridizations

Extraction of genomic DNA and PCR amplification of 16S rRNA gene was done as described by Li et al. (2007). The two sequences obtained were compared with reference 16S rRNA gene sequences retrieved from GenBank/EMBL by means of a BLAST search and the EzTaxon server 2.0 (Chun et al. 2007). Multiple alignments with sequences of the most closely related bacteria and calculations of levels of sequence similarity were carried out using CLUSTAL_X (Thompson et al. 1997). Phylogenetic analyses were performed using three tree-making algorithms: neighbor-joining (Saitou and Nei 1987), maximum likelihood (Felsenstein 1981), and maximum parsimony (Fitch 1971). A neighbor-joining phylogenetic tree was constructed from Knuc values (Kimura 1980) using MEGA, version 4.0 (Tamura et al. 2007), a maximum-parsimony phylogenetic tree was constructed by using MEGA, version 4.0 (Tamura et al. 2007), and a maximum likelihood phylogenetic tree was constructed by using PHYML (Guindon and Gascuel 2003). The topology of the phylogenetic tree was evaluated using the bootstrap resampling method, with 1000 replicates (Felsenstein 1985). The sequence of Marinospirillum alkaliphilum Z4T (AF275713) was used as an outgroup. DNA–DNA relatedness were studied by using the optical renaturation method (De Ley et al. 1970; Huß et al. 1983; Jahnke 1992) on a UV–Vis spectrophotometer (model UV1601; Shimadzu) under optimal hybridization conditions. Every hybridization experiment was performed with three replicates and the DNA–DNA relatedness values expressed as the means of the three values.

Results and discussion

Phenotypic characteristics

Two novel strains were isolated from samples of shore sediments obtained from Qijiaojing Lake, a salt lake in Xinjiang province, north-west China. Strains YIM 93003T and YIM 94343T had typical morphological characteristics consistent with members of the genus Halomonas. The cells of the novel strains YIM 93003T and YIM 94343T were both aerobic, Gram-negative rods of sizes 0.4–0.7 × 1.8–2.8 and 0.4–0.5 × 1.7–2.4 μm, respectively. Neither strain was motile. After incubation at 37°C for 48 h, the colonies on TSA medium were circular with entire margins, convex, smooth and yellow; and approximately 1–2 and 1–1.5 mm in diameter, respectively. The two strains grew well on TSA medium, with growth observed for the temperature range 20–40°C (optimal growth for each at 37°C) an the pH range 6–9 (optimal growth for each at pH 7.5). The NaCl range for growth of strains YIM 93003T and YIM 94343T was 0.5–24% (w/v) NaCl and 0.5–23% (w/v) NaCl, respectively, with optimal growth for both at 5–7% (w/v) NaCl. The phenotypic characteristic of strains YIM 93003T and YIM 94343T were distinctly different from H. anticariensis DSM 16096T, which is shown in detail in Table 1. The detailed physiological and biochemical characteristics of the strain are given in the species description.

Table 1 Different characteristics between the novel species and its closest phylogenetic neighbor, H. anticariensis DSM 16096T
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Chemotaxonomic characteristics

The predominant respiratory quinone found in strain YIM 93003T and YIM 94343T were Q-9, and the major cellular fatty acids of strain YIM 93003T were C18:1ω7c (43.5%), C16:0 (22.1%) and Summed feature 3 (C16:1 ω7c/iso-C15:02-OH) (12.3%); in strain YIM 94343T are C18:1ω7c (48.5%), C16:0 (23.3%) and Summed feature 3 (C16:1 ω7c/iso-C15:02-OH) (12.6%). The fatty acids that account for more than 0.1% total fatty acids in both strains YIM 93003T, YIM 94343T and DSM 16096T are detailed in Table 2. The chemotaxonomic properties of strains YIM 93003T and YIM 94343T, such as the predominant respiratory quinine and the major fatty acids were similar to those reported for H. anticariensis DSM 16096T (Martínez-Cánovas et al. 2004). The G+C contents of the strains YIM 93003T and YIM 94343T were as 63.4 and 64.0 mol%, respectively.

Table 2 Fatty acid profiles of strains YIM 93003T, YIM 94343T and H.anticariensis DSM 16096T
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Phylogenetic analysis

The almost complete 16S rRNA gene sequences of strain YIM 93003T (1474 bp) and YIM 94343T (1561 bp) were determined in this study. The GenBank accession numbers of the 16S rRNA gene sequences of strains YIM 93003T and YIM 94343T are HQ832735 and HQ832736, respectively. Alignment data showed that strains YIM 93003T and YIM 94343T had the highest level of 16S rRNA gene sequence similarity with respect to members of the Gammaproteobacteria, in particular with respect to members of the genus Halomonas. In the phylogenetic tree based on the neighbor-joining algorithm, strains YIM 93003T and YIM 94343T clustered together with the type strain of H. anticariensis DSM 16096T (Fig. 1); this relationship was supported by all of the tree-making methods used in this study (Fig. 1, Figs. S2, S3). The 16S rRNA gene sequences indicated that the closest relative strain with YIM 93003T and YIM 94343T was the type strain H. anticariensis DSM 16096T (97.5 and 97.4% sequence similarity, respectively). Levels of the 16S rRNA gene sequences similarity between strains YIM 93003T, YIM 94343T and the other Halomonas species were less than 97%, ranging from 96.7 [Halomonas rifensis HK31T (HM026177)] to 91.9% [Halomonas magadiensis 21M1T (X92150)], and the similarity between strains YIM 93003T and YIM 94343T was 99.4%. The results of the 16S rRNA gene sequence comparisons clearly demonstrated that strains YIM 93003T and YIM 94343T were two members of the genus Halomonas. DNA–DNA relatedness were studied by using the optical renaturation method on a UV–Vis spectrophotometer (model UV1601; Shimadzu) under optimal hybridization conditions. The DNA–DNA hybridization relatedness values were expressed as the means of the three values. The DNA–DNA hybridization values for strains YIM 93003T and YIM 94343T, YIM 93003T and DSM 16096T, YIM 94343T and DSM 16096T were 38.1 ± 3.0, 18.3 ± 4.7, and 20.8 ± 4.6%, respectively, which are well below the 70% cut-off point recommended for assignment of strains to the same genomic species (Wayne et al. 1987), thus suggesting that the strains YIM 93003T and YIM 94343T should be considered as two different genomic species of the genus Halomonas.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strains YIM 93003T and YIM 94343T among species of genus Halomonas. The sequence of Marinospirillum alkaliphilum Z4T (AF275713) was used as an outgroup. Asterisks indicate branches of the tree that were also found using the maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) tree-making algorithms. Numbers on branch nodes are bootstrap values (1000 resamplings, only values over 50% are given). Bar, 0.01 substitution per 100 nucleotide positions

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Taxonomic conclusion

The combination of phylogenetic, chemotaxonomic and phenotypic data indicated that strains YIM 93003T and YIM 94343T were two members of the genus Halomonas. However on the basis of phenotypic, phylogenetic differences (Table 1) and the DNA–DNA hybridization values between these novel strains and previously described species H. anticariensis DSM 16096T within the genus Halomonas, the strains YIM 93003T and YIM 94343T should be considered to represent two different novel species of the genus Halomonas, for which the names Halomonas qijiaojingensis sp. nov., and Halomonas flava sp. nov., are proposed.

Description of Halomonas qijiaojingensis

Halomonas qijiaojingensis (qi.jiao’jing.en’sis. N.L. fem. adj. qijiaojingensis pertaining to Qijiaojing Lake, Xinjiang Province, north-west China, where the sample from which the type strain was isolated and was collected).

Cells are aerobic, Gram-negative rods (0.4–0.7 × 1.8–2.8 μm), not motile. Colonies on TSA medium are circular with entire margins, convex, smooth and yellow, and approximately 1–2 mm in diameter after incubation at 37°C for 2 days. The type strain grow well on the TSA medium. Growth ranges for temperature, pH, and NaCl are 20–40°C, pH 6–9 and 0.5–23% (w/v) NaCl, with optimal growth at 37°C, pH 7.5, and 5–7% (w/v) NaCl. Nitrate is reduced. Oxidase- and catalase-positive. Hydrolyzes urea, while aesculin, gelatin, casein, ONPG, starch and, Tweens 20, 40, 60, and 80 are not hydrolyzed. Poly-β-hydroxyalkanoates, exopolysaccharide, H2S, indole production, methyl red and Voges–Proskauer tests give negative results. Oxidation/fermentation of d-glucose; respiration in fumarate, nitrate and nitrite; and DNAse activity all give negative results. Utilizes d-galactose, l-arabinose, citrate, dulcitol, glycerol, d-glucose, d-mannitol, d-xylose, maltose, sodium citrate, d-sorbitol, sucrose, trehalose, xylitol as sole carbon sources; inositol, lactose, l-rhamnose, raffinose are not utilized. Utilizes alanine, dl-α-alanine, glutamic acid, histidine, hypoxanthine, l-arginine, l-asparagine, l-threonine, lysine, ornithine, phenylalanine, proline, tyrosine, valine, xanthine as sole nitrogen sources; dl-methionine, glycine are not utilized. In the API 50 CHB system, acid is produced from d-adonitol, d-fructose, d-fucose, d-glucose, glycerol, maltose, d-mannose, sucrose, trehalose, d-turanose, but not from N-acetylglucosamine, aesculin, amygdalin, arbutin, d-arabinose, l-arabinose, d-arabitol, l-arabitol, cellobiose, dulcitol, erythritol, l-fucose, d-galactose, gentiobiose, glycogen, inositol, inulin, d-lactose, d-lyxose, d-mannitol, melezitose, melibiose, methyl α-d-glucopyranoside, methyl α-d-mannopyranoside, methyl β-d-xylopyranoside, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, raffinose, -ribose, rhamnose, salicin, d-sorbitol, sorbose, starch, d-tagatose, xylitol, d-xylose and l-xylose. In the API ZYM system, alkaline phosphatase and α-glucosidase give positive results; acid phosphatase, N-acetyl-β-glucosaminidase, arylamidase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), α-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, leucine arylamidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin, valine arylamidase are negative. The type strain is sensitive to the following antibiotics (μg per disc, unless indicated otherwise): amikacin (30), ampicillin (10), chloramphenicol (30), ciprofloxacin (5), gentamicin (10), norfloxacin (10), tobramycin (10), sulfamethoxazole (23.75). Resistance is exhibited only to vancomycin (30), erythromycin (15), penicillin G (10 Iu/disc) and clindamycin (3). Q-9 is the predominant ubiquinone. The major cellular fatty acids (>10%) are C18:1ω7c (43.5%), C16:0 (22.1%) and Summed feature 3 (C16:1 ω7c/iso-C15:02-OH) (12.3%). The DNA G+C content is 63.4 mol%.

The type strain, YIM 93003T (= CCTCC AB 208133T = KCTC 22228T), was isolated from Qijiaojing Lake, Xinjiang province, north-west China.

Description of H. flava

Halomonas flava (fla.va. L. fem. adj. flava, yellow, reflecting the colour of colonies).

Cells are aerobic, Gram-negative rods (0.4–0.5 × 1.7–2.4 μm), not motile. Colonies on TSA medium are circular with entire margins, convex, smooth and yellow, and approximately 1–1.5 mm in diameter after incubation at 37°C for 2 days. The type strain grows well on TSA medium. Growth ranges for temperature, pH and NaCl are 20–40°C, pH 6–9 and 0.5–23% (w/v) NaCl, with optimal growth at 37°C, pH 7.5 and 5–7% (w/v) NaCl. Nitrate is reduced. Oxidase- and catalase-positive. Hydrolyezs aesculin, while casein, gelatin, ONPG, starch, urea and Tweens 20, 40, 60 and 80 are not hydrolysed. Poly-β-hydroxyalkanoates, exopolysaccharide, H2S, indole production and methyl red tests give negative results. Positive for Voges-Proskauer test. Oxidation/fermentation of d-glucose; respiration in fumarate, nitrate and nitrite; DNAse activity each give negative results. Utilizes dulcitol, glycerol, d-glucose, l-arabinose, d-mannitol, maltose, sodium citrate, d-sorbitol, sucrose, trehalose, xylitol as sole carbon sources; d-galactose, d-xylose, inositol, lactose, l-rhamnose, raffinose are not utilized. Utilizes alanine, citrate, glutamic acid, histidine, hypoxanthine, l-arginine, l-asparagine, l-threonine, lysine, ornithine, phenylalanine, proline, tyrosine, valine, xanthine as sole nitrogen sources; dl-methionine, glycine, dl-α-alanine are not utilized. In the API 50 CHB system, acid is produced from aesculin, l-arabinose, arbutin, cellobiose, d-fructose, d-glucose, glycerol, d-mannitol, d-mannose, d-ribose, salicin, sucrose, d-tagatose, trehalose, but not from amygdalin, d-adonitol, d-arabinose, d-arabitol, l-arabitol, dulcitol, erythritol, d-fucose, l-fucose, d-galactose, gentiobiose, glycogen, inositol, inulin, d-lactose, d-lyxose, maltose, melezitose, melibiose, methyl α-d-glucopyranoside, methyl α-d-mannopyranoside, methyl β-d-xylopyranoside, N-acetylglucosamine, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, raffinose, rhamnose, d-sorbitol, sorbose, starch, d-turanose, d-xylose, xylitol and l-xylose. In the API ZYM system, acid phosphatase, alkaline phosphatase, α-glucosidase, β-glucosidase give positive results; N-acetyl-β-glucosaminidase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), α-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase, leucine arylamidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin, valine arylamidase are negative. The type strain is sensitive to the following antibiotics (μg per disk, unless indicated otherwise): amikacin (30), ampicillin (10), chloramphenicol (30), ciprofloxacin (5), erythromycin (15), gentamicin (10), penicillin G (10 Iu/disc), norfloxacin (10), sulfamethoxazole (23.75). Resistance is exhibited only to tobramycin (10), vancomycin (30) and clindamycin (3). Q-9 is the predominant ubiquinone. The major cellular fatty acids (>10%) are C18:1ω7c (48.5%), C16:0 (23.3%) and Summed feature 3 (C16:1 ω7c/iso-C15:02-OH) (12.6%). The DNA G+C content is 64.0 mol%.

The type strain, YIM 94343T (=CCTCC AB 2010382T =KCTC 23356T), was isolated from Qijiaojing Lake, Xinjiang province, north-west China.