Antonie van Leeuwenhoek (2015) 108:483–489 DOI 10.1007/s10482-015-0501-8
ORIGINAL PAPER
Halosimplex litoreum sp. nov., isolated from a marine solar saltern Pan-Pan Yuan . Jia-Qi Xu . Wen-Mei Xu . Zhao Wang . Shuai Yin . Dong Han . Wen-Jiao Zhang . Heng-Lin Cui
Received: 31 March 2015 / Accepted: 2 June 2015 / Published online: 10 June 2015 Ó Springer International Publishing Switzerland 2015
Abstract A halophilic archaeal strain, YGH94T, was isolated from the Yinggehai marine solar saltern near the Shanya city of Hainan Province, China. Cells of the strain were observed to be short rods, stain Gram-negative and to form red-pigmented colonies on solid media. Strain YGH94T was found to grow at 25–50 °C (optimum 40 °C), at 0.9–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.5). The cells were found to lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was determined to be 5 % (w/v). The major polar lipids were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids (disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and two unidentified glycolipids chromatographically identical to glycolipids in Halosimplex carlsbadense JCM 11222T). Strain YGH94T was found to possess two heterogeneous 16S rRNA genes (rrnA and rrnB) and both are related
to those of Hsx. carlsbadense JCM 11222T (92.7–98.6 % similarities), Halosimplex pelagicum R2T (94.6–99.2 % similarities) and Halosimplex rubrum R27T (92.9–98.8 % similarities). The rpoB0 gene similarity between strain YGH94T and Hsx. carlsbadense JCM 11222T, Hsx. pelagicum R2T and Hsx. rubrum R27T are 95.4, 94.9 and 95.1 %, respectively. The DNA G?C content of strain YGH94T was determined to be 64.0 mol%. Strain YGH94T showed low DNA–DNA relatedness (35–39 %) with the current three members of the genus Halosimplex. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain YGH94T (=CGMCC 1.12235T = JCM 18647T) represents a new species of the genus Halosimplex, for which the name Halosimplex litoreum sp. nov. is proposed. Keywords Halosimplex litoreum sp. nov. Halophilic archaeon Marine solar saltern Polyphasic taxonomy
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0501-8) contains supplementary material, which is available to authorized users. P.-P. Yuan J.-Q. Xu W.-M. Xu Z. Wang S. Yin D. Han W.-J. Zhang H.-L. Cui (&) School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Jingkou District, Zhenjiang 212013, People’s Republic of China e-mail: [email protected]
Marine solar salterns are artificial thalassohaline environments hosting diverse halophilic archaea, members of the family Halobacteriaceae within the order Halobacteriales (Oren et al. 2009; Oren 2012; Ventosa et al. 2014). The family Halobacteriaceae encompasses 50 genera containing over 196 species as of May 2015 (Oren 2014; Koh et al. 2015;
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Kondo et al. 2015; Minegishi et al. 2015a, b; Zhang and Cui 2015). During our survey on halophilic archaeal diversity of Yinggehai marine solar saltern near the Sanya city of Hainan Province, China, we obtained a halophilic archaeal strain YGH94T, which was found to be most closely related to the members of Halosimplex of the family Halobacteriaceae, as judged from 16S rRNA gene sequence analysis. The genus Halosimplex of the family Halobacteriaceae was first proposed by Vreeland et al. to accommodate the species Halosimplex carlsbadense, which is an extremely halophilic archaeon isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico (Vreeland et al. 2002). Currently, there are three validly named species, Hsx. carlsbadense, Halosimplex pelagicum and Halosimplex rubrum within the genus Halosimplex (Han and Cui 2014). Hsx. pelagicum and Hsx. rubrum were isolated from the red brine of salted Laminaria produced at Dalian, Liaoning Province, China. These two species can utilise diverse sugars as single carbon, nitrogen or energy sources for growth while Hsx. carlsbadense cannot use any of the sugars tested to date. The members of Halosimplex are different from other members of the Halobacteriaceae, contain two unidentified glycolipids and have dissimilar 16S rRNA genes (Boucher et al. 2004; Vreeland et al. 2002; Han and Cui 2014). In this study, we characterize strain YGH94T as a new species of the genus Halosimplex, for which the name Halosimplex litoreum sp. nov. is proposed.
Materials and methods Isolation and cultivation Strain YGH94T was isolated from a brine sample taken in 2010 from the Yinggehai marine solar saltern near Sanya city, Hainan Province, China (18°310 5200 N, 108°430 3900 E; elevation, sea level). Strain YGH94T has been deposited in the China General Microbiological Culture Collection (CGMCC) and the Japan Collection of Microorganisms (JCM) and was assigned the accession numbers, CGMCC 1.12235T and JCM 18647T, respectively. During transport of the sample to the laboratory it was kept at 4 °C. The pH of the brine was 7.2 and the salinity 226 g/L. Neutral haloarchaeal medium (NHM) was used for the
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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, MgSO47H2O 26.8, MgCl26H2O 23.0, NaCl 184.0 (pH adjusted to 7.0–7.2 with 1 M NaOH solution). The medium was solidified with 15 g/L agar. The strain was routinely grown aerobically at 37 °C for 7 days in NHM and preserved at -20 °C as a suspension in NHM broth supplemented with glycerol (150 g/L). Phenotypic determination Phenotypic tests were performed according to the proposed minimal standards for description of novel taxa in the order Halobacteriales (Oren et al. 1997). Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests and sensitivity to antimicrobial agents were performed as described and cited previously (Cui et al. 2010). The type strains Hsx. pelagicum R2T, Hsx. rubrum R27T and Hsx. carlsbadense JCM 11222T were selected as reference strains in phenotypic tests. These reference strains were routinely grown aerobically at 37 °C in NHM medium. Chemotaxonomic characterisation Three strains, Hsx. carlsbadense JCM 11222T, Halorientalis regularis CGMCC 1.10123T and Halobacterium salinarum CGMCC 1.2367 (=ATCC 33170 = NRC 34001), were selected as reference strains in the polar lipid analyses. Incubation was carried out at 37 °C in an orbital shaker at 150 r.p.m. for 7 days. Polar lipids of halophilic archaeal strains were extracted using a chloroform–methanol system and analysed using one- and two-dimensional TLC, as described and cited previously (Cui et al. 2010). Two specific detection spray reagents, phosphate stain reagent for phospholipids and a-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. Phylogenetic and genomic analysis Genomic DNA for determination of the DNA base composition and PCR-mediated amplification
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experiments was prepared from the strain as described previously (Cui et al. 2011). The 16S rRNA genes were amplified with the forward primer 0018F (50 -ATTCCGGTTGATCCTGCC-30 ) and reverse primer 1518R (50 -AGGAGGTGATCCAGCCGC-30 ) (Cui et al. 2009). The PCR products were recovered from the agarose gel by using a gel extraction kit (CW2302, Beijing ComWin Biotech Co., Ltd.), were cloned into the pUC-T vector (CW2591) and transformed into Escherichia coli DH5a. Positive clones harbouring cloned 16S rRNA gene fragments were sequenced by SinoGenoMax Company Limited (Beijing, China). The rpoB0 gene was amplified using the primer pair HrpoB2 1420F (50 -TGTGGGCTNGTGA AGAACTT-30 ) and HrpoA 153R (50 -GGG TCCATCAGCCCCATGTC-30 ) (Minegishi et al. 2010), and the PCR product was sequenced using the following primers: HrpoB2 1420F, HrpoA 153R and B1-628F (50 -CCNGCNGSVCAGAACTTC-30 ). Multiple sequ ence alignments were performed using the ClustalW program integrated in the MEGA 5 software (Tamura et al. 2011). Phylogenetic trees were reconstructed using the maximum-likelihood (ML) and neighbourjoining (NJ) algorithms in the MEGA 5 software. Gene sequence similarity among halophilic archaea was calculated using the Pairwise-Distance computing function of MEGA 5. The DNA G?C content was determined from the mid-point value (Tm) of the thermal denaturation method (Marmur and Doty 1962) at 260 nm with a Beckman-Coulter DU800TM spectrophotometer equipped with a high-performance temperature controller. E. coli K12 was selected as the reference strain for the analyses, and the formula G? C mol% unknown strain = G?C mol% reference strain ? 2.08 9 (Tm unknown strain-Tm reference strain) was used to calculate the G?C content from the known Tm value (Owen and Pitcher, 1985). DNA–DNA hybridizations were performed in a Beckman-Coulter DU800TM 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). DNA–DNA hybridizations were carried out in 2 9 SSC at 79 °C and each determination was carried out in triplicate.
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Results and discussion Cells of strain YGH94T were observed to be motile and short rods when grown in NHM liquid medium (Supplementary Fig. S1). The cells were found to stain Gram-negative and the colonies were observed to be red-pigmented. Strain YGH94T was found to grow at 25–50 °C (optimum 40 °C), at 0.9–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.5). The cells were found to lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was determined to be 50 g/L (w/v). The strain was found to be able to grow under anaerobic conditions using nitrate, DMSO and Larginine. The strain is positive for H2S formation and negative for indole formation. Strain YGH94T was found to be unable to hydrolyse starch, casein, gelatin and Tween 80. Strain YGH94T is sensitive to the following antimicrobial compounds (lg per disc, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disc), rifampin (5), mycostatin (100), trimethoprim (5) and nitrofurantoin (300). The strain is resistant to the following antimicrobial compounds: 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 YGH94T from Hsx. pelagicum R2T, Hsx. rubrum R27T and Hsx. carlsbadense JCM 11222T are the optimum NaCl concentration for growth, Mg2? required for growth, anaerobic growth with nitrate, arginine and DMSO, reduction of nitrate to nitrite, utilisation of specific carbon sources and H2S formation (Table 1). More detailed results of phenotypic features of strain YGH94T are given in the species description. The major polar lipids of strain YGH94T were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids (disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and two unidentified glycolipids chromatographically identical to those of Hsx. carlsbadense JCM 11222T; Supplementary Fig. S2). Sequence comparisons indicated that strain YGH94T possesses two different 16S rRNA genes
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Table 1 Differential characteristics between YGH94T and members of the genus Halosimplex Characteristic
1
2
3
4
Optimum NaCl (M)
3.1
3.4
3.1
4.3
Mg2? required
-
?
-
?
Anaerobic growth with nitrate Reduction of nitrate to nitrite
? ?
-
-
-
Anaerobic growth with arginine
?
-
-
-
Anaerobic growth with DMSO
?
-
-
-
D-glucose
?
?
?
-
D-mannose
?
?
?
-
D-galactose
?
?
?
-
Maltose
?
-
-
-
Sucrose
?
?
?
-
Lactose
?
?
-
-
D-mannitol
-
?
?
-
D-sorbitol
?
?
?
-
Acetate
-
?
-
?
DL-lactate
?
?
?
-
Utilization of
Succinate
-
?
-
-
L-malate
Fumarate
-
? ?
?
-
Citrate
-
?
-
-
Glycine
?
-
-
-
L-alanine
-
?
-
-
L-arginine
-
?
-
-
L-aspartate
-
?
?
-
L-glutamate
-
-
?
-
L-ornithine
-
?
?
-
H2S formation
?
-
?
?
Taxa 1 YGH94T, 2 Hsx. pelagicum R2T, 3 Hsx. rubrum R27T, 4 Hsx. carlsbadense JCM 11222 ?, positive; -, negative. All data are from this study
(denoted rrnA and rrnB) that differ in sequence by 3.9 %. The rrnA and rrnB genes of strain YGH94T were found to be phylogenetically related to those of Hsx. carlsbadense JCM 11222T (92.7–98.6 % similarities), Hsx. pelagicum R2T (94.6–99.2 % similarities) and Hsx. rubrum R27T (92.9–98.8 % similarities). Phylogenetic tree reconstructions using the ML algorithm revealed that strain YGH94T tightly clustered with the current three members of Halosimplex (Fig. 1a). The phylogenetic position was also confirmed in the tree generated using the NJ algorithm (Supplementary Fig. S3a). The rpoB0 gene of strain YGH94T was found to be closely similar to the corresponding gene of Hsx.
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carlsbadense JCM 11222T (95.4 % similarity), Hsx. pelagicum R2T (94.9 % similarity) and Hsx. rubrum R27T (95.1 % similarity); these similarity values are higher than the recommended threshold (86.2 %) which is used to distinguish genera (Minegishi et al. 2010). In the phylogenetic tree reconstruction using rpoB0 (Fig. 1b), strain YGH94T tightly clustered with Hsx. carlsbadense JCM 11222T, Hsx. pelagicum R2T and Hsx. rubrum R27T. The phylogenetic position was also confirmed by the tree generated using the NJ algorithm (Supplementary Fig. S3b). The 16S rRNA gene-based and rpoB0 gene-based phylogenetic analysis results support the placement of strain YGH94T in the genus Halosimplex.
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(a) 16S rRNA gene Halosimplex pelagicum R2T rrnA (HM159602) Halosimplex rubrum R27T rrnA (HM159603) 98
0.05
97
Halosimplex carlsbadense JCM 11222T rrn1 HQ263561) Halosimplex litoreum YGH94T rrnA (JQ937362)
Halosimplex carlsbadense JCM 11222T rrn2 HQ263563) Halosimplex rubrum R27T rrnB (KF434757)
74
Halosimplex pelagicum R2T rrnB (KF434756) Halosimplex litoreum YGH94T rrnB (KM386641) Halorhabdus tiamatea JCM 14471T (AB576122)
100
Halorhabdus utahensis DSM 12940T (NC_013158)
Halorientalis regularis TNN28T (GQ282621) Haloterrigena turkmenica DSM 5511T (NC_013743) Halobacterium salinarum CGMCC 1.1958T (KC854794) Methanospirillum hungatei JF-1T (NC_007796)
(b) rpoB′ gene
Halosimplex carlsbadense JCM 11222T (AB477192) Halosimplex pelagicum R2T (KF434759)
84 100
0.05
Halosimplex rubrum R27T (KF434760) Halosimplex litoreum YGH94T (KJ921126) Halorientalis regularis TNN28T (KF434758) Halorhabdus tiamatea SARL4BT (NC_021921) 100
Halorhabdus utahensis DSM 12940T (NC_013158)
Haloterrigena turkmenica JCM 9101T (AB477198) Halobacterium salinarum JCM 8978T (AB477150) Methanospirillum hungatei JF-1T (NC_007796)
Fig. 1 Maximum-Likelihood phylogenetic trees based on 16S rRNA gene (a) and rpoB0 gene (b) sequences showing the relationships between strain YGH94T 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 changes per site
The DNA G?C content of strain YGH94T was determined to be 64.0 mol%, which is higher than that of Hsx. pelagicum R2T (62.5 mol%), lower than that of Hsx. carlsbadense JCM 11222T (64.4 mol%) and equal to that of Hsx. rubrum R27T. The DNA–DNA hybridization values between strain YGH94T and Hsx. pelagicum R2T, H sx. carlsbadense JCM 11222T, Hsx. rubrum R27T were 39 ± 4, 38 ± 1 and 35 ± 2 %, respectively, much lower than the accepted threshold value (70 %) to separate prokaryotic species (Stackebrandt and Goebel 1994). Based on these phenotypic, chemotaxonomic and phylogenetic properties, a novel species of the genus
Halosimplex is proposed to accommodate strain YGH94T, Halosimplex litoreum sp. nov. Characteristics that distinguish strain YGH94T from Hsx. pelagicum R2T, Hsx. rubrum R27T and Hsx. carlsbadense JCM 11222T are shown in Table 1.
Description of Halosimplex litoreum sp. nov. Halosimplex litoreum (li.to’re.um. L. neut. adj. litoreum, of or belonging to the sea-shore). Cells are motile, short rods under optimal growth conditions and stain Gram-negative. Colonies on
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NHM agar plates containing 3.1 M NaCl are red, elevated and round. Chemoorganotrophic and aerobic. Growth occurs at 25–50 °C (optimum 40 °C), at 0.9–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.5). Cells lyse in distilled water and the minimal NaCl concentration to prevent cell lysis is 50 g/L. Catalase and oxidase positive. Grows anaerobically in the presence of nitrate, arginine and DMSO. Nitrate reduction to nitrite is observed but gas formation from nitrate does not occur. H2S formation is positive and indole formation is negative. Does not hydrolyse starch, casein, gelatin or Tween 80. The following substrates are utilised for growth: D-glucose, D-mannose, D-galactose, maltose, sucrose, lactose, glycerol, D-sorbitol, pyruvate, DL-lactate, glycine, L-arginine and L-glutamate. No growth occurs on D-fructose, Lsorbose, D-ribose, D-xylose, starch, D-mannitol, acetate, succinate, L-malate, fumarate, citrate, L-alanine, Laspartate, L-lysine or L-ornithine. Acid is produced from D-glucose, D-mannose, D-galactose, maltose, sucrose and lactose. The major polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids, two of them chromatographically identical to disulfated mannosyl glucosyl diether and sulfated mannosyl glucosyl diether; the remaining two glycolipids are unidentified. The DNA G?C content of the type strain is 64.0 mol% (Tm). The type strain YGH94T (=CGMCC T T 1.12235 = JCM 18647 ) was obtained from the Yinggehai marine solar saltern near Sanya city of Hainan Province, China. The GenBank/EMBL/DDBJ accession numbers for the rrnA and rrnB 16S rRNA and rpoB0 gene sequences of strain YGH94T are JQ937362, KM386641 and KJ921126, respectively.
Supplementary information A phase-contrast micrograph of strain YGH94T, thinlayer chromatograms of the polar lipids extracted from strain YGH94T as well as those from some related haloarchaea, and Neighbour-Joining phylogenetic tree reconstructions based on 16S rRNA gene and rpoB0 gene sequences are available as supplementary materials. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31370054), the 11th
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Antonie van Leeuwenhoek (2015) 108:483–489 ‘‘Six Talents Peak’’ (No. 2014-SWYY-021), Province and a project Program Development Institutions (PAPD).
Project of Jiangsu Province the Qinglan Project of Jiangsu funded by the Priority Academic of Jiangsu Higher Education
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Antonie van Leeuwenhoek (2015) 108:483–489 Minegishi H, Echigo A, Shimane Y, Kamekura M, Itoh T, Ohkuma M, Usami R (2015b) Halococcus agarilyticus sp. nov., an agar degrading haloarchaeon isolated from commercial salt. Int J Syst Evol Microbiol (in press) Oren A (2012) Taxonomy of the family Halobacteriaceae: a paradigm for changing concepts in prokaryote systematics. Int J Syst Evol Microbiol 62:263–271 Oren A (2014) Taxonomy of halophilic Archaea: current status and future challenges. Extremophiles 18:825–834 Oren A, Ventosa A, Grant WD (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47:233–238 Oren A, Arahal DR, Ventosa A (2009) Emended descriptions of genera of the family Halobacteriaceae. Int J Syst Evol Microbiol 59:637–642 Owen RJ, Pitcher D (1985) Current methods for estimating DNA base composition and levels of DNA-DNA hybridization. In: Goodfellow M, Minnikin DE (eds) Chemical methods in bacterial systematics. Academic Press, London, pp 67–93 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis
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ORIGINAL PAPER
Halosimplex litoreum sp. nov., isolated from a marine solar saltern Pan-Pan Yuan . Jia-Qi Xu . Wen-Mei Xu . Zhao Wang . Shuai Yin . Dong Han . Wen-Jiao Zhang . Heng-Lin Cui
Received: 31 March 2015 / Accepted: 2 June 2015 / Published online: 10 June 2015 Ó Springer International Publishing Switzerland 2015
Abstract A halophilic archaeal strain, YGH94T, was isolated from the Yinggehai marine solar saltern near the Shanya city of Hainan Province, China. Cells of the strain were observed to be short rods, stain Gram-negative and to form red-pigmented colonies on solid media. Strain YGH94T was found to grow at 25–50 °C (optimum 40 °C), at 0.9–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.5). The cells were found to lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was determined to be 5 % (w/v). The major polar lipids were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids (disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and two unidentified glycolipids chromatographically identical to glycolipids in Halosimplex carlsbadense JCM 11222T). Strain YGH94T was found to possess two heterogeneous 16S rRNA genes (rrnA and rrnB) and both are related
to those of Hsx. carlsbadense JCM 11222T (92.7–98.6 % similarities), Halosimplex pelagicum R2T (94.6–99.2 % similarities) and Halosimplex rubrum R27T (92.9–98.8 % similarities). The rpoB0 gene similarity between strain YGH94T and Hsx. carlsbadense JCM 11222T, Hsx. pelagicum R2T and Hsx. rubrum R27T are 95.4, 94.9 and 95.1 %, respectively. The DNA G?C content of strain YGH94T was determined to be 64.0 mol%. Strain YGH94T showed low DNA–DNA relatedness (35–39 %) with the current three members of the genus Halosimplex. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain YGH94T (=CGMCC 1.12235T = JCM 18647T) represents a new species of the genus Halosimplex, for which the name Halosimplex litoreum sp. nov. is proposed. Keywords Halosimplex litoreum sp. nov. Halophilic archaeon Marine solar saltern Polyphasic taxonomy
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0501-8) contains supplementary material, which is available to authorized users. P.-P. Yuan J.-Q. Xu W.-M. Xu Z. Wang S. Yin D. Han W.-J. Zhang H.-L. Cui (&) School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Jingkou District, Zhenjiang 212013, People’s Republic of China e-mail: [email protected]
Marine solar salterns are artificial thalassohaline environments hosting diverse halophilic archaea, members of the family Halobacteriaceae within the order Halobacteriales (Oren et al. 2009; Oren 2012; Ventosa et al. 2014). The family Halobacteriaceae encompasses 50 genera containing over 196 species as of May 2015 (Oren 2014; Koh et al. 2015;
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Kondo et al. 2015; Minegishi et al. 2015a, b; Zhang and Cui 2015). During our survey on halophilic archaeal diversity of Yinggehai marine solar saltern near the Sanya city of Hainan Province, China, we obtained a halophilic archaeal strain YGH94T, which was found to be most closely related to the members of Halosimplex of the family Halobacteriaceae, as judged from 16S rRNA gene sequence analysis. The genus Halosimplex of the family Halobacteriaceae was first proposed by Vreeland et al. to accommodate the species Halosimplex carlsbadense, which is an extremely halophilic archaeon isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico (Vreeland et al. 2002). Currently, there are three validly named species, Hsx. carlsbadense, Halosimplex pelagicum and Halosimplex rubrum within the genus Halosimplex (Han and Cui 2014). Hsx. pelagicum and Hsx. rubrum were isolated from the red brine of salted Laminaria produced at Dalian, Liaoning Province, China. These two species can utilise diverse sugars as single carbon, nitrogen or energy sources for growth while Hsx. carlsbadense cannot use any of the sugars tested to date. The members of Halosimplex are different from other members of the Halobacteriaceae, contain two unidentified glycolipids and have dissimilar 16S rRNA genes (Boucher et al. 2004; Vreeland et al. 2002; Han and Cui 2014). In this study, we characterize strain YGH94T as a new species of the genus Halosimplex, for which the name Halosimplex litoreum sp. nov. is proposed.
Materials and methods Isolation and cultivation Strain YGH94T was isolated from a brine sample taken in 2010 from the Yinggehai marine solar saltern near Sanya city, Hainan Province, China (18°310 5200 N, 108°430 3900 E; elevation, sea level). Strain YGH94T has been deposited in the China General Microbiological Culture Collection (CGMCC) and the Japan Collection of Microorganisms (JCM) and was assigned the accession numbers, CGMCC 1.12235T and JCM 18647T, respectively. During transport of the sample to the laboratory it was kept at 4 °C. The pH of the brine was 7.2 and the salinity 226 g/L. Neutral haloarchaeal medium (NHM) was used for the
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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, MgSO47H2O 26.8, MgCl26H2O 23.0, NaCl 184.0 (pH adjusted to 7.0–7.2 with 1 M NaOH solution). The medium was solidified with 15 g/L agar. The strain was routinely grown aerobically at 37 °C for 7 days in NHM and preserved at -20 °C as a suspension in NHM broth supplemented with glycerol (150 g/L). Phenotypic determination Phenotypic tests were performed according to the proposed minimal standards for description of novel taxa in the order Halobacteriales (Oren et al. 1997). Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests and sensitivity to antimicrobial agents were performed as described and cited previously (Cui et al. 2010). The type strains Hsx. pelagicum R2T, Hsx. rubrum R27T and Hsx. carlsbadense JCM 11222T were selected as reference strains in phenotypic tests. These reference strains were routinely grown aerobically at 37 °C in NHM medium. Chemotaxonomic characterisation Three strains, Hsx. carlsbadense JCM 11222T, Halorientalis regularis CGMCC 1.10123T and Halobacterium salinarum CGMCC 1.2367 (=ATCC 33170 = NRC 34001), were selected as reference strains in the polar lipid analyses. Incubation was carried out at 37 °C in an orbital shaker at 150 r.p.m. for 7 days. Polar lipids of halophilic archaeal strains were extracted using a chloroform–methanol system and analysed using one- and two-dimensional TLC, as described and cited previously (Cui et al. 2010). Two specific detection spray reagents, phosphate stain reagent for phospholipids and a-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. Phylogenetic and genomic analysis Genomic DNA for determination of the DNA base composition and PCR-mediated amplification
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experiments was prepared from the strain as described previously (Cui et al. 2011). The 16S rRNA genes were amplified with the forward primer 0018F (50 -ATTCCGGTTGATCCTGCC-30 ) and reverse primer 1518R (50 -AGGAGGTGATCCAGCCGC-30 ) (Cui et al. 2009). The PCR products were recovered from the agarose gel by using a gel extraction kit (CW2302, Beijing ComWin Biotech Co., Ltd.), were cloned into the pUC-T vector (CW2591) and transformed into Escherichia coli DH5a. Positive clones harbouring cloned 16S rRNA gene fragments were sequenced by SinoGenoMax Company Limited (Beijing, China). The rpoB0 gene was amplified using the primer pair HrpoB2 1420F (50 -TGTGGGCTNGTGA AGAACTT-30 ) and HrpoA 153R (50 -GGG TCCATCAGCCCCATGTC-30 ) (Minegishi et al. 2010), and the PCR product was sequenced using the following primers: HrpoB2 1420F, HrpoA 153R and B1-628F (50 -CCNGCNGSVCAGAACTTC-30 ). Multiple sequ ence alignments were performed using the ClustalW program integrated in the MEGA 5 software (Tamura et al. 2011). Phylogenetic trees were reconstructed using the maximum-likelihood (ML) and neighbourjoining (NJ) algorithms in the MEGA 5 software. Gene sequence similarity among halophilic archaea was calculated using the Pairwise-Distance computing function of MEGA 5. The DNA G?C content was determined from the mid-point value (Tm) of the thermal denaturation method (Marmur and Doty 1962) at 260 nm with a Beckman-Coulter DU800TM spectrophotometer equipped with a high-performance temperature controller. E. coli K12 was selected as the reference strain for the analyses, and the formula G? C mol% unknown strain = G?C mol% reference strain ? 2.08 9 (Tm unknown strain-Tm reference strain) was used to calculate the G?C content from the known Tm value (Owen and Pitcher, 1985). DNA–DNA hybridizations were performed in a Beckman-Coulter DU800TM 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). DNA–DNA hybridizations were carried out in 2 9 SSC at 79 °C and each determination was carried out in triplicate.
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Results and discussion Cells of strain YGH94T were observed to be motile and short rods when grown in NHM liquid medium (Supplementary Fig. S1). The cells were found to stain Gram-negative and the colonies were observed to be red-pigmented. Strain YGH94T was found to grow at 25–50 °C (optimum 40 °C), at 0.9–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.5). The cells were found to lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was determined to be 50 g/L (w/v). The strain was found to be able to grow under anaerobic conditions using nitrate, DMSO and Larginine. The strain is positive for H2S formation and negative for indole formation. Strain YGH94T was found to be unable to hydrolyse starch, casein, gelatin and Tween 80. Strain YGH94T is sensitive to the following antimicrobial compounds (lg per disc, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disc), rifampin (5), mycostatin (100), trimethoprim (5) and nitrofurantoin (300). The strain is resistant to the following antimicrobial compounds: 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 YGH94T from Hsx. pelagicum R2T, Hsx. rubrum R27T and Hsx. carlsbadense JCM 11222T are the optimum NaCl concentration for growth, Mg2? required for growth, anaerobic growth with nitrate, arginine and DMSO, reduction of nitrate to nitrite, utilisation of specific carbon sources and H2S formation (Table 1). More detailed results of phenotypic features of strain YGH94T are given in the species description. The major polar lipids of strain YGH94T were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids (disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and two unidentified glycolipids chromatographically identical to those of Hsx. carlsbadense JCM 11222T; Supplementary Fig. S2). Sequence comparisons indicated that strain YGH94T possesses two different 16S rRNA genes
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Table 1 Differential characteristics between YGH94T and members of the genus Halosimplex Characteristic
1
2
3
4
Optimum NaCl (M)
3.1
3.4
3.1
4.3
Mg2? required
-
?
-
?
Anaerobic growth with nitrate Reduction of nitrate to nitrite
? ?
-
-
-
Anaerobic growth with arginine
?
-
-
-
Anaerobic growth with DMSO
?
-
-
-
D-glucose
?
?
?
-
D-mannose
?
?
?
-
D-galactose
?
?
?
-
Maltose
?
-
-
-
Sucrose
?
?
?
-
Lactose
?
?
-
-
D-mannitol
-
?
?
-
D-sorbitol
?
?
?
-
Acetate
-
?
-
?
DL-lactate
?
?
?
-
Utilization of
Succinate
-
?
-
-
L-malate
Fumarate
-
? ?
?
-
Citrate
-
?
-
-
Glycine
?
-
-
-
L-alanine
-
?
-
-
L-arginine
-
?
-
-
L-aspartate
-
?
?
-
L-glutamate
-
-
?
-
L-ornithine
-
?
?
-
H2S formation
?
-
?
?
Taxa 1 YGH94T, 2 Hsx. pelagicum R2T, 3 Hsx. rubrum R27T, 4 Hsx. carlsbadense JCM 11222 ?, positive; -, negative. All data are from this study
(denoted rrnA and rrnB) that differ in sequence by 3.9 %. The rrnA and rrnB genes of strain YGH94T were found to be phylogenetically related to those of Hsx. carlsbadense JCM 11222T (92.7–98.6 % similarities), Hsx. pelagicum R2T (94.6–99.2 % similarities) and Hsx. rubrum R27T (92.9–98.8 % similarities). Phylogenetic tree reconstructions using the ML algorithm revealed that strain YGH94T tightly clustered with the current three members of Halosimplex (Fig. 1a). The phylogenetic position was also confirmed in the tree generated using the NJ algorithm (Supplementary Fig. S3a). The rpoB0 gene of strain YGH94T was found to be closely similar to the corresponding gene of Hsx.
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carlsbadense JCM 11222T (95.4 % similarity), Hsx. pelagicum R2T (94.9 % similarity) and Hsx. rubrum R27T (95.1 % similarity); these similarity values are higher than the recommended threshold (86.2 %) which is used to distinguish genera (Minegishi et al. 2010). In the phylogenetic tree reconstruction using rpoB0 (Fig. 1b), strain YGH94T tightly clustered with Hsx. carlsbadense JCM 11222T, Hsx. pelagicum R2T and Hsx. rubrum R27T. The phylogenetic position was also confirmed by the tree generated using the NJ algorithm (Supplementary Fig. S3b). The 16S rRNA gene-based and rpoB0 gene-based phylogenetic analysis results support the placement of strain YGH94T in the genus Halosimplex.
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(a) 16S rRNA gene Halosimplex pelagicum R2T rrnA (HM159602) Halosimplex rubrum R27T rrnA (HM159603) 98
0.05
97
Halosimplex carlsbadense JCM 11222T rrn1 HQ263561) Halosimplex litoreum YGH94T rrnA (JQ937362)
Halosimplex carlsbadense JCM 11222T rrn2 HQ263563) Halosimplex rubrum R27T rrnB (KF434757)
74
Halosimplex pelagicum R2T rrnB (KF434756) Halosimplex litoreum YGH94T rrnB (KM386641) Halorhabdus tiamatea JCM 14471T (AB576122)
100
Halorhabdus utahensis DSM 12940T (NC_013158)
Halorientalis regularis TNN28T (GQ282621) Haloterrigena turkmenica DSM 5511T (NC_013743) Halobacterium salinarum CGMCC 1.1958T (KC854794) Methanospirillum hungatei JF-1T (NC_007796)
(b) rpoB′ gene
Halosimplex carlsbadense JCM 11222T (AB477192) Halosimplex pelagicum R2T (KF434759)
84 100
0.05
Halosimplex rubrum R27T (KF434760) Halosimplex litoreum YGH94T (KJ921126) Halorientalis regularis TNN28T (KF434758) Halorhabdus tiamatea SARL4BT (NC_021921) 100
Halorhabdus utahensis DSM 12940T (NC_013158)
Haloterrigena turkmenica JCM 9101T (AB477198) Halobacterium salinarum JCM 8978T (AB477150) Methanospirillum hungatei JF-1T (NC_007796)
Fig. 1 Maximum-Likelihood phylogenetic trees based on 16S rRNA gene (a) and rpoB0 gene (b) sequences showing the relationships between strain YGH94T 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 changes per site
The DNA G?C content of strain YGH94T was determined to be 64.0 mol%, which is higher than that of Hsx. pelagicum R2T (62.5 mol%), lower than that of Hsx. carlsbadense JCM 11222T (64.4 mol%) and equal to that of Hsx. rubrum R27T. The DNA–DNA hybridization values between strain YGH94T and Hsx. pelagicum R2T, H sx. carlsbadense JCM 11222T, Hsx. rubrum R27T were 39 ± 4, 38 ± 1 and 35 ± 2 %, respectively, much lower than the accepted threshold value (70 %) to separate prokaryotic species (Stackebrandt and Goebel 1994). Based on these phenotypic, chemotaxonomic and phylogenetic properties, a novel species of the genus
Halosimplex is proposed to accommodate strain YGH94T, Halosimplex litoreum sp. nov. Characteristics that distinguish strain YGH94T from Hsx. pelagicum R2T, Hsx. rubrum R27T and Hsx. carlsbadense JCM 11222T are shown in Table 1.
Description of Halosimplex litoreum sp. nov. Halosimplex litoreum (li.to’re.um. L. neut. adj. litoreum, of or belonging to the sea-shore). Cells are motile, short rods under optimal growth conditions and stain Gram-negative. Colonies on
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NHM agar plates containing 3.1 M NaCl are red, elevated and round. Chemoorganotrophic and aerobic. Growth occurs at 25–50 °C (optimum 40 °C), at 0.9–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.5). Cells lyse in distilled water and the minimal NaCl concentration to prevent cell lysis is 50 g/L. Catalase and oxidase positive. Grows anaerobically in the presence of nitrate, arginine and DMSO. Nitrate reduction to nitrite is observed but gas formation from nitrate does not occur. H2S formation is positive and indole formation is negative. Does not hydrolyse starch, casein, gelatin or Tween 80. The following substrates are utilised for growth: D-glucose, D-mannose, D-galactose, maltose, sucrose, lactose, glycerol, D-sorbitol, pyruvate, DL-lactate, glycine, L-arginine and L-glutamate. No growth occurs on D-fructose, Lsorbose, D-ribose, D-xylose, starch, D-mannitol, acetate, succinate, L-malate, fumarate, citrate, L-alanine, Laspartate, L-lysine or L-ornithine. Acid is produced from D-glucose, D-mannose, D-galactose, maltose, sucrose and lactose. The major polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids, two of them chromatographically identical to disulfated mannosyl glucosyl diether and sulfated mannosyl glucosyl diether; the remaining two glycolipids are unidentified. The DNA G?C content of the type strain is 64.0 mol% (Tm). The type strain YGH94T (=CGMCC T T 1.12235 = JCM 18647 ) was obtained from the Yinggehai marine solar saltern near Sanya city of Hainan Province, China. The GenBank/EMBL/DDBJ accession numbers for the rrnA and rrnB 16S rRNA and rpoB0 gene sequences of strain YGH94T are JQ937362, KM386641 and KJ921126, respectively.
Supplementary information A phase-contrast micrograph of strain YGH94T, thinlayer chromatograms of the polar lipids extracted from strain YGH94T as well as those from some related haloarchaea, and Neighbour-Joining phylogenetic tree reconstructions based on 16S rRNA gene and rpoB0 gene sequences are available as supplementary materials. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31370054), the 11th
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Antonie van Leeuwenhoek (2015) 108:483–489 ‘‘Six Talents Peak’’ (No. 2014-SWYY-021), Province and a project Program Development Institutions (PAPD).
Project of Jiangsu Province the Qinglan Project of Jiangsu funded by the Priority Academic of Jiangsu Higher Education
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