Curr Microbiol (2015) 70:91–95 DOI 10.1007/s00284-014-0685-6
Halovenus rubra sp. nov., Isolated from Salted Brown Alga Laminaria Dong Han • Wen-Jiao Zhang • Heng-Lin Cui Zheng-Rong Li
•
Received: 20 June 2014 / Accepted: 29 July 2014 / Published online: 9 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Halophilic archaeal strain R28T was isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. The cells of the strain were pleomorphic and lysed in distilled water, stained Gramnegative, and formed red-pigmented colonies. Strain R28T was able to grow at 25–50 °C (optimum 42 °C), in the presence of 3.1–5.1 M NaCl (optimum 3.9 M NaCl), with 0.005–1.0 M MgCl2 (optimum 0.01 M MgCl2) and at pH 6.0–9.5 (optimum pH 7.0–7.5). The minimal NaCl concentration to prevent cell lysis was 15 % (w/v). The major polar lipids of the strain were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and two glycolipids chromatographically identical to those of Halovenus aranensis CGMCC 1.11001T. The 16S rRNA gene and rpoB0 gene of strain R28T were phylogenetically related to the corresponding genes of Hvn. aranensis CGMCC 1.11001T (91.9–97.2 and 82.9 % nucleotide identity, respectively). The DNA G?C
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and rpoB0 gene sequences of strain R28T are HM159605 and KF573409, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0685-6) contains supplementary material, which is available to authorized users. 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] Z.-R. Li (&) Department of Cell Biology, Nanjing Medical University, 140 Hanzhong Road, Gulou District, Nanjing 210029, People’s Republic of China e-mail: [email protected]
content of strain R28T was determined to be 56.3 mol%. The phenotypic, chemotaxonomic, and phylogenetic properties suggest that strain R28T (=CGMCC 1.10592T = JCM 17269T) represents a novel species of the genus Halovenus, for which the name Halovenus rubra sp. nov. is proposed.
Introduction Saturated brines, crude solar salt, and salted products are potential sources of halophilic archaea, members of the family Halobacteriaceae within the order Halobacteriales [1, 8, 18, 20]. Since most salted products were prepared with crude salt which may be contaminated with halophilic or halotolerant microorganisms; subsequently, these microorganisms may lead to the fermentation and spoilage. During our surveys on the microbiological nature of the peculiar reddening of salted brown alga Laminaria, we obtained a halophilic archaeal strain R28T, which was most closely related to the member of Halovenus, as judged from 16S rRNA gene sequences. The genus Halovenus was first proposed to accommodate the species Halovenus aranensis, which is a nonmotile, pleomorphic-shaped and red-pigmented halophilic archaeon, and was isolated from Aran-Bidgol salt lake, a hypersaline playa in Iran [13]. Halovenus aranensis EB27T was able to grow at 25–50 °C (optimum 40 °C), in the presence of 2.5–5.0 M NaCl (optimum 4.0 M NaCl), with 0.2–1.0 M MgCl2 (optimum 0.5 M MgCl2) and at pH 6.0–8.0 (optimum pH 7.0). It contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, common phospholipids found in haloarchaea, together with two minor phospholipids. Halovenus aranensis EB27T, a
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non-alkaliphilic haloarchaeon, showed a simple pattern of polar lipids and did not contain any glycolipid. In this study, we characterized strain R28T as a new species of the genus Halovenus.
Materials and Methods Isolation and Cultivation of Halophilic Archaeal Strains Strain R28T was isolated from the red brine of salted Laminaria produced from Dalian, Liaoning Province, China (38°550 5700 N, 121°120 2500 E; elevation, sea level). The brine had a pH of 7.5 and a total salinity of 293 g/L. 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, MgCl26H2O 23.0, and 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 for 7 days in NHM and preserved at -20 °C as a suspension in NHM broth supplemented with glycerol (15 %, w/v). Phenotypic Determination Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests, and sensitivity to antimicrobial agents were performed for all species in NHM medium, according to the proposed minimal standards for description of new taxa in the order Halobacteriales [16]. The type strains Hvn. aranensis CGMCC 1.11001T, Halosimplex pelagicum CGMCC 1.10586T, Halomicrobium mukohataei JCM 9738T , and Halorientalis regularis CGMCC 1.10123T were selected as reference strains in phenotypic tests. These reference strains were routinely grown aerobically at 37 °C in NHM medium. The NaCl range for growth was determined by incubating the strain at 0.9, 1.4, 1.7, 2.1, 2.6, 3.1, 3.4, 3.9, 4.3, 4.8, and 5.1 M. The pH range for growth was determined at pH 5.0–10.0 (with intervals of 0.5) using following buffers: MES (pH 5.5–6.7), PIPES (pH 6.1–7.5), MOPS (pH 6.5–7.9), HEPES (pH 6.8–8.2), Tricine (pH 7.4–8.8), and CHES (pH 8.6–10.0) at a concentration of 25 mM. The temperature range for growth was determined by incubating the strain at 10, 15, 20, 25, 30, 37, 40, 42, 45, 50, 55, and 60 °C. The Gram stain was performed as described previously [7]. Cell morphology and motility in exponentially growing liquid cultures were examined using a Nikon microscope equipped with phase contrast optics (model:
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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 NHM 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 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 [12]. Esterase activity was measured as outlined by Gutie´rrez and Gonza´lez [10]. Tests for catalase and oxidase activities were performed as described by Gonzalez et al. [9]. Production of H2S was tested by growing the isolate 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 [3]. To test for growth on single-carbon sources, fish peptone and sodium pyruvate were omitted from the NHM medium 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 Polar lipids were extracted using a chloroform/methanol system and analyzed using one- and two-dimensional TLC, as described previously [5]. Merck silica gel 60 F254 aluminum-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 for one-dimensional TLC. 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. The presence of phospholipids and glycolipids on the two-dimensional TLC was confirmed by comparing with one-dimensional TLC on which the polar lipid profile of reference strains was developed. Isoprenoid quinones were extracted, purified, and analyzed by HPLC [2]. The isoprenoid quinones were confirmed by HPLC–MS analysis.
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Phylogenetic and Genomic Analysis
Table 1 Characteristics that distinguish strain R28T from Hvn. aranensis CGMCC 1.11001T
Genomic DNA from halophilic archaeal strains was prepared as described previously [6]. The 16S rRNA genes were amplified, cloned, and sequenced according to a previous protocol [4]. PCR-mediated amplification and sequencing of the rpoB0 genes were performed as described previously [15]. Multiple sequence alignments were performed using the ClustalW program integrated in the MEGA 5 software (http://www.megasoftware.net/). Phylogenetic tree was reconstructed using Maximum-Likelihood algorithm in the MEGA 5 software [19]. The gene sequence similarity was calculated using the PairwiseDistance computing function of MEGA 5 in comparison with those of related halophilic archaea. The DNA G?C content was determined from the mid-point value (Tm) of the thermal denaturation method [14] at 260 nm with a Beckman-Coulter DU800TM spectrophotometer equipped with a high performance temperature controller.
Characteristic
1
2
Motility
?
–
NaCl range (M)
3.1–5.1
2.5–5.0
Optimum NaCl (M)
3.9
4.0
Mg2? range (M)
0.005–1.0
0.2–1.0
Optimum Mg2?(M)
0.01
0.5
D-Galactose
– –
? ?
Glycine
Results and Discussions Morphological, Physiological, and Biochemical Characteristics Cells of strain R28T were motile and pleomorphic when grown in NHM liquid medium (Supplementary Fig. S1). Strain R28T was sensitive to the following antimicrobial compounds (lg per disk, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disk), rifampin (5), mycostatin (100), and nitrofurantoin (300). It was resistant to the following antimicrobial compounds: trimethoprim (5), 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 R28T from Hvn. aranensis CGMCC 1.11001T were motility, NaCl range, optimum NaCl, Mg2? range, optimum Mg2?, utilization of specific carbon sources (D-glucose, D-galactose), hydrolysis of gelatin, and H2S formation (Table 1). More detailed results of phenotypic features of strain R28T are given in the species description. Chemotaxonomic Characteristics The polar lipids of strain R28T were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) [11], and two glycolipids (GL1, GL2), in a pattern chromatographically identical to the polar lipid profile of Hvn. aranensis CGMCC 1.11001T (Supplementary
Utilization of D-Glucose
?
–
Gelatin hydrolysis
?
–
H2S formation
?
–
G?C content (mol%) T
56.3
61.0 T
Taxa: 1 Strain R28 ; 2 Hvn. aranensis CGMCC 1.11001 . Symbols: ?, positive; –, negative
Fig. S2). Of the two glycolipids, the minor one (GL2) was chromatographically identical to mannosyl glucosyl diether (DGD-1) [17], while the major one (GL1) was unidentified. This result is somewhat not consistent with the characterization of Hvn. aranensis [13]. In this study, two glycolipids were definitely detected in both Hvn. aranensis CGMCC 1.11001T and strain R28T. The spot of PL2 on TLC plate detected by Makhdoumi-Kakhki et al. [13] seems to be chromatographically similar to that of the major glycolipid GL1, detected in this study, which is not a phospholipid but a glycolipid. In spite of this, the polar lipid composition supported classification of strain R28T in the genus Halovenus. Unsaturated and dihydrogenated menaquinones with eight isoprene units, MK-8 (minor), and MK-8 (H2) (major) were detected in strain R28T and Hvn. aranensis CGMCC 1.11001T. The DNA G?C content of strain R28T was 56.3 mol%. The value was lower than that of Hvn. aranensis CGMCC 1.11001T (61.0 %). Phylogenetic Analysis Twenty complete 16S rRNA gene sequences of strain R28T were obtained (1470 bp in length) and these indicated that the strain has a single 16S rRNA gene sequence (GenBank/ EMBL/DDBJ accession number HM159605). Strain R28T was determined to be closely related to Hvn. aranensis CGMCC 1.11001T (91.9 and 97.2 % nucleotide identity, respectively) which possessed two different 16S rRNA genes (rrnA, KJ534548; rrnB, KJ534549) that differed in sequence by 6.8 % (Fig. 1a). The rrnA of Hvn. aranensis CGMCC 1.11001T was identical to that of Hvn. aranensis EB27T (HQ197980), which confirmed the authenticity of Hvn. aranensis CGMCC 1.11001T. Our result related to the intraspecific polymorphism of 16S rRNA genes in Hvn. aranensis CGMCC 1.11001T was also confirmed by
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94 Fig. 1 Maximum-Likelihood phylogenetic trees based on 16S rRNA gene (a) and rpoB0 gene (b) sequences showing the relationships between strain R28T and related members within the family Halobacteriaceae. Bootstrap values (%) are based on 1,000 replicates and are shown for branches with more 70 % bootstrap support. Bar represents expected changes per site. a 16S rRNA gene. b rpoB0 gene
D. Han et al.: H. rubra Isolated from Laminaria
a
Halovenus aranensis EB27T (HQ197980) 100
99
Halovenus aranensis CGMCC 1.11001T rrnA (KJ534548)
Halovenus aranensis CGMCC 1.11001T rrnB (KJ534549) 98
Halovenus rubra R28T (HM159605) Halomicrobium mukohataei DSM 12286T rrn1 (NC_013202)
0.05
Halomicrobium mukohataei DSM 12286T rrn2 (NC_013202)
86 78
100
Halomicrobium mukohataei DSM 12286T rrn3 (NC_013201)
Halorhabdus utahensis DSM 12940T (NC_013158) Halorientalis regularis TNN28T (GQ282621) Halosimplex carlsbadense JCM 11222T rrn1 (HQ263561)
82 99
Halosimplex pelagicum R2T rrnA (HM159602)
Halosimplex carlsbadense JCM 11222T rrn2 (HQ263563)
99
Halosimplex pelagicum R2T rrnB (KF434756) Natronomonas pharaonis DSM 2160T (NC_007426)
79
Salinirubrum litoreum XD46T (JQ237118) Haloquadratum walsbyi C23T (NC_017459) Salinigranum rubrum GX10T (GU951431) Methanospirillum hungatei JF-1T (NC_007796)
Halomicrobium mukohataei JCM 9738T (AB477172)
b
Halorhabdus utahensis JCM 11049T (AB477175) Halorientalis regularis TNN28T (KF434758) Halosimplex carlsbadense JCM 11222T (AB477192)
0.05 74
100
Halosimplex pelagicum R2T (KF434759) Natronomonas pharaonis DSM 2160T (NC_007426) Halovenus aranensis CGMCC 1.11001T (KJ534550)
100 79
Halovenus rubra R28T (KF573409) Salinigranum rubrum GX10T (KF540217)
Salinirubrum litoreum XD46T (KF316330) Haloquadratum walsbyi C23T (NC_017459) Methanospirillum hungatei JF-1T (NC_007796)
the GenBank gene sequence data (AB840771 & AB840772) submitted by Takashi Itoh from Japan Collection of Microorganisms (Supplementary Fig. S3). Phylogenetic tree reconstruction using the maximumlikelihood (ML) algorithm revealed that strain R28T tightly clustered with Hvn. aranensis CGMCC 1.11001T (Fig. 1a). The rpoB0 gene of strain R28T (1827 nt, KF573409) was found to be similar to the corresponding gene of Hvn. aranensis CGMCC 1.11001T (82.9 % nucleotide identity). In phylogenetic tree reconstructions using rpoB0 , strain R28T clustered with Hvn. aranensis CGMCC 1.11001T (Fig. 1b). The 16S rRNA gene and rpoB0 gene-based phylogenetic analysis results supported the placement of strain R28T in the genus Halovenus.
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The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain R28T represents a novel species of the genus Halovenus, for which the name Halovenus rubra sp. nov. is proposed. Description of Halovenus rubra sp. nov. Halovenus rubra (ru0 bra. L. fem. adj. rubra Red-Colored, Red) Cells were motile, pleomorphic under optimal growth conditions, and stained Gram-negative. Colonies on agar plates containing 3.9 M NaCl were red, elevated, and round. chemoorganotrophic and aerobic. Growth occurred at 25–50 °C
D. Han et al.: H. rubra Isolated from Laminaria
(optimum 42 °C), in the presence of 3.1–5.1 M NaCl (optimum 3.9 M NaCl), with 0.005–1.0 M MgCl2 (optimum 0.01 M MgCl2) and at pH 6.0–9.5 (optimum pH 7.0–7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 15 % (w/v). Catalase and oxidase were positive. They did not grow under anaerobic conditions with nitrate, arginine, or DMSO. Nitrate reduction to nitrite and gas formation from nitrate were not observed. H2S was produced from sodium thiosulfate. Indole formation was negative. Hydrolyzed gelatin but did not hydrolyze starch, casein, or Tween 80. The following substrates were utilized as single carbon and energy sources for growth: sucrose, glycerol, D-mannitol, D-sorbitol, acetate, pyruvate, DL-lactate, succinate, L-malate, fumarate, and citrate. The following substrates were utilized as single carbon, nitrogen, or energy sources for growth: glycine, Larginine, L-aspartate, L-glutamate and L-ornithine. No growth occurred on D-glucose, D-mannose, D-galactose, Dfructose, L-sorbose, D-ribose, D-xylose, maltose, lactose, starch, L-alanine, or L-lysine. The polar lipids were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and two glycolipids (GL1, GL2), the major one (GL1) was unidentified, and the minor one (GL2) was chromatographically identical to mannosyl glucosyl diether (DGD-1). Unsaturated and dihydrogenated menaquinones with eight isoprene units, MK-8, and MK-8 (H2) were present. The DNA G?C content of the type strain was 56.3 mol% (Tm). The type strain R28T (=CGMCC 1.10592T = JCM 17269T) was obtained from the salted brown alga Laminaria produced at Dalian, Liaoning Province, China. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31370054), the Grant from China Ocean Mineral Resources Research and Development 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).
References 1. Amoozegar MA, Makhdoumi-Kakhki A, Mehrshad M, Shahzadeh Fazeli SA, Ventosa A (2013) Halopenitus malekzadehii sp. nov., a novel extremely halophilic archaeon from a salt lake. Int J Syst Evol Microbiol 63:3232–3236 2. Collins MD (1985) Isoprenoid quinone analysis in bacterial classification and identification. In: Goodfellow M, Minnikin DE (eds) Chemical methods in bacterial systematics. Academic Press, London, pp 267–287
95 3. Cui H-L, Lin Z-Y, Dong Y, Zhou P-J, Liu S-J (2007) Halorubrum litoreum sp. nov., an extremely halophilic archaeon from a solar saltern. Int J Syst Evol Microbiol 57:2204–2206 4. Cui H-L, Zhou P-J, Oren A, Liu S-J (2009) Intraspecific polymorphism of 16S rRNA genes in two halophilic archaeal genera, Haloarcula and Halomicrobium. Extremophiles 13:31–37 5. Cui H-L, Gao X, Yang X, Xu X-W (2010) Halorussus rarus gen. nov., sp. nov., a new member of the family Halobacteriaceae isolated from a marine solar saltern. Extremophiles 14:493–499 6. Cui H-L, Yang X, Mou Y-Z (2011) Salinarchaeum laminariae gen. nov., sp. nov.: a new member of the family Halobacteriaceae isolated from salted brown alga Laminaria. Extremophiles 15:625–631 7. Dussault HP (1955) An improved technique for staining red halophilic bacteria. J Bacteriol 70:484–485 8. Echigo A, Minegishi H, Shimane Y, Kamekura M, Itoh T, Usami R (2013) Halomicroarcula pellucida gen. nov., sp. nov., a nonpigmented, transparent-colony-forming, halophilic archaeon isolated from solar salt. Int J Syst Evol Microbiol 63:3556–3562 9. Gonzalez C, Gutierrez C, Ramirez C (1978) Halobacterium vallismortis sp. nov. an amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 24:710–715 10. Gutie´rrez C, Gonza´lez C (1972) Method for simultaneous detection of proteinase and esterase activities in extremely halophilic bacteria. Appl Microbiol 24:516–517 11. Kates M, Kushwaha SC (1995) Isoprenoids and polar lipids of extreme halophiles. In: DasSarma S, Fleischmann EM (eds) Archaea, a laboratory manual. Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 35–54 12. McDade JJ, Weaver RH (1959) Rapid methods for the detection of gelatin hydrolysis. J Bacteriol 77:60–64 13. Makhdoumi-Kakhki A, Amoozegar MA, Ventosa A (2012) Halovenus aranensis gen. nov., sp. nov., an extremely halophilic archaeon from Aran-Bidgol salt lake. Int J Syst Evol Microbiol 62:1331–1336 14. Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5:109–118 15. Minegishi H, Kamekura M, Itoh T, Echigo A, Usami R, Hashimoto T (2010) Further refinement of Halobacteriaceae phylogeny based on the full-length RNA polymerase subunit B0 (rpoB0 ) gene. Int J Syst Evol Microbiol 60:2398–2408 16. 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 17. Oren A, Arahal DR, Ventosa A (2009) Emended descriptions of genera of the family Halobacteriaceae. Int J Syst Evol Microbiol 59:637–642 18. Oren A (2012) Taxonomy of the family Halobacteriaceae: a paradigm for changing concepts in prokaryote systematics. Int J Syst Evol Microbiol 62:263–271 19. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 20. Yim KJ, Cha I-T, Lee H-W, Song HS, Kim K-N, Lee S-J, Nam Y-D, Hyun D-W, Bae J-W, Rhee S-K, Seo M-J, Choi J-S, Choi H-J, Roh SW, Kim D (2014) Halorubrum halophilum sp. nov., an extremely halophilic archaeon isolated from a salt-fermented seafood. Antonie Van Leeuwenhoek 105:603–612
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Halovenus rubra sp. nov., Isolated from Salted Brown Alga Laminaria Dong Han • Wen-Jiao Zhang • Heng-Lin Cui Zheng-Rong Li
•
Received: 20 June 2014 / Accepted: 29 July 2014 / Published online: 9 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Halophilic archaeal strain R28T was isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. The cells of the strain were pleomorphic and lysed in distilled water, stained Gramnegative, and formed red-pigmented colonies. Strain R28T was able to grow at 25–50 °C (optimum 42 °C), in the presence of 3.1–5.1 M NaCl (optimum 3.9 M NaCl), with 0.005–1.0 M MgCl2 (optimum 0.01 M MgCl2) and at pH 6.0–9.5 (optimum pH 7.0–7.5). The minimal NaCl concentration to prevent cell lysis was 15 % (w/v). The major polar lipids of the strain were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and two glycolipids chromatographically identical to those of Halovenus aranensis CGMCC 1.11001T. The 16S rRNA gene and rpoB0 gene of strain R28T were phylogenetically related to the corresponding genes of Hvn. aranensis CGMCC 1.11001T (91.9–97.2 and 82.9 % nucleotide identity, respectively). The DNA G?C
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and rpoB0 gene sequences of strain R28T are HM159605 and KF573409, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0685-6) contains supplementary material, which is available to authorized users. 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] Z.-R. Li (&) Department of Cell Biology, Nanjing Medical University, 140 Hanzhong Road, Gulou District, Nanjing 210029, People’s Republic of China e-mail: [email protected]
content of strain R28T was determined to be 56.3 mol%. The phenotypic, chemotaxonomic, and phylogenetic properties suggest that strain R28T (=CGMCC 1.10592T = JCM 17269T) represents a novel species of the genus Halovenus, for which the name Halovenus rubra sp. nov. is proposed.
Introduction Saturated brines, crude solar salt, and salted products are potential sources of halophilic archaea, members of the family Halobacteriaceae within the order Halobacteriales [1, 8, 18, 20]. Since most salted products were prepared with crude salt which may be contaminated with halophilic or halotolerant microorganisms; subsequently, these microorganisms may lead to the fermentation and spoilage. During our surveys on the microbiological nature of the peculiar reddening of salted brown alga Laminaria, we obtained a halophilic archaeal strain R28T, which was most closely related to the member of Halovenus, as judged from 16S rRNA gene sequences. The genus Halovenus was first proposed to accommodate the species Halovenus aranensis, which is a nonmotile, pleomorphic-shaped and red-pigmented halophilic archaeon, and was isolated from Aran-Bidgol salt lake, a hypersaline playa in Iran [13]. Halovenus aranensis EB27T was able to grow at 25–50 °C (optimum 40 °C), in the presence of 2.5–5.0 M NaCl (optimum 4.0 M NaCl), with 0.2–1.0 M MgCl2 (optimum 0.5 M MgCl2) and at pH 6.0–8.0 (optimum pH 7.0). It contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, common phospholipids found in haloarchaea, together with two minor phospholipids. Halovenus aranensis EB27T, a
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non-alkaliphilic haloarchaeon, showed a simple pattern of polar lipids and did not contain any glycolipid. In this study, we characterized strain R28T as a new species of the genus Halovenus.
Materials and Methods Isolation and Cultivation of Halophilic Archaeal Strains Strain R28T was isolated from the red brine of salted Laminaria produced from Dalian, Liaoning Province, China (38°550 5700 N, 121°120 2500 E; elevation, sea level). The brine had a pH of 7.5 and a total salinity of 293 g/L. 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, MgCl26H2O 23.0, and 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 for 7 days in NHM and preserved at -20 °C as a suspension in NHM broth supplemented with glycerol (15 %, w/v). Phenotypic Determination Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests, and sensitivity to antimicrobial agents were performed for all species in NHM medium, according to the proposed minimal standards for description of new taxa in the order Halobacteriales [16]. The type strains Hvn. aranensis CGMCC 1.11001T, Halosimplex pelagicum CGMCC 1.10586T, Halomicrobium mukohataei JCM 9738T , and Halorientalis regularis CGMCC 1.10123T were selected as reference strains in phenotypic tests. These reference strains were routinely grown aerobically at 37 °C in NHM medium. The NaCl range for growth was determined by incubating the strain at 0.9, 1.4, 1.7, 2.1, 2.6, 3.1, 3.4, 3.9, 4.3, 4.8, and 5.1 M. The pH range for growth was determined at pH 5.0–10.0 (with intervals of 0.5) using following buffers: MES (pH 5.5–6.7), PIPES (pH 6.1–7.5), MOPS (pH 6.5–7.9), HEPES (pH 6.8–8.2), Tricine (pH 7.4–8.8), and CHES (pH 8.6–10.0) at a concentration of 25 mM. The temperature range for growth was determined by incubating the strain at 10, 15, 20, 25, 30, 37, 40, 42, 45, 50, 55, and 60 °C. The Gram stain was performed as described previously [7]. Cell morphology and motility in exponentially growing liquid cultures were examined using a Nikon microscope equipped with phase contrast optics (model:
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D. Han et al.: H. rubra Isolated from Laminaria
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 NHM 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 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 [12]. Esterase activity was measured as outlined by Gutie´rrez and Gonza´lez [10]. Tests for catalase and oxidase activities were performed as described by Gonzalez et al. [9]. Production of H2S was tested by growing the isolate 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 [3]. To test for growth on single-carbon sources, fish peptone and sodium pyruvate were omitted from the NHM medium 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 Polar lipids were extracted using a chloroform/methanol system and analyzed using one- and two-dimensional TLC, as described previously [5]. Merck silica gel 60 F254 aluminum-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 for one-dimensional TLC. 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. The presence of phospholipids and glycolipids on the two-dimensional TLC was confirmed by comparing with one-dimensional TLC on which the polar lipid profile of reference strains was developed. Isoprenoid quinones were extracted, purified, and analyzed by HPLC [2]. The isoprenoid quinones were confirmed by HPLC–MS analysis.
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Phylogenetic and Genomic Analysis
Table 1 Characteristics that distinguish strain R28T from Hvn. aranensis CGMCC 1.11001T
Genomic DNA from halophilic archaeal strains was prepared as described previously [6]. The 16S rRNA genes were amplified, cloned, and sequenced according to a previous protocol [4]. PCR-mediated amplification and sequencing of the rpoB0 genes were performed as described previously [15]. Multiple sequence alignments were performed using the ClustalW program integrated in the MEGA 5 software (http://www.megasoftware.net/). Phylogenetic tree was reconstructed using Maximum-Likelihood algorithm in the MEGA 5 software [19]. The gene sequence similarity was calculated using the PairwiseDistance computing function of MEGA 5 in comparison with those of related halophilic archaea. The DNA G?C content was determined from the mid-point value (Tm) of the thermal denaturation method [14] at 260 nm with a Beckman-Coulter DU800TM spectrophotometer equipped with a high performance temperature controller.
Characteristic
1
2
Motility
?
–
NaCl range (M)
3.1–5.1
2.5–5.0
Optimum NaCl (M)
3.9
4.0
Mg2? range (M)
0.005–1.0
0.2–1.0
Optimum Mg2?(M)
0.01
0.5
D-Galactose
– –
? ?
Glycine
Results and Discussions Morphological, Physiological, and Biochemical Characteristics Cells of strain R28T were motile and pleomorphic when grown in NHM liquid medium (Supplementary Fig. S1). Strain R28T was sensitive to the following antimicrobial compounds (lg per disk, unless otherwise indicated): novobiocin (30), bacitracin (0.04 IU per disk), rifampin (5), mycostatin (100), and nitrofurantoin (300). It was resistant to the following antimicrobial compounds: trimethoprim (5), 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 R28T from Hvn. aranensis CGMCC 1.11001T were motility, NaCl range, optimum NaCl, Mg2? range, optimum Mg2?, utilization of specific carbon sources (D-glucose, D-galactose), hydrolysis of gelatin, and H2S formation (Table 1). More detailed results of phenotypic features of strain R28T are given in the species description. Chemotaxonomic Characteristics The polar lipids of strain R28T were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) [11], and two glycolipids (GL1, GL2), in a pattern chromatographically identical to the polar lipid profile of Hvn. aranensis CGMCC 1.11001T (Supplementary
Utilization of D-Glucose
?
–
Gelatin hydrolysis
?
–
H2S formation
?
–
G?C content (mol%) T
56.3
61.0 T
Taxa: 1 Strain R28 ; 2 Hvn. aranensis CGMCC 1.11001 . Symbols: ?, positive; –, negative
Fig. S2). Of the two glycolipids, the minor one (GL2) was chromatographically identical to mannosyl glucosyl diether (DGD-1) [17], while the major one (GL1) was unidentified. This result is somewhat not consistent with the characterization of Hvn. aranensis [13]. In this study, two glycolipids were definitely detected in both Hvn. aranensis CGMCC 1.11001T and strain R28T. The spot of PL2 on TLC plate detected by Makhdoumi-Kakhki et al. [13] seems to be chromatographically similar to that of the major glycolipid GL1, detected in this study, which is not a phospholipid but a glycolipid. In spite of this, the polar lipid composition supported classification of strain R28T in the genus Halovenus. Unsaturated and dihydrogenated menaquinones with eight isoprene units, MK-8 (minor), and MK-8 (H2) (major) were detected in strain R28T and Hvn. aranensis CGMCC 1.11001T. The DNA G?C content of strain R28T was 56.3 mol%. The value was lower than that of Hvn. aranensis CGMCC 1.11001T (61.0 %). Phylogenetic Analysis Twenty complete 16S rRNA gene sequences of strain R28T were obtained (1470 bp in length) and these indicated that the strain has a single 16S rRNA gene sequence (GenBank/ EMBL/DDBJ accession number HM159605). Strain R28T was determined to be closely related to Hvn. aranensis CGMCC 1.11001T (91.9 and 97.2 % nucleotide identity, respectively) which possessed two different 16S rRNA genes (rrnA, KJ534548; rrnB, KJ534549) that differed in sequence by 6.8 % (Fig. 1a). The rrnA of Hvn. aranensis CGMCC 1.11001T was identical to that of Hvn. aranensis EB27T (HQ197980), which confirmed the authenticity of Hvn. aranensis CGMCC 1.11001T. Our result related to the intraspecific polymorphism of 16S rRNA genes in Hvn. aranensis CGMCC 1.11001T was also confirmed by
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94 Fig. 1 Maximum-Likelihood phylogenetic trees based on 16S rRNA gene (a) and rpoB0 gene (b) sequences showing the relationships between strain R28T and related members within the family Halobacteriaceae. Bootstrap values (%) are based on 1,000 replicates and are shown for branches with more 70 % bootstrap support. Bar represents expected changes per site. a 16S rRNA gene. b rpoB0 gene
D. Han et al.: H. rubra Isolated from Laminaria
a
Halovenus aranensis EB27T (HQ197980) 100
99
Halovenus aranensis CGMCC 1.11001T rrnA (KJ534548)
Halovenus aranensis CGMCC 1.11001T rrnB (KJ534549) 98
Halovenus rubra R28T (HM159605) Halomicrobium mukohataei DSM 12286T rrn1 (NC_013202)
0.05
Halomicrobium mukohataei DSM 12286T rrn2 (NC_013202)
86 78
100
Halomicrobium mukohataei DSM 12286T rrn3 (NC_013201)
Halorhabdus utahensis DSM 12940T (NC_013158) Halorientalis regularis TNN28T (GQ282621) Halosimplex carlsbadense JCM 11222T rrn1 (HQ263561)
82 99
Halosimplex pelagicum R2T rrnA (HM159602)
Halosimplex carlsbadense JCM 11222T rrn2 (HQ263563)
99
Halosimplex pelagicum R2T rrnB (KF434756) Natronomonas pharaonis DSM 2160T (NC_007426)
79
Salinirubrum litoreum XD46T (JQ237118) Haloquadratum walsbyi C23T (NC_017459) Salinigranum rubrum GX10T (GU951431) Methanospirillum hungatei JF-1T (NC_007796)
Halomicrobium mukohataei JCM 9738T (AB477172)
b
Halorhabdus utahensis JCM 11049T (AB477175) Halorientalis regularis TNN28T (KF434758) Halosimplex carlsbadense JCM 11222T (AB477192)
0.05 74
100
Halosimplex pelagicum R2T (KF434759) Natronomonas pharaonis DSM 2160T (NC_007426) Halovenus aranensis CGMCC 1.11001T (KJ534550)
100 79
Halovenus rubra R28T (KF573409) Salinigranum rubrum GX10T (KF540217)
Salinirubrum litoreum XD46T (KF316330) Haloquadratum walsbyi C23T (NC_017459) Methanospirillum hungatei JF-1T (NC_007796)
the GenBank gene sequence data (AB840771 & AB840772) submitted by Takashi Itoh from Japan Collection of Microorganisms (Supplementary Fig. S3). Phylogenetic tree reconstruction using the maximumlikelihood (ML) algorithm revealed that strain R28T tightly clustered with Hvn. aranensis CGMCC 1.11001T (Fig. 1a). The rpoB0 gene of strain R28T (1827 nt, KF573409) was found to be similar to the corresponding gene of Hvn. aranensis CGMCC 1.11001T (82.9 % nucleotide identity). In phylogenetic tree reconstructions using rpoB0 , strain R28T clustered with Hvn. aranensis CGMCC 1.11001T (Fig. 1b). The 16S rRNA gene and rpoB0 gene-based phylogenetic analysis results supported the placement of strain R28T in the genus Halovenus.
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The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain R28T represents a novel species of the genus Halovenus, for which the name Halovenus rubra sp. nov. is proposed. Description of Halovenus rubra sp. nov. Halovenus rubra (ru0 bra. L. fem. adj. rubra Red-Colored, Red) Cells were motile, pleomorphic under optimal growth conditions, and stained Gram-negative. Colonies on agar plates containing 3.9 M NaCl were red, elevated, and round. chemoorganotrophic and aerobic. Growth occurred at 25–50 °C
D. Han et al.: H. rubra Isolated from Laminaria
(optimum 42 °C), in the presence of 3.1–5.1 M NaCl (optimum 3.9 M NaCl), with 0.005–1.0 M MgCl2 (optimum 0.01 M MgCl2) and at pH 6.0–9.5 (optimum pH 7.0–7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 15 % (w/v). Catalase and oxidase were positive. They did not grow under anaerobic conditions with nitrate, arginine, or DMSO. Nitrate reduction to nitrite and gas formation from nitrate were not observed. H2S was produced from sodium thiosulfate. Indole formation was negative. Hydrolyzed gelatin but did not hydrolyze starch, casein, or Tween 80. The following substrates were utilized as single carbon and energy sources for growth: sucrose, glycerol, D-mannitol, D-sorbitol, acetate, pyruvate, DL-lactate, succinate, L-malate, fumarate, and citrate. The following substrates were utilized as single carbon, nitrogen, or energy sources for growth: glycine, Larginine, L-aspartate, L-glutamate and L-ornithine. No growth occurred on D-glucose, D-mannose, D-galactose, Dfructose, L-sorbose, D-ribose, D-xylose, maltose, lactose, starch, L-alanine, or L-lysine. The polar lipids were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and two glycolipids (GL1, GL2), the major one (GL1) was unidentified, and the minor one (GL2) was chromatographically identical to mannosyl glucosyl diether (DGD-1). Unsaturated and dihydrogenated menaquinones with eight isoprene units, MK-8, and MK-8 (H2) were present. The DNA G?C content of the type strain was 56.3 mol% (Tm). The type strain R28T (=CGMCC 1.10592T = JCM 17269T) was obtained from the salted brown alga Laminaria produced at Dalian, Liaoning Province, China. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31370054), the Grant from China Ocean Mineral Resources Research and Development 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).
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