Introduction

Diverse halophilic archaea, members of the class Halobacteria, are always found to be present in marine solar salterns (Ghai et al. 2011; Narasingarao et al. 2012; Oren 2014). The past several years have witnessed the description of novel species and genera based on the haloarchaeal strains isolated from these kinds of hypersaline environments, such as Halobellus inordinatus (Qiu et al. 2013a), Natronomonas gomsonensis (Kim et al. 2013), Halobacterium rubrum (Han and Cui 2014), Haloarchaeobius litoreus (Zhang and Cui 2014b), Salinigranum rubrum (Cui and Zhang 2014) and Halovenus salina (Infante-Domínguez et al. 2015). During our survey on halophilic archaeal diversity of the Yangjiang marine solar saltern (Guangxi Province, China), we obtained a halophilic archaeal isolate, YJ-18-S1T, that was most closely related to the members of Halorubrum, as judged from 16S rRNA gene sequences.

The genus Halorubrum, belonging to the family Haloferacaceae (Gupta et al. 2015), was first established by McGenity and Grant (1995) and currently contains 30 named species (Fig. 1) (Yim et al. 2014; Zhang and Cui 2014a; Han and Cui 2015; Kondo et al. 2015). Among these Halorubrum species, five of them (H. alkaliphilum, H. gandharaense, H. luteum, H. tibetense and H. vacuolatum) are haloalkaliphilic species, while the remaining 25 members are neutrophilic. The polar lipids of the neutrophilic members of Halorubrum are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The alkaliphilic species lack phosphatidylglycerol sulfate and glycolipids (Oren et al. 2009). In this study, we characterized strain YJ-18-S1T as a new species of the genus Halorubrum.

Fig. 1
figure 1

Maximum-likelihood phylogenetic tree reconstructions based on 16S rRNA gene (a) and rpoB′ gene (b) sequences, showing the relationships between strains YJ-18-S1T and related members within the family Halobacteriaceae. Bootstrap values (%) are based on 1000 replicates and are shown for branches with more 70 % bootstrap support. Bar represents expected substitutions per nucleotide position

Full size image

Materials and methods

Isolation and cultivation of halophilic archaeal strain

Strain YJ-18-S1T was isolated from a sediment sample from Yangjiang marine solar saltern, at Yangjiang, Guangxi Province, China (21°31′48′N, 111°28′5′E; elevation, sea level), in 2012. The sediment sample was picked up from the surface of the bed of a crystallizer pond in which the brine had a temperature of 28 °C at the time of sampling, a pH of 7.2 and salinity of 28 °Bé. The neutral haloarchaeal medium (NHM) was used for the isolation procedure and contained the following ingredients (g/L): yeast extract (Oxoid) 0.05, fish peptone (Sinopharm Chemical Reagent Co., Ltd.) 0.25, sodium pyruvate 1.0, KCl 5.4, K2HPO4 0.3, CaCl2 0.29, NH4Cl 0.27, MgSO4·7H2O 26.8, MgCl2·6H2O 23.0, NaCl 184.0 (pH adjusted to 7.0–7.2 with 1 M NaOH solution). One gram of the sediment sample was suspended in 9 ml of NHM, and the turbid liquid was serially diluted in liquid NHM and spread onto NHM agar plates. The inoculated plates were incubated for 3 months at 37 °C. After this initial cultivation, colonies were successively re-streaked on NHM agar plates at least three times to obtain pure colonies.

Phenotypic determination

The type strains H. aidingense CGMCC 1.2670T, H. halophilum JCM 18963T, H. kocurii CGMCC 1.7018T, H. lacusprofundi CGMCC 1.3490T, H. lipolyticum CGMCC 1.5332T, H. rubrum CGMCC 1.12124T, H. saccharovorum CGMCC 1.2147T were selected as reference strains in phenotypic tests. These reference strains were routinely grown aerobically at 37 °C in NHM.

Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests and sensitivity to antimicrobial agents were performed for all species in NHM, according to the proposed minimal standards for description of new taxa in the order Halobacteriales (Oren et al. 1997). The Gram staining was performed as described previously (Dussault 1955). Cell morphology and motility in exponentially growing liquid cultures were examined using a light microscope equipped with phase-contrast optics. The minimum salt concentration preventing cell lysis was determined by suspending cells in serial dilutions of sterile saline with NaCl concentrations ranging from 0 to 150 g/L, and the stability of the cells was detected by light microscopic examination. Growth and gas formation with nitrate as an electron acceptor were tested in 9-mL stoppered tubes (with Durham tubes) completely filled with liquid NHM and to which NaNO3 (5 g/L) had been added. The formation of gas from nitrate was detected by the presence of gas bubbles in the Durham tubes, and the formation of nitrite was monitored colorimetrically. Anaerobic growth in the presence of l-arginine or DMSO (5 g/L) was tested in completely filled 9-mL stoppered tubes. Starch hydrolysis was determined on NHM agar plates supplemented with 2 g/L soluble starch and detected by flooding the plates with Lugol’s iodine solution. Gelatin hydrolysis was performed by growing colonies on NHM agar plates amended with 5 g/L gelatin and detected by flooding the plates with Frazier’s reagent (McDade and Weaver 1959). Tests for catalase and oxidase activities were performed as described by Gonzalez et al. (1978). Esterase activity was detected as described by Gutiérrez and González (1972). Production of H2S was tested by growing the isolates and reference strains in a tube containing NHM liquid medium supplemented with 5 g/L sodium thiosulfate and detected using a filter-paper strip impregnated with lead acetate (Cui et al. 2007). To test for growth on single carbon sources, fish peptone and sodium pyruvate were omitted from the NHM and the compound to be tested was added at a concentration of 5 g/L. Antimicrobial susceptibilities were determined on NHM agar plates with antimicrobial compound disks.

Chemotaxonomic characterization

Strain YJ-18-S1T and H. saccharovorum CGMCC 1.2147T were routinely grown aerobically at 37 °C in NHM. Halophilic archaeal polar lipids were extracted using a chloroform–methanol system and analyzed using one- and two-dimensional TLC, as described previously (Cui et al. 2010). Two specific detection spray reagents, phosphate stain reagent for phospholipids and α-naphthol stain for glycolipids, were used. The general detection reagent, sulfuric acid–ethanol (1:2, by vol.), was also used to detect total polar lipids. The presence of phospholipids and glycolipids on the two-dimensional TLC was confirmed by comparison with one-dimensional TLC on which the polar lipid profile of reference strains was developed.

Phylogenetic and genotypic analysis

Halophilic archaeal genomic DNA was prepared as described previously (Cui et al. 2011), and the 16S rRNA genes were amplified with the forward primer 0018F (5′-ATTCCGGTTGATCCTGCC-3′) and reverse primer 1518R (5′-AGGAGGTGATCCAGCCGC-3′), then cloned and sequenced according to a previous protocol (Cui et al. 2009). The rpoB′ gene was amplified using the primer pair HrpoB2 1420F (5′-TGTGGGCTNGTGAAGAACTT-3′) and HrpoA 153R (5′-GGG TCCATCAGCCCCATGTC-3′) (Minegishi et al. 2010), and the PCR product was sequenced using the following primers: HrpoB2 1420F, HrpoA 153R and B1-628F (5′-CCNGCNGSVCAGAACTTC-3′). These sequences were aligned using the ClustalW program integrated in the MEGA 5 software (Tamura et al. 2011), and the phylogenetic trees were reconstructed using maximum-likelihood, maximum-parsimony and neighbor-joining algorithms in the MEGA 5 software. The DNA G+C content was determined from the midpoint value (T m) of the thermal denaturation method (Marmur and Doty 1962) at 260 nm with a Beckman Coulter DU800™ spectrophotometer equipped with a high-performance temperature controller.

Results and discussion

Cells of strain YJ-18-S1T were observed to be motile and pleomorphic rods when grown in NHM liquid medium (Supplementary Fig. S1). They stained Gram-negative, and the colonies were observed to be red-pigmented. Strain YJ-18-S1T was found to be able to grow at 20–55 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 2.6 M NaCl), at 0.005–1.0 M MgCl2 (optimum 0.3 MgCl2) and at pH 5.5–8.5 (optimum pH 7.0). The cells were lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was found to be 5 % (w/v). The strain was able to grow under anaerobic conditions using nitrate or l-arginine, but not with DMSO. It was found to be positive for H2S formation and indole formation. Strain YJ-18-S1T did not hydrolyze starch, gelatin, Tween 80 or casein. Strain YJ-18-S1T was sensitive to the following antimicrobial compounds (µg per disk, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disk), rifampin (5), nitrofurantoin (300), mycostatin (100) and trimethoprim (5). It was resistant to the following antimicrobial compounds: erythromycin (15), penicillin G (10 IU per disk), ampicillin (10), chloramphenicol (30), neomycin (30), norfloxacin (10), ciprofloxacin (5), streptomycin (10), kanamycin (30), tetracycline (30), vancomycin (30), gentamicin (10) and nalidixic acid (30). The main phenotypic characteristics differentiating strain YJ-18-S1T from the related members of the genus Halorubrum are anaerobic growth with nitrate, reduction of nitrate to nitrite, utilization of specific carbon sources, indole formation, hydrolysis of casein, gelatin, starch and Tween 80 (Table 1). More detailed results of phenotypic features of strain YJ-18-S1T are given in the species description.

Table 1 Differential characteristics between strain YJ-18-S1T and closely related members of Halorubrum
Full size table

The major polar lipids of strain YJ-18-S1T were identified as phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and a major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-3) (Supplementary Fig. S2). Since the polar lipid profile of strain YJ-18-S1T was identical to those of the neutrophilic members of the genus Halorubrum (Oren et al. 2009), the major polar lipid composition supports the classification of strain YJ-18-S1T in the genus Halorubrum.

Complete 16S rRNA gene sequence comparisons indicated that strain YJ-18-S1T has one kind of 16S rRNA gene sequence (1472 bp in length). The 16S rRNA gene of the strain was phylogenetically related to H. lipolyticum CGMCC 1.5332T (98.0 % similarity), H. saccharovorum CGMCC 1.2147T (97.8 % similarity), H. kocurii CGMCC 1.7018T (97.8 % similarity) and other members of genus Halorubrum, showing 94.3–97.7 % similarity. These 16S rRNA gene similarities are well lower than the recently recommended thresholds (98.65 %) to separate two prokaryotic species (Kim et al. 2014). Phylogenetic tree reconstructions using the maximum-likelihood (ML) algorithm revealed that strain YJ-18-S1T tightly clustered with the current 30 members of Halorubrum (Fig. 1a). This phylogenetic position was also confirmed in other trees generated using the maximum-parsimony (MP) and neighbor-joining (NJ) algorithms (Supplementary Fig. S3a and Fig. S4a).

The rpoB′ gene of strain YJ-18-S1T was closely similar to the corresponding gene of H. halophilum JCM 18963T (96.1 % similarity), H. saccharovorum CGMCC 1.2147T (96.0 % similarity) and other members of genus Halorubrum, showing 86.7–95.8 % similarity. In phylogenetic tree reconstructions using the rpoB′ (Fig. 1b), strain YJ-18-S1T tightly clustered with the members of Halorubrum. The phylogenetic position was also confirmed in trees generated using the maximum-parsimony (MP) and neighbor-joining (NJ) algorithms (Supplementary Fig. S3b and Fig. S4b).

The 16S rRNA gene and rpoB′ gene-based phylogenetic analysis results supported the placement of strain YJ-18-S1T in the genus Halorubrum.

The DNA G+C content of strain YJ-18-S1T was determined to be 66.2 mol%, within the range of the DNA G+C content of the members of the genus Halorubrum (60.2–71.2 mol%; Oren et al. 2009). The DNA G+C content of strain YJ-18-S1T was higher than those of H. lipolyticum CGMCC 1.5332T (65.9 mol%) (Cui et al. 2006), H. halophilum JCM 18963T (64.6 mol%) (Yim et al. 2014), H. rubrum CGMCC 1.12124T (64.9 mol%) (Qiu et al. 2013b), H. lacusprofundi CGMCC 1.3490T (65.3 mol%) (McGenity and Grant 1995), H. aidingense CGMCC 1.2670T (64.2 mol%) (Cui et al. 2006) but lower than those of H. saccharovorum CGMCC 1.2147T (71.2 mol%) (Tomlinson and Hochstein 1976; McGenity and Grant 1995) and H. kocurii CGMCC 1.7018T (69.4 mol%) (Gutiérrez et al. 2008).

Based on these phenotypic, chemotaxonomic and phylogenetic properties, a novel species of the genus Halorubrum is proposed to accommodate the strain, Halorubrum rutilum sp. nov. Characteristics that distinguish strain YJ-18-S1T from the related members of the genus Halorubrum are shown in Table 1.

Description of Halorubrum rutilum sp. nov.

Halorubrum rutilum (ru.ti’lum. L. neut. adj. rutilum red-colored, pertaining to the colony pigmentation of the type strain).

Cells are motile, pleomorphic rods under optimal growth conditions and Gram-negative. Colonies on agar plates containing 2.6 M NaCl are red, elevated and round. The type strain is chemoorganotrophic and aerobic. Growth occurs at 20–55 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 2.6 M NaCl), at 0.005–1.0 M MgCl2 (optimum 0.3 MgCl2) and at pH 5.5–8.5 (optimum pH 7.0). Cells were lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis is 5 % w/v. Catalase and oxidase positive. Anaerobic growth occurs in the presence of nitrate and arginine but not with DMSO. Nitrate reduction to nitrite and gas formation from nitrate were observed. H2S formation and indole formation are positive. The type strain does not hydrolyze casein, starch, gelatin or Tween 80. The following substrates are utilized as single carbon and energy sources for growth: d-glucose, d-mannose, l-sorbose, sucrose, glycerol, d-sorbitol, acetate, pyruvate, dl-lactate, succinate, l-malate, fumarate and citrate. The following substrates are utilized as single carbon, nitrogen or energy sources for growth: l-arginine, l-aspartate and l-glutamate. No growth occurs on d-galactose, d-fructose, d-ribose, d-xylose, maltose, lactose, d-mannitol, glycine, l-alanine, l-lysine or l-ornithine. The major polar lipids are phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and sulfated mannosyl glucosyl diether (S-DGD-3). The DNA G+C content of the type strain was 66.2 mol% (T m).

The type strain is YJ-18-S1T (= CGMCC 1.12554T = JCM 30030T) and was isolated from Yangjiang marine solar saltern, Guangxi Province, China.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and rpoB′ gene sequences of strain YJ-18-S1T are KC918819 and KT184687, respectively.