Introduction

The genus Zhihengliuella, belonging to the suborder Micrococcineae (Stackebrandt et al. 1997), was created by Zhang et al. (2007) with the description of Z. halotolerans as the sole recognized species of the genus. The genus has been recently emended by Tang et al. (2009) along with the proposal of Z. alba. The genus was defined as mesophilic, aerobic, catalase-positive, oxidase-negative, non-motile, non-sporulating, short rod-shaped, Gram-positive bacterium, having cell-wall peptidoglycan type A4α (l-Lys–l-Ala–l-Glu), with tyvelose and mannose as the major cell-wall sugars, MK-9 and MK-10 as the predominant menaquinones, anteiso-C15:0 and iso-C15:0 as the major fatty acids and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol as the major polar lipids. The type strains of the two recognized Zhihengliuella species were all isolated from saline soil in north-west China. In a recent study of the microbial diversity in the ancient salt deposit of the Xiangli Salt Mine in Hunan Province, China (Chen et al. 2009), a moderately halophilic bacterium, designated strain JSM 071043T, was isolated from a subterranean brine sample (g L−1: Na+ 81.5, Mg2+ 28.3, K+ 4.5, Ca2+ 0.1, pH 7.6). Based on the results of the present polyphasic taxonomic study, this strain was proposed to represent a novel species of the genus Zhihengliuella.

Materials and methods

Strains and culture conditions

Strain JSM 071043T was isolated from a subterranean brine sample by plating 1:10 serial dilutions of the sample on marine agar 2216 (MA; Difco) supplemented with 5% (w/v) NaCl (hereafter MA5) at 30°C for 2 weeks. After primary isolation, the strain was purified by repeated streaking and subculturing on MA5 plates and examining the cultures by light microscopy. It was maintained either as serial transfers on MA5 slants, or lyophilized cultures at 4°C, or deep-frozen in 20% (v/v) glycerol at −80°C. For comparison, two type strains, Z. halotolerans YIM 70185T and Z. alba YIM 90734T, were obtained from the Yunnan Institute of Microbiology (YIM, 650091 Kunming, China). Unless indicated otherwise, morphological, physiological, molecular and chemotaxonomic studies were performed with cells grown on MA5 (pH 8.5) at 28°C.

Phenotypic characterization

Cell morphology was examined by using light microscopy (model DM3000; Leica). The Gram staining and the KOH lysis test were carried out according to Doetsch (1981) and Gregersen (1978), respectively. Growth in the absence of salt was investigated on nutrient agar (NA) prepared according to the formula of Atlas and Parks (1993) except that NaCl was excluded. Tolerance of salt was tested on NA in different salt (NaCl, KCl, MgCl2·6H2O) concentrations [0.1 and 0.5% (w/v), and 1–30% (w/v) at increments of 1%]. Growth was tested at various temperatures (4, 5–55°C, at increments of 5°C) and at different pH (5.0–11.0, at increments of 0.5 pH units) on MA5 as well as in nutrient broth (Atlas and Parks 1993) supplemented with 7% (w/v) NaCl. For pH tolerance experiments, the buffer solutions described by Chen et al. (2004) were used. Growth under anaerobic conditions was determined on MA5 supplemented with 0.5% (w/v) glucose and with or without 0.1% (w/v) nitrate by using the GasPak Anaerobic Systems (BBL) according to the manufacturer’s instructions. Methyl red and Voges–Proskauer tests and determination of hydrolysis of aesculin, indole and H2S production, nitrate and nitrite reduction, and lysine decarboxylase, phenylalanine deaminase and ornithine decarboxylase were assessed as recommended by Smibert and Krieg (1994). Hydrolysis of casein, cellulose, chitin, DNA, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, urea and xanthine was determined as described by Cowan and Steel (1965). Determination of acid production from carbohydrates and utilization of carbon and nitrogen sources was performed as recommended by Ventosa et al. (1982). Observation of motility and tests of catalase and oxidase activities were determined as described previously (Chen et al. 2007). Other enzymic activities were also assayed by using API ZYM strips (bioMérieux) according to the manufacturer’s instructions with 7.5% (w/v) NaCl.

Determination of 16S rRNA gene sequence, phylogenetic analysis and DNA–DNA hybridization

The 16S rRNA gene sequence was amplified by PCR and sequenced as described by Cui et al. (2001). Pairwise sequence similarities were calculated using a global alignment algorithm, implemented at the EzTaxon server (Chun et al. 2007). Phylogenetic analysis was performed by using the software package MEGA version 3.1 (Kumar et al. 2004) after multiple alignment of sequence data by CLUSTAL_X (Thompson et al. 1997). Distances were calculated using distance options according to Kimura’s two-parameter model (Kimura 1980) and clustering was performed with the neighbour-joining method (Saitou and Nei 1987). Maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Kluge and Farris 1969) trees were generated by using the treeing algorithms contained in the PHYLIP package (Felsenstein 2002). Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by means of 1,000 resamplings (Felsenstein 1985). DNA–DNA hybridization experiments were carried out using the optical renaturation method (De Ley et al. 1970; Huß et al. 1983; Jahnke 1992).

Chemotaxonomic characterization

Purified cell-wall preparations were obtained and hydrolyzed as described by Schleifer and Kandler (1972). Sugars, amino acids and peptides in cell-wall hydrolysates were analyzed by HPLC as described by Tang et al. (2009). Polar lipids were extracted according to the method of Minnikin et al. (1984) and identified by two-dimensional TLC and spraying with appropriate detection reagents (Collins and Jones 1980). Isoprenoid quinones were analyzed by means of HPLC as described by Groth et al. (1996). Fatty acid compositions were determined according to Sasser (1990) by using the Microbial Identification System (Microbial ID). Genomic DNA was isolated according to Hopwood et al. (1985) and the G + C content was determined using the HPLC method (Mesbah et al. 1989).

Results and discussion

Phenotypic characteristics

Strain JSM 071043T was moderately halophilic and alkaliphilic, growth occurring in the presence of 0.5–20% (w/v) NaCl (optimum 5–10%), at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl2·6H2O (optimum 5–10%). Colonies were light yellow-pigmented, circular, somewhat convex and non-translucent with glistening surfaces and entire margins and 2–3 mm in diameter after incubation for 3–5 days at 28°C on MA5. Detailed phenotypic properties that differentiate strain JSM 071043T from recognized Zhihengliuella species are summarized in Table 1 and also mentioned in the species description below.

Table 1 Characteristics used to distinguish strain JSM 071043T from recognized Zhihengliuella species
Full size table

Phylogenetic analysis based on 16S rRNA gene sequence comparison and DNA–DNA relatedness

The almost-complete 16S rRNA gene sequence (1,421 bp) of the organism was determined. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JSM 071043T was a member of the suborder Micrococcineae and was most closely related to the type strains of the two recognized species of the genus Zhihengliuella, Z. halotolerans YIM 70185T (sequence similarity 98.9%) and Z. alba YIM 90734T (98.2%), and the three strains formed a distinct clade supported by a significant bootstrap resampling value (100%) in the phylogenetic tree (Fig. 1). The topology was similar to those of the phylogenetic trees constructed by using maximum-likelihood and maximum-parsimony methods (not shown). Levels of DNA–DNA relatedness between strain JSM 071043T and the type strains of Z. halotolerans and Z. alba were 30.9 and 20.5%, respectively, values that are well below the threshold value (70%) recommended by Wayne et al. (1987) for assigning strains to the same species. Therefore, it would appear that, on the basis of the phylogenetic and DNA–DNA hybridization data, strain JSM 071043T represents a new species of the genus Zhihengliuella according to accepted criteria (Wayne et al. 1987; Stackebrandt and Goebel 1994).

Fig. 1
figure 1

Phylogenetic tree showing the phylogenetic positions of strain JSM 071043T and related taxa based on 16S rRNA gene sequence analysis constructed by using the neighbour-joining method. Labels m or p indicate branches that are also found with the maximum-likelihood (Felsenstein 1981) or parsimony (Kluge and Farris 1969) algorithms, respectively; asterisks indicate branches that are recovered with all three methods. Numbers at nodes indicate bootstrap percentages (>50%) based on a neighbour-joining analysis of 1,000 resampled datasets. The scale bar indicates 0.01 substitutions per nucleotide position

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Chemotaxonomic characteristics and DNA base composition

Chemotaxonomic data for strain JSM 071043T were consistent with the assignment of the strain to the genus Zhihengliuella. The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose, which are characteristic of the genus Zhihengliuella (Zhang et al. 2007; Tang et al. 2009). MK-9 (56.1%) and MK-8 (26.4%) were detected as the major menaquinones, with MK-9 (H2) (3.0%), MK-9 (H4) (5.7%) and MK-10 (8.8%) present in minor amounts. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and an unknown glycolipid. The major fatty acids (>10% of the total) of strain JSM 071043T were anteiso-C15:0 (56.1%), iso-C16:0 (17.0%) and anteiso-C17:0 (12.4%); iso-C14:0 (3.0%), iso-C15:0 (3.9%), C16:0 (4.1%), iso-C17:0 (0.5%), C18:0 (0.5%) and C18:1 2-OH (0.5%) were present in minor amounts. This fatty acid profile was similar to those of recognized Zhihengliuella species, although there were differences in the proportions of some components (Zhang et al. 2007; Tang et al. 2009). The DNA G + C content of strain JSM 071043T was 67.8 mol%.

Taxonomic conclusion

The results of the phylogenetic analysis and of morphological and chemotaxonomic investigations supported the affiliation of strain JSM 071043T to the genus Zhihengliuella. However, the novel strain could be clearly distinguished from the two recognized Zhihengliuella species by differences in several phenotypic and chemotaxonomic characteristics, including cell morphology, growth condition, utilization of and acid production from several substrates, activity of some enzymes and megaquinone composition (Table 1). Although strain JSM 071043T exhibited comparatively high 16S rRNA gene sequence similarity with Arthrobacter oryzae (97.5%; Kageyama et al. 2008) and some other related Arthrobacter species (<96.9%), the new isolate, as well as the two recognized Zhihengliuella species, differed clearly from related Arthrobacter species by sufficient taxonomic markers as discussed previously (Zhang et al. 2007; Tang et al. 2009). In conclusion, the results of phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization experiments and the phenotypic and chemotaxonomic data presented here supported the proposal of strain JSM 071043T representing a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed.

Description of Z. salsuginis sp. nov.

Z. salsuginis (sal.su’gi.nis. L. gen. n. salsuginis of/from brine)

Cells are non-motile, non-sporulating, aerobic, Gram-positive, coccoid and about 0.6–0.9 mm in diameter and form pairs, tetrads and clusters. Oxidase-negative and catalase-positive. Colonies are light yellow-pigmented, circular, somewhat convex and non-translucent with glistening surfaces and entire margins and 2–3 mm in diameter. Moderately halophilic, alkaliphilic and mesophilic, growth occurring with 0.5–20% (w/v) NaCl (optimum 5–10%), at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurs in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl2·6H2O (optimum 5–10%). Positive for hydrolysis of gelatin, starch, Tweens 20, 40, 60 and 80, but negative for nitrate and nitrite reduction, H2S and indole production, methyl red and Voges-Proskauer test and hydrolysis of aesculin, casein, cellulose, chitin, DNA, hypoxanthine, urea and xanthine. Produces acid from cellubiose, d-fructose, d-glucose, glycerol, glycogen, maltose, l-rhamnose, d-ribose, starch, sucrose, xylitol and d-xylose, but not from l-arabinose, d-galactose, inulin, lactose, d-mannose, melibiose, melezitose, raffinose, trehalose, adonitol, dulcitol, myo-inositol, d-mannitol, salicin and d-sorbitol. The following compounds are utilized as sole sources of carbon and energy or sole sources of carbon, nitrogen and energy: dextrin, d-glucose, maltose, raffinose, d-ribose, sucrose, glycerol, d-mannitol, acetate, gluconate and l-glutamic acid; the following are not utilized: l-arabinose, cellobiose, d-fructose, d-galactose, glycogen, inulin, lactose, d-mannose, melezitose, melibiose, l-rhamnose, trehalose, d-xylose, adonitol, myo-inositol, d-sorbitol, salicin, butyrate, citrate, formate, fumarate, malate, malonate, propionate, succinate, acetamide, l-alanine, l-arginine, l-asparagine, l-glycine, l-histidine, hydroxy-l-proline, l-isoleucine, l-leucine, l-methionine, l-proline, l-serine and l-valine. Constitutive enzymes expressed are N-acetyl-β-glucosaminidase, alkaline phosphatase, α-chymotrypsin, esterase (C4), α-fucosidase, β-glucosidase, β-glucuronidase, lipase (C8) and trypsin; acid phosphatase, cystine arylamidase, α- and β-galactosidase, α-glucosidase, leucine arylamidase, lipase (C14), lysine decarboxylase, α-mannosidase, naphthol-AS-BI-phosphohydrolase, ornithine decarboxylase, phenylalanine deaminase or valine arylamidase are not observed. The peptidoglycan type is A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars are tyvelose and mannose. Possesses MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. Major cellular fatty acids (>10% of the total) are anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The DNA G + C content of the type strain is 67.8 mol% (HPLC method).

The type strain, JSM 071043T (= DSM 21149T = KCTC 19466T), was isolated from a subterranean brine sample collected from the Xiangli Salt Mine in Hunan Province, China.