Halophiles Research Institute

Noda, Japan

Halophiles Research Institute

Noda, Japan
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Gutierrez M.C.,University of Seville | Castillo A.M.,University of Seville | Corral P.,University of Seville | Kamekura M.,Halophiles Research Institute | Ventosa A.,University of Seville
International Journal of Systematic and Evolutionary Microbiology | Year: 2011

Two halophilic archaea, strains EN-2T and SH-4, were isolated from the saline lakes Erliannor and Shangmatala, respectively, in Inner Mongolia, China. Cells were strictly aerobic, motile rods. Colonies were red. Strains EN-2T and SH-4 were able to grow at 25-50 6° (optimum 35-40 6C), with 2.5-5.0 M NaCl (optimum 3.4 M NaCl) and at pH 6.0-9.0 (optimum pH 7.5). MgCl2 was not required for growth. Cells lysed in distilled water and the lowest NaCl concentration that prevented cell lysis was 12% (w/v). On the basis of 16S rRNA gene sequence analysis, strains EN-2T and SH-4 were closely related to Halorubrum cibi B31T (97.9 and 98.0% similarity, respectively), Hrr. tibetense 8W8T (97.3 and 97.7%), Hrr. alkaliphilum DZ-1T (96.8 and 97.1%), Hrr. luteum CGSA15T (96.8 and 97.0%) and Hrr. lipolyticum 9-3T (96.8 and 97.0%). DNA-DNA hybridization showed that strains EN-2T and SH-4 did not belong to the same species as any of these strains (≤45% DNA-DNA relatedness) but that they are members of the same species (>70% DNA-DNA relatedness). Polar lipid analysis revealed that strains EN-2T and SH-4 contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diethers and several unidentified glycolipids. The DNA G+C content of both isolates was 62.1 mol%. It was concluded that strains EN-2T and SH-4 represent a novel species of the genus Halorubrum, for which the name Halorubrum aquaticum sp. nov. is proposed. The type strain is EN-2T (=CECT 7174T =CGMCC 1.6377T =JCM 14031T). © 2011 IUMS Printed in Great Britain.


Papke R.T.,University of Connecticut | White E.,University of Connecticut | Reddy P.,University of Connecticut | Weigel G.,University of Connecticut | And 4 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2011

Members of the order Halobacteriales are obligate extreme halophiles that belong to the domain Archaea. The classification of the Halobacteriales currently relies on a polyphasic approach, which integrates phenotypic, genotypic and chemotaxonomic characterization. However, the most utilized genetic marker for phylogeny, the 16S rRNA gene, has multiple drawbacks for use with the Halobacteriales: the species of many genera exhibit large intragenic differences between multiple ribosomal RNA operons, the gene is too conserved to discriminate reliably at the species level and it appears to be the most frequently recombined gene between closely related species. Moreover, the Halobacteriales is a rapidly expanding group due to recent successes at cultivating novel strains from a diverse set of hypersaline environments; a fast, reliable, inexpensive, portable molecular method for discriminating species is required for their investigation. Recently, multilocus sequence analysis (MLSA) has been shown to be an effective tool for strain identification and taxonomic designation, even for those taxa that experience frequent lateral gene transfer and homologous recombination. In this study, MLSA was utilized for evolutionary and taxonomic investigation of the Halobacteriales. Efficacy of the MLSA approach was tested across a hierarchical gradient using 52 halobacterial strains, representing 33 species (including names without standing in nomenclature) and 14 genera. A subset of 21 strains from the genus Haloarcula was analysed separately to test the sensitivity and relevance of the MLSA approach among closely related strains and species. The results demonstrated that MLSA differentiated individual strains, reliably grouped strains into species and species into genera and identified potential novel species and also family-like relationships. This study demonstrates that MLSA is a rapid and informative molecular method that will probably accommodate strain analysis at any taxonomic level within the Halobacteriales. © 2011 IUMS.


Burns D.G.,University of Melbourne | Janssen P.H.,Grasslands Research Center | Itoh T.,RIKEN | Kamekura M.,Halophiles Research Institute | And 2 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2010

Strains 1.15.5T, 2.27.5, 5.24.4 and 6.14.5 were isolated from a solar saltern. They have flattened, rod-shaped cells and are aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media around neutral pH and containing 20-24% (w/v) (strains 1.15.5T and 2.27.5) or 22-24% (w/v) (5.24.4 and 6.14.5) salts. Mg2+ was not required. The DNA G+C contents of these isolates were all close to 58 mol%, and DNA-DNA cross-hybridization showed a mean relatedness of 77 %. Their 16S rRNA gene sequences differed by no more than 1.6% from each other. Phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halorubrum saccharovorum (86.6-87.6% 16S rRNA gene sequence similarity to the type strain). The only major polar lipid of all four isolates was the sulfated diglycosyl diether lipid S-DGD-1. By phase-contrast microscopy, the long, flattened cells of these strains often displayed a 'wing-like' shape. The phenotypic and phylogenetic data support the placement of these isolates into a novel species in a new genus within the Halobacteriaceae, for which we propose the name Halonotius pteroides gen. nov., sp. nov. The type strain of Halonotius pteroides is 1.15.5T (=JCM 14355T =CECT 7525T 5DSM 18729T), with the additional reference strains 2.27.5 (=JCM 14356 =DSM 18671), 5.24.4 (=JCM 14357 =DSM 18673) and 6.14.5 (=JCM 14358 =DSM 18692). © 2010 IUMS.


Echigo A.,Toyo University | Minegishi H.,Toyo University | Shimane Y.,Japan Agency for Marine - Earth Science and Technology | Kamekura M.,Halophiles Research Institute | And 2 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2013

A novel halophilic strain, BNERC31T, was isolated from solar salt, 'Sel marin de Guérande', imported from France. Colonies on agar medium containing soluble starch, sodium citrate, sodium glutamate and inorganic salts were non-pigmented and transparent, while cells obtained by centrifuging liquid cultures were red-pigmented. Cells of strain BNERC31T were non-motile, pleomorphic, stained Gram-negative and lysed in distilled water. Growth occurred with 20-30% (w/v) NaCl (optimum, 25 %, w/v), with 0-500 mM MgCl2 (optimum, 10 mM), at pH 6.0-8.5 (optimum, pH 7.0) and at 25-55 °C (optimum, 40 °C). Growth was dependent on soluble starch, and inhibited completely by 0.5% organic nutrients, such as Casamino acids or yeast extract. The DNA G+C content was 64.1 mol%. Strain BNERC31T possessed at least two heterogeneous 16S rRNA genes, and the sequence of the orthologous gene (preceded by the dihydroorotate oxidase gene, pyrD) showed the highest similarity (96.5 %) to that of Haloarcula marismortui JCM 8966T. The RNA polymerase subunit B' gene sequence showed the highest similarity (91.7 %) to that of Haloarcula amylolytica JCM 13557T. The polar lipids of strain BNERC31T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, diglycosyl diether and sulfated diglycosyl diether, similar to those of species of the genus Halomicrobium. The phenotypic and phylogenetic characteristics showed that strain BNERC31T differed from species of the genera Haloarcula and Halomicrobium and indicated that it represents a novel species in a new genus, for which the name Halomicroarcula pellucida gen. nov., sp. nov. is proposed. The type strain of the type species is BNERC31T (=JCM 17820T=CECT 7537T). © 2013 IUMS.


Echigo A.,Toyo University | Minegishi H.,Toyo University | Shimane Y.,Toyo University | Kamekura M.,Halophiles Research Institute | Usami R.,Toyo University
International Journal of Systematic and Evolutionary Microbiology | Year: 2012

A moderately halophilic and alkalitolerant bacterium, designated strain HN30T, was isolated from garden soil in Japan. Cells of strain HN30T were motile, endospore-forming, aerobic, rod-shaped and Gram-positive, and contained A1γ meso-diaminopimelic acid-type murein. Growth occurred in 7-23% (w/v) NaCl (optimum, 10-15%, w/v), at pH 6.5-10.0 (optimum, pH 8.0-8.5) and at 20-40 6C (optimum, 30 °C). The isoprenoid quinone was menaquinone-7. The polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major cellular fatty acids were anteiso-C15: 0, anteiso-C17: 0, iso-C16: 0 and C16: 0. The DNA G+C content of strain HN30T was 47 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HN30T was most closely related to Geomicrobium halophilum BH1T (93% sequence similarity). 16S rRNA gene sequence similarities with other recognized species were less than 89%. Phylogenetic and phenotypic characteristics indicated that strain HN30T represents a novel species in a new genus, for which the name Natribacillus halophilus gen. nov., sp. nov. is proposed; the type strain is HN30T (=JCM 15649T=DSM 21771T). © 2012 IUMS.


Nagaoka S.,Toyo University | Minegishi H.,Toyo University | Echigo A.,Toyo University | Shimane Y.,Toyo University | And 2 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2011

A Gram-negative, pleomorphic, aerobic, haloalkaliphilic archaeon, strain 167-74 T, was isolated from commercial rock salt imported into Japan from China. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain 167-74 T is closely related to Halostagnicola larsenii XH-48 T (98.3%) and Halostagnicola kamekurae 194-10 T (97.2%). The major polar lipids of the isolate were C 20C 20 and C 20C 25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. A glycolipid was not detected, in contrast to the two existing, neutrophilic species of the genus Halostagnicola. The DNA G+C content of strain 167-74 T was 60.7 mol%. and it gave DNA-DNA reassociation values of 19.5 and 18.8%, respectively, with Hst. larsenii JCM 13463 T and Hst. kamekurae 194-10 T. Therefore, strain 167-74 T represents a novel species, for which the name Halostagnicola alkaliphila sp. nov. is proposed, with the type strain 167-74 T (=JCM 16592 T =CECT 7631 T). © 2011 IUMS Printed in Great Britain.


Echigo A.,Toyo University | Minegishi H.,Toyo University | Mizuki T.,Toyo University | Kamekura M.,Halophiles Research Institute | Usami R.,Toyo University
International Journal of Systematic and Evolutionary Microbiology | Year: 2010

Two moderately halophilic and alkaliphilic bacteria, designated strains BH1T and HN5, were isolated from forest soil and garden soil, respectively, in Japan. Cells of strains BH1T and HN5 were non-motile, aerobic, bean-shaped, formed irregular clusters with 2-20 cells, Gram-positive and contained A1γ, meso-diaminopimelic acid-type murein. Spore formation was not detected. Growth occurred in 5-25 % (w/v) NaCl (optimum, 10-15%, w/v), at pH 6.0-10.0 (optimum, pH 8.5-9.0) and at 20-40 °C (optimum, 30 °C). The predominant isoprenoid quinones were menaquinone-7 and menaquinone-6. The phospholipids were diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids were i-C15:0, i-C17:0 and i-C18:0. The DNA G+C content of strains BH1T and HN5 was 45 and 46 mol%, respectively. The 16S rRNA gene sequences of the two strains were 99.9% similar. DNA-DNA hybridization results indicated high levels of relatedness (88 and 85 % reciprocally). Similarities with recognized species were less than 90.2 %. The phylogenetic and phenotypic characteristics indicated that strains BH1T and HN5 represent a novel species in a new genus, for which the name Geomicrobium halophilum gen. nov., sp. nov. is proposed. The type strain is BH1T (=JCM 15647 T=DSM 21769T). © 2010 IUMS.


Minegishi H.,Toyo University | Kamekura M.,Halophiles Research Institute | Itoh T.,RIKEN | Echigo A.,Toyo University | And 2 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2010

A considerable number of species of the Halobacteriaceae possess multiple copies of the 16S rRNA gene that exhibit more than 5% divergence, complicating phylogenetic interpretations. Two additional problems have been pointed out: (i) the genera Haloterrigena and Natrinema show a very close relationship, with some species being shown to overlap in phylogenetic trees reconstructed by the neighbour-joining method, and (ii) alkaliphilic and neutrophilic species of the genus Natrialba form definitely separate clusters in neighbour-joining trees, suggesting that these two clusters could be separated into two genera. In an attempt to solve these problems, the RNA polymerase B′ subunit has been used as an additional target molecule for phylogenetic analysis, using partial sequences of 1305 bp. In this work, a primer set was designed that consistently amplified the full-length RNA polymerase B′ subunit gene (rpoB′) (1827-1842 bp) from 85 strains in 27 genera of the Halobacteriaceae. Differences in sequence length were found within the first 15 to 31 nt, and their downstream sequences (1812 bp) were aligned unambiguously without any gaps or deletions. Phylogenetic trees reconstructed from nucleotide sequences and deduced amino acid sequences by the maximum-likelihood method demonstrated that multiple species/strains in most genera individually formed cohesive clusters. Two discrepancies were observed: (i) the two species of Natronolimnobius were placed in definitely different positions, in that Natronolimnobius innermongolicus was placed in the Haloterrigena/Natrinema cluster, while Natronolimnobius baerhuensis was closely related to Halostagnicola larsenii, and (ii) Natronorubrum tibetense was segregated from the three other Natronorubrum species in the protein tree, while all four species formed a cluster in the gene tree, although supported by a bootstrap value of less than 50%. The six Haloterrigena species/strains and the five species of Natrinema formed a large cluster in both trees, with Halopiger xanaduensis and Nln. innermongolicus located in the cluster in the protein tree and Nln. innermongolicus in the gene tree. Hpg. xanaduensis broke into the cluster of the genus Halobiforma, instead of the Haloterrigena/Natrinema cluster, in the gene tree. The six Natrialba species formed a tight cluster with two subclusters, of neutrophilic species and alkaliphilic species, in both trees. Overall, our data strongly suggest that (i) Nln. innermongolicus is a member of Haloterrigena/Natrinema, (ii) Nrr. tibetense might represent a new genus and (iii) the two genera Haloterrigena and Natrinema might constitute a single genus. Asmore and more novel species and genera are proposed in the family Halobacteriaceae, the full sequence of the rpoB′ gene may provide a supplementary tool for determining the phylogenetic position of new isolates. © 2010 IUMS.


Minegishi H.,Toyo University | Echigo A.,Toyo University | Nagaoka S.,Toyo University | Kamekura M.,Halophiles Research Institute | Usami R.,Toyo University
International Journal of Systematic and Evolutionary Microbiology | Year: 2010

A novel halophilic archaeon, strain MH1-52-1 T, was isolated from solar salt imported from Australia. Cells were pleomorphic, non-motile and Gram-negative. Strain MH1-52-1 T required at least 3.0 M NaCl and 1 mM Mg 2+ for growth. Strain MH1-52-1 T was able to grow at pH 4.0-6.0 (optimum, pH 4.4-4.5) and 15-45 °C (optimum, 37 °C). The diether phospholipids phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, derived from both C 20C 20 and C 20C 25 archaeol, were present. Four unidentified glycolipids were also detected. The 16S rRNA gene sequence showed the highest similarity to that of Halobacterium noricense A1 T (91.7 %); there were lower levels of similarity to other members of the family Halobacteriaceae. The G+C content of its DNA was 61.4 mol%. Based on our phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolate should be classified as a representative of a new genus and species, for which the name Halarchaeum acidiphilum gen. nov., sp. nov. is proposed. The type strain of Halarchaeum acidiphilum is MH1-52-1 T (5JCM 16109 T =DSM 22442 T =CECT 7534 T). © 2010 IUMS.


Minegishi H.,Toyo University | Echigo A.,Toyo University | Shimane Y.,Toyo University | Kamekura M.,Halophiles Research Institute | And 3 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2012

Halobacterium piscisalsi was proposed by Yachai et al. (2008), with a single strain, HPC1-2T (5BCC 24372T 5JCM 14661T 5PCU 302T), which was isolated from fermented fish (pla-ra) in Thailand. According to Yachai et al. (2008), the strain was closely related to Halobacterium salinarum based on 16S rRNA gene sequence comparisons and could be differentiated by low DNA-DNA relatedness values and different biochemical profiles compared with other species of the genus. The reanalysis of the 16S rRNA gene sequences and the DNA-DNA relatedness among H. piscisalsi JCM 14661T and H. salinarum strains JCM 8978T, R1 and NRC-1 revealed that they all had exactly the same 16S rRNA gene sequence and shared more than 70% DNA- DNA relatedness. In addition, the full-length DNA-dependent RNA polymerase subunit B (RpoB) protein sequence of H. piscisalsi JCM 14661T (607 amino acids) was the same as that of H. salinarum JCM 8978T and showed 94.7 and 96.7% similarities with those of Halobacterium noricense JCM 15102T and Halobacterium jilantaiense JCM 13558T, respectively. Despite the different biochemical properties described by Yachai et al. (2008), the characteristic phenotypic properties of H. piscisalsi agreed with those in the description of H. salinarum emended byGruber et al. (2004). Therefore, H. piscisalsi Yachai et al. (2008) should be regarded as a later heterotypic synonym of H. salinarum Elazari-Volcani 1957 © 2012 IUMS.

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