Halomonas antri sp. nov., a carotenoid-producing bacterium isolated from surface seawater.

A Gram-negative, moderately halophilic bacterium, designated as strain Y3S6T, was isolated from a surface seawater sample collected from Dongangyoeng cave, Udo-myeon, Jeju-si, Jeju-do, Repulic of Korea. Cells of strain Y3S6T were aerobic, rod-shaped, non-sporulated, yellow, catalase- negative, oxidase-negative and motile with one polar flagellum. Growth of strain Y3S6T occurred at 15-40 °C (optimum: 25-30 °C), at pH 6.0-9.0 (optimum: pH 7.0) and in the presence of 0-13% NaCl (optimum: 1-6 %, w/v). The novel strain was able to produce carotenoids. Its chemotaxonomic and morphological characteristics were consistent with those of members of the genus Halomonas. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Y3S6T formed a clade with Halomonas pellis L5T (98.97 %) and Halomonas saliphila LCB169T(98.90%). The average nucleotide identity and digital DNA-DNA hybridization values of strain Y3S6T with the most closely related strains for which whole genomes are publicly available were 82.3-85.2% and 62.8-66.1 %, respectively. The major fatty acids in strain Y3S6T were C16 : 0, C19 : 0 cyclo ω8c and summed feature 8 (composed of C18 : 1 ω7c and/or C18 : 1 ω6c), and the predominant quinone was Q-9. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phosphoglycolipid, one unidentified phosphoaminoglycolipid and one unidentified phospholipid. The genomic DNA G+C content based on the draft genome sequence was 64.2 mol%. The results of physiological and biochemical tests and 16S rRNA sequence analysis clearly revealed that strain Y3S6T represents a novel species in the genus Halomonas, for which the name Halomonas antri sp. nov. has been proposed. The type strain is Y3S6T (=KACC 21536T=NBRC 114315=TBRC 15164T).

Carotenoids produced by halophilic bacterial strains on mural paintings and laboratory conditions.

Due to the presence of efflorescences and improper microclimate conditions for conservation, pink-pigmented areas were reported in two historic monuments in Northern and Central part of Romania. The aims of the present study were to find the nature of pink pigments observed on the pictorial layer, original and infilling mortar, to investigate the presence of carotenoids both on mural paintings and in the isolated halophilic bacterial strains and to preliminary characterize and identify the producing strains. Their role in the aesthetical biodeterioration of historic monuments was also pointed out. Obtained Raman spectra of the pink pigments extracted both from the isolated bacterial cultures (molecularly identified as mostly related to Halobacillus hunanensis and Halobacillus naozhouensis) and from the mural painting samples contain diagnostic bands of carotenoids. These results were confirmed by FTIR spectroscopy. The strong Raman signal of bacterial carotenoids detected on mural painting indicated their potential use as biomarker molecules in the evaluation of contamination and state of conservation of mural paintings and lithic monuments. Our results contribute to opening a new direction in cultural heritage restoration to assess the conservation status on the basis of interdisciplinary research, starting with spectroscopic methods (Raman, FTIR) and confirmed by microbiological analysis.

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov.

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PET (DSM 105487T, CIP 111404T) and 5HT (DSM 105,488T, CIP 111405T), respectively.

Diversity of culturable moderately halophilic bacteria producing extracellular hydrolytic enzymes from marine sediments / Diversidade de bactérias cultiváveis moderadamente halofílicas que produzem enzimas hidrolíticas extracelulares de sedimentos marinhos

A total of 114 moderately halophilic bacteria were isolated from marine sediment environments. The isolates are belonged to 23 species based on the 16S rRNA sequence analysis. 63, 52, 47, 57, 74, 15 and 4 isolates are able to produce protease, amylase, lipase, pectinase, pulluanase, xylanase, cellulase, respectively. Combined hydrolytic enzyme activity analysis show that 15 strains present 1 hydrolytic activity, 32 strains present 2 hydrolytic activities, 21 strains present 3 hydrolytic activities, 26 strains present 4 hydrolytic activities, 11 strains present 5 hydrolytic activities and 2 strains present 6 hydrolytic activities. Hydrolase activities are widely distributed in a variety of species. The highest rates for production of protease, amylase, lipase, pectinase, pullanase, xylanase and cellulase were observed in species of B. baekryungensis, Hallobacillus sp., B. pumilus, B. megaterium or P. chungwhensis, B. amyloliquefaciens, B. pumilus, B. baekryungensis, respectively. However, the higher activities of protease, pectinase and pulluanase are frequently produced by the species of Halomonas sp. B. amyloliquefaciens or P. chungwhensis, and Vibrio sp. respectively. This investigation show that the diversity of halophilic bacteria from marine sediments could serve as a potential source of hydrolytic enzymes for industrial applications. (AU)

Um total de 114 bactérias moderadamente halofílicas foram isoladas de ambientes de sedimentos marinhos. Os isolados pertencem a 23 espécies com base na análise da sequência 16S rRNA. 63, 52, 47, 57, 74, 15 e 4 isolados são capazes de produzir protease, amilase, lipase, pectinase, pululanase, xilanase, celulase, respectivamente. A análise da atividade enzimática hidrolítica combinada mostra que 15 cepas apresentam 1 atividade hidrolítica, 32 cepas apresentam 2 atividades hidrolíticas, 21 cepas apresentam 3 atividades hidrolíticas, 26 cepas apresentam 4 atividades hidrolíticas, 11 cepas apresentam 5 atividades hidrolíticas e 2 cepas apresentam 6 atividades hidrolíticas. Atividades de hidrolase são amplamente distribuídas em uma variedade de espécies. As maiores taxas de produção de protease, amilase, lipase, pectinase, pululanase, xilanase e celulase foram observadas em espécies de B. baekryungensis, Hallobacillus sp., B. pumilus, B. megaterium ou P. chungwhensis, B. amyloliquefaciens, B. pumilus, B. baekryungensis, respectivamente. No entanto, as atividades mais elevadas de protease, pectinase e pululanase são freqüentemente produzidas pelas espécies de Halomonas sp. B. amyloliquefaciens ou P. chungwhensis e Vibrio sp. respectivamente. Esta investigação mostra que a diversidade de bactérias halofílicas de sedimentos marinhos pode servir como uma fonte potencial de enzimas hidrolíticas para aplicações industriais. (AU)

Emended description of Salinivibrio proteolyticus, including Salinivibrio costicola subsp. vallismortis and five new isolates.

We carried out a comparative taxonomic study of Salinivibrio proteolyticus and Salinivibrio costicola subsp. vallismortis, as well as of five halophilic strains (IB574, IB872, PR5, PR919 and PR932), isolated from salterns in Spain and Puerto Rico that were closely related to these bacteria. Multilocus sequence analysis of concatenated gyrB, recA, rpoA and rpoD housekeeping genes showed that they constituted a single cluster separate from the other species and subspecies of Salinivibrio. Experimental and in silico DNA-DNA hybridization studies indicated that they are members of the same species, with relatedness of 100-74 % and 97.8-70.0 %, respectively. The average nucleotide identity (ANI) determined for these strains was 99.7-95.6 % for ANIb and 99.7-95.7 % for OrthoANI. However, the ANI values for S. costicolasubsp.vallismortis DSM 8285T with respect to S. costicolasubsp.costicola DSM 11403T and S. costicolasubsp.alcaliphilus DSM 16359T were 78.7 and 78.9 % (ANIb) and 79.4 and 79.4 % (OrthoANI), respectively. The phylogenomic tree based on 1072 concatenated orthologous single-copy core genes confirmed that S. proteolyticus, S. costicolasubsp.vallismortis and the five new isolates constitute a coherent single phylogroup, separated from the other species and subspecies of Salinivibrio. All these data indicate that S. costicolasubsp.vallismortis is a heterotypic synonym of S. proteolyticus and we propose an emended description of this species.

Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns.

Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37°C, at pH 7.2-7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAGT (99.6-98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508T (99.0-98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472T (96.8-95.4% and 94.9-94.7%, respectively) and S. sharmensis DSM 18182T (94.0-92.6% and 92.9-92.7%, respectively). In silico DNA-DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3-44.8% and 80.2-91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184T were C16:0 and C18:1. The DNA G+C content range was 51.9-52.5mol% (Tm) and 50.2-50.9mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184T (=CECT 9177T=LMG 29817T).

Lentibacillus sediminis sp. nov., isolated from a marine saltern.

A novel, Gram-stain-positive, moderately halophilic, endospore-forming, motile, facultatively anaerobic and rod-shaped strain, designated 0W14T, was isolated from a marine saltern of Wendeng, China. Optimal growth occurred at 37 °C, pH 7.5 and with 6.0 % (w/v) NaCl. MK-7 was the sole respiratory quinone and the peptidoglycan type of 0W14T was A4ßl-Orn-d-Glu. The major cellular fatty acid (>10.0 %) in strain 0W14T was anteiso-C15 : 0. The polar lipid profile of strain 0W14T consisted of diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids and four unknown phospholipids. The genomic DNA G+C content of the strain was 44.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 0W14T forms a phylogenetic lineage with members of the genus Lentibacillus within the family Bacillaceae. Based on data from the current polyphasic study, the isolate is proposed to represent a novel species of genus Lentibacillus, for which the name Lentibacillus sediminis sp. nov. is proposed. The type strain is 0W14T (=KCTC 33835T=MCCC 1H00171T).

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Virgibacillus senegalensis sp. nov., a new moderately halophilic bacterium isolated from human gut.

Virgibacillus senegalensis SK-1(T) (= CSUR P1101 = DSM 28585) is the type strain of V. senegalensis sp. nov. It is an aerobic, Gram positive, moderately halophilic, motile bipolar flagellum isolated from a healthy Senegalese man. Here we describe the genomic and phenotypic characteristics of this isolate. The 3 755 098 bp long genome (one chromosome, no plasmid) exhibits a G + C content of 42.9% and contains 3738 protein-coding and 95 RNA genes.

Noncontiguous finished genome sequence and description of Virgibacillus massiliensis sp. nov., a moderately halophilic bacterium isolated from human gut.

Strain Vm-5(T) was isolated from the stool specimen of a 10-year-old Amazonian boy. This bacterium is a Gram-positive, strictly aerobic rod, motile by a polar flagellum. Here we describe its phenotypic characteristics and complete genome sequence. The 4 353 177 bp long genome exhibits a G + C content of 36.87% and contains 4394 protein-coding and 125 predicted RNA genes. Phylogenetically and genetically, strain Vm-c is a member of the genus Virgibacillus but is distinct enough to be classified as a new species. We propose the creation of V. massiliensis sp. nov., whose type strain is strain Vm-5(T) (CSUR P971 = DSM 28587).

Partial characterization of an extracellular polysaccharide produced by the moderately halophilic bacterium Halomonas xianhensis SUR308.

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) was isolated from a multi-pond solar saltern at Surala, Ganjam district, Odisha, India. The isolate produced a significant amount (7.87 g l(-1)) of extracellular polysaccharides (EPS) when grown in malt extract-yeast extract medium supplemented with 2.5% NaCl, 0.5% casein hydrolysate and 3% glucose. The EPS was isolated and purified following the conventional method of precipitation and dialysis. Chromatographic analysis (paper, GC and GC-MS) of the hydrolyzed EPS confirmed its heteropolymeric nature and showed that it is composed mainly of glucose (45.74 mol%), galactose (33.67 mol %) and mannose (17.83 mol%). Fourier-transform infrared spectroscopy indicated the presence of methylene and carboxyl groups as characteristic functional groups. In addition, its proton nuclear magnetic resonance spectrum revealed functional groups specific for extracellular polysaccharides. X-ray diffraction analysis revealed the amorphous nature (CIxrd, 0.56) of the EPS. It was thermostable up to 250 °C and displayed pseudoplastic rheology and remarkable stability against pH and salts. These unique properties of the EPS produced by H. xianhensis indicate its potential to act as an agent for detoxification, emulsification and diverse biological activities.

High-throughput screening for a moderately halophilic phenol-degrading strain and its salt tolerance response.

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg · L(-1) starting concentration) over a range of 3%-10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

Investigation of mercury tolerance in Chromohalobacter israelensis DSM 6768T and Halomonas zincidurans B6T by comparative genomics with Halomonas xinjiangensis TRM 0175T.

Chromohalobacter israelensis DSM 6768(T), Halomonas zincidurans B6(T), and Halomonas xinjiangensis TRM 0175(T) are three phylogenetically close strains belonging to the class Gammaproteobacteria. Both strains DSM 6768(T) and B6(T) can grow on plate containing 0.5mM HgCl2. Strain TRM 0175(T) could not grow on plates containing 0.1mM or more HgCl2. Here we report the draft genomes of strains DSM 6768(T) and TRM 0175(T) for comparative genomic analysis. Gene cluster with putative function in mercury resistance in strain DSM 6768(T) includes a mercuric ion reductase, whose homologues distribute among several marine microbes. Strain B6(T), which was isolated from the Atlantic Ocean, has one more gene cluster with putative function in mercury resistance than strain DSM 6768(T). This study will enhance our understanding of the mercury tolerance and further investigation in marine microbes.

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis.

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18(T) and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3-25% (w/v) NaCl (optimal growth at 7.5-10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0-7.0) and at temperatures between 15 and 40°C (optimal at 37°C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985(T) (95.2-94.8%) and Chromohalobacter salexigens DSM 3043(T) (95.0-94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA-DNA hybridization between both strains was 82%, whereas the values between strain M1-18(T) and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C18:1ω7c/C18:1ω6c, C16:0, and C16:1ω7c/C16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18(T) (=CCM 8464=CECT 8192(T)=IBRC-M 10767(T)=LMG 27461(T)).