Abstract
Two extremely halophilic archaeal strains GX1T and GX60 were isolated from the Gangxi marine solar saltern, China. Cells from the two strains were observed to be rod-shaped and stained Gram-negative, with red-pigmented colonies. Strains GX1T and GX60 were found to be able to grow at 25–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.6 M), at pH 5.5–9.5 (optimum pH 7.0) and neither strain required Mg2+ for growth. The cells lysed in distilled water and the minimal NaCl concentration to prevent cell-lysis was found to be 8 % (w/v). The major polar lipids of the two strains were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and three glycolipids chromatographically identical to those of Haloarchaeobius iranensis IBRC-M 10013T. 16S rRNA gene analysis revealed that each strain had two dissimilar 16S rRNA genes and both strains were phylogenetically related to Hab. iranensis IBRC-M 10013T (94.9–98.9 % nucleotide identity). The rpoB′ gene similarity between strains GX1T and GX60, and between these strains and Hab. iranensis IBRC-M 10013T were found to be 99.6, 96.0 and 95.8 %, respectively. The DNA G + C content of strain GX1T and GX60 were determined to be 67.7 and 67.8 mol %, respectively. The DNA–DNA hybridization value of strains GX1T and GX60 was 86 % and the two strains showed low DNA–DNA relatedness with Hab. iranensis IBRC-M 10013T (38 and 32 %). It was concluded that strain GX1T (= CGMCC 1.10390T = JCM 17114T) and strain GX60 (= CGMCC 1.10389 = JCM 17120) represent a new species of Haloarchaeobius, for which the name Haloarchaeobius litoreus sp. nov. is proposed.
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Introduction
The genus Haloarchaeobius of the family Halobacteriaceae was first proposed by Makhdoumi-Kakhki et al. to accommodate the species Haloarchaeobius iranensis, which is an extremely halophilic archaeon isolated from a brine sample from Aran-Bidgol salt lake, a saline playa in Iran (Makhdoumi-Kakhki et al. 2012). Currently, there is only one species, Hab. iranensis, within the genus Haloarchaeobius. Hab. iranensis, harbouring two distinct 16S rRNA genes differing by 1.6 %, was distinctly different from other members of the family Halobacteriaceae, required at least 2 M NaCl but not MgCl2 for growth, contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one minor phospholipid and three unidentified glycolipids. During our survey on halophilic archaeal diversity of a marine solar saltern of Eastern China, two halophilic archaeal isolates related to Hab. iranensis were obtained. In this study, we characterize strains GX1T and GX60 as a new species of the genus Haloarchaeobius, for which the name Haloarchaeobius litoreus sp. nov. is proposed.
Materials and methods
Isolation and cultivation of halophilic archaeal strains
Strains GX1T and GX60 were isolated from brine samples taken from Gangxi marine solar saltern near Weihai city of Shandong Province, China (37°22′57″ N, 122°27′49″ E) and stored at 4 °C during transport to the laboratory in 2009. The pH of the brine was 7.1 and the salinity 283 g/L. The neutral oligotrophic haloarchaeal medium (NOM) 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 0.25, NH4Cl 0.25, MgSO4·7H2O 26.8, MgCl2·6H2O 23.0, NaCl 184.0 (pH adjusted to 7.0–7.2 with 1 M NaOH solution). The medium was solidified with 2.0 % agar. The strains were routinely grown aerobically at 37 °C in NOM medium. Cultures were kept on NOM agar slopes at 4 °C and were subcultured every 6 months for short-time storage. For long-term storage, preparations of cells suspended in NOM supplemented with 15 % (w/v) glycerol were stored at −20 °C.
Phenotypic determination
Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests and sensitivity to antimicrobial agents were performed for all species in the same basic medium, NOM, according to the proposed minimal standards for description of new taxa in the order Halobacteriales (Oren et al. 1997). The type strains Hab. iranensis IBRC-M 10013T, Halomicrobium mukohataei JCM 9738T and Halobacterium jilantaiense CGMCC 1.5337T were selected as reference strains in the phenotypic tests.
The Gram stain was performed following the method outlined by Dussault (1955). Cell morphology and motility in exponentially growing liquid cultures were examined using a Nikon microscope equipped with phase-contrast optics (model: E400). The minimum salt concentration preventing cell lysis was determined by suspending washed cells in serial sterile saline solutions containing NaCl 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 NOM medium 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 NOM 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 NOM agar plates amended with 5 g/L gelatin and detected by flooding the plates with Frazier’s reagent (McDade and Weaver 1959). Esterase activity was measured as outlined by Gutiérrez and González (1972). Tests for catalase and oxidase activities were performed as described by Gonzalez et al. (1978). Production of H2S was tested by growing the isolates and reference strains in a tube containing NOM 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 NOM medium and the compound to be tested was added at a concentration of 5 g/L. Antimicrobial susceptibilities were determined on NOM agar plates with antimicrobial compound discs.
Chemotaxonomic characterization
Polar lipids were extracted using a chloroform/methanol system and analysed using one- and two-dimensional TLC, as described previously (Cui et al. 2010). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used for TLC analyses. In two-dimensional TLC, the first solvent was chloroform–methanol–water (65:25:4, by vol.) and the second solvent was chloroform–methanol–acetic acid–water (80:12:15:4, by vol.). The latter solvent mixture was also used in one-dimensional TLC. Two specific detection spray reagents, phosphate stain reagent for phospholipids and α-naphthol strain for glycolipids, were used. The general detection reagent, sulfuric acid–ethanol (1:2, by vol.), was also used to detect total polar lipids.
Phylogenetic analysis
Genomic DNA from halophilic archaeal strains was prepared as described previously (Cui et al. 2011). The 16S rRNA genes were amplified, cloned and sequenced according to the protocol described previously (Cui et al. 2009). PCR-mediated amplification and sequencing of the rpoB′ genes were performed as described previously (Minegishi et al. 2010). Multiple sequence alignments were performed using the ClustalW program integrated in the MEGA 5 software (http://www.megasoftware.net/). Phylogenetic trees were reconstructed using the neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) algorithms in the MEGA 5 software (Tamura et al. 2011). Gene sequence similarity among halophilic archaea was calculated using the Pairwise-Distance computing function of MEGA 5.
Genomic DNA analyses
The DNA G+C content was determined from the mid-point 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. DNA–DNA hybridizations were performed in a Beckman-Coulter DU800™ spectrophotometer equipped with a high performance temperature controller, and were carried out according to the thermal denaturation and renaturation method (De Ley et al. 1970; Huß et al. 1983).
Results and discussion
Cells of strains GX1T and GX60 were observed to be motile and rod-shaped when grown in NOM liquid medium (Supplementary Fig. S1). They stained Gram-negative and their colonies were observed to be red-pigmented. Both strains were found to be able to grow at 25–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.6 M NaCl), at 0–1.0 M MgCl2 (optimum 0.05 M MgCl2) and at pH 5.5–9.5 (optimum pH 7.0). The cells of both strains were observed to lyse in distilled water and the minimal NaCl concentration to prevent lysis was found to be 8 % (w/v). Both strains were unable to grow under anaerobic conditions using nitrate or l-arginine, but they could grow under anaerobic conditions using DMSO. Both strains were found to be positive for catalase and oxidase. Strain GX1T was observed to form indole but GX60 did not. Strain GX1T and GX60 produced H2S from sodium thiosulfate, hydrolyzed starch, gelatin, casein and Tween 80. Both strains were sensitive to the following antimicrobial compounds (µg per disc, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disc), rifampin (5), mycostatin (100) and nitrofurantoin (300). They were resistant to the following antimicrobial compounds: trimethoprim (5), erythromycin (15), penicillin G (10 IU per disc), 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 GX1T and strain GX60 from Hab. iranensis IBRC-M 10013T are optimum NaCl, optimum pH, reduction of nitrate to nitrite, anaerobic growth with DMSO, utilization of specific carbon sources, formation of indole and H2S, hydrolysis of starch, casein and Tween 80 (Table 1). More detailed results of phenotypic tests and nutritional features of these strains are given in the species description.
The major polar lipids of strains GX1T and GX60 were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and three major glycolipids, in a pattern chromatographically identical to the polar lipid profiles of Hab. iranensis IBRC-M 10013T. Two of the three major glycolipids (GL2 and GL3) were chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), the remaining one (GL1) was unidentified (see Supplementary Fig. S2). The major polar lipid composition supports the classification of strains GX1T and GX60 in the genus Haloarchaeobius.
Sequence comparisons indicated that each strain possessed two different 16S rRNA genes (denoted rrnA and rrnB) that differed in sequence by 5 %. When the corresponding rrnA and rrnB genes of the two isolates were compared, they showed >99 % identity. Comparison of the rrnA and rrnB genes of the two isolates to the corresponding 16S rRNA genes of Hab. iranensis gave similarity values of 94.9–98.9 %. Apart from this, strains GX1T and GX60 were found to be related to the members of the genera Halorubellus (90.0–93.4 %), Halobiforma (89.9–92.5 %) and Halalkalicoccus (89.4–91.7 %). Phylogenetic analysis using the NJ algorithm revealed that each of the 16S rRNA genes of strain GX1T and strain GX60 formed a tight clade clustered with sequences of Hab. iranensis IBRC-M 10013T, separated from the clades containing members of the genera Halorubellus, Halobiforma and Halalkalicoccus (Fig. 1a). The phylogenetic position was also confirmed in other trees generated using the MP and ML algorithms (data not shown). The 16S rRNA gene-based phylogenetic analysis results support the placement of strains GX1T and GX60 in the genus Haloarchaeobius.
The rpoB′ genes of both strains were sequenced and found to be identical in length 1830 bp. The nucleotide sequences are 99.6 % similar to each other and also closely similar to the corresponding gene of Hab. iranensis IBRC-M 10013T (96.0 and 95.8 %), followed by showing 87.3–87.7 % similarities to the sequences of members of Halorubellus, 85.6–86.2 % similarities to the members of Halobiforma and 83.5–85.2 % similarities to the members of Halalkalicoccus. In phylogenetic tree reconstructions, strains GX1T and GX60 tightly clustered and formed a monophyletic group with Hab. iranensis IBRC-M 10013T, separated from the clades of Halorubellus, Halobiforma and Halalkalicoccus (Fig. 1b). The phylogenetic position was also confirmed in other trees generated using the MP and ML algorithms (data not shown). The rpoB′ gene-based phylogenetic analysis results also support the placement of strains GX1T and GX60 in the genus Haloarchaeobius.
The DNA G+C content of strain GX1T and strain GX60 was determined to be 67.7 and 67.8 mol %, values that are very close to that of Hal. iranensis IBRC-M 10013T (67.7 mol %). The DNA–DNA hybridization value between strain GX1T and strain GX60 was 86 ± 3 %, and these two strains showed low DNA–DNA relatedness with Hab. iranensis IBRC-M 10013T (38 ± 5 % and 32 ± 2 % relatedness, respectively), lower than the accepted threshold value (70 %) to separate two species (Stackebrandt and Goebel 1994).
Based on these phenotypic, chemotaxonomic and phylogenetic properties, a novel species of the genus Haloarchaeobius is proposed to accommodate these two strains, Haloarchaeobius litoreus sp. nov. Characteristics that distinguish strains GX1T and GX60 from Hab. iranensis IBRC-M 10013T are shown in Table 1.
Description of Haloarchaeobius litoreus sp. nov.
Haloarchaeobius litoreus (li.to’re.us. L. masc. adj. litoreus, living near the sea, of or belonging to, the seashore).
Cells are motile, rod-shaped (0.4–0.5 × 1–6 µm) under optimal growth conditions and stain Gram-negative. Colonies on agar plates containing 2.6 M NaCl are red, elevated and round. Chemoorganotrophic and aerobic. Growth occurs at 25–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.6 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.5–9.5 (optimum pH 7.0). Cells lyse in distilled water and the minimal NaCl concentration to prevent cell lysis is 8 %. Catalase- and oxidase-positive. Grows under anaerobic conditions with DMSO but does not with nitrate or arginine. Nitrate reduction to nitrite is observed but formation of gas from nitrate is not observed. Indole formations is variable (the type strain is negative) and H2S formation is positive. Hydrolyzes starch, gelatin, casein and Tween 80. The following substrates are utilized as single carbon and energy sources for growth: d-glucose, d-mannose, d-galactose, sucrose, starch, glycerol and pyruvate. The following substrates are utilized as single carbon, nitrogen or energy sources for growth: l-alanine and l-glutamate. No growth occurs on d-fructose, l-sorbose, d-ribose, d-xylose, maltose, lactose, d-mannitol, d-sorbitol, acetate, dl-lactate, succinate, l-malate, fumarate, citrate, glycine, l-arginine, l-aspartate, l-lysine or l-ornithine. Acid is produced from d-glucose, d-mannose, d-galactose and sucrose. Sensitive to the following antimicrobial compounds (µg per disc, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disc), rifampin (5), mycostatin (100) and nitrofurantoin (300). Resistant to the following antimicrobial compounds: trimethoprim (5), erythromycin (15), penicillin G (10 IU per disc), 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 polar lipids are phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and three major glycolipids; two of three major glycolipids are chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), the remaining one (GL1) is unidentified. The DNA G+C content of the type strain is 67.7 mol % (T m).
The type strain is GX1T (= CGMCC 1.10390T = JCM 17114T). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains GX1T and GX60 are GU951427 (rrnA), JF421971 (rrnB), GU951428 (rrnA) and JF421972 (rrnB), respectively. Those for the rpoB′ gene sequences of strain strains GX1T and GX60 are KF540215 and KF540216.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 31370054), the grant from China Ocean Mineral Resources R & D Association (COMRA) Special Foundation (DY125-15-R-03), the Qinglan Project of Jiangsu Province and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to Dr. M. A. Amoozegar for kindly providing strain Haloarchaeobius iranensis IBRC-M 10013T.
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Zhang, WJ., Cui, HL. Haloarchaeobius litoreus sp. nov., isolated from a marine solar saltern. Antonie van Leeuwenhoek 105, 1085–1090 (2014). https://doi.org/10.1007/s10482-014-0166-8
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DOI: https://doi.org/10.1007/s10482-014-0166-8