Genome mining and physiological analyses uncover adaptation strategies and biotechnological potential of Virgibacillus dokdonensis T4.6 isolated from high-salt shrimp paste.

Virgibacillus spp. stand out as a potent starter culture for accelerating the fermention of fish sauces and shrimp pastes. However, the underlying molecular mechanisms responsible for their adaptation and biotechnological potential remain elusive. Therefore, the present study focuses on phenotypic and genomic analyses of a halophilic bacterium Virgibacillus dokdonensis T4.6, derived from Vietnamese high-salt fermented shrimp paste. The draft genome contained 4,096,868 bp with 3780 predicted coding sequences. Genome mining revealed the presence of 143 genes involved in osmotic adaptation explaining its resistant phenotype to 24% (w/v) NaCl. Among them, 37 genes making up the complete ectoine metabolism pathway, confirmed its ability to produce 4.38 ± 0.29 wt% ectoine under 12.5% NaCl stress. A significant finding was the identification of 39 genes responsible for an entire degradation pathway of the toxic biogenic amine histamine, which was in agreement with its histamine degradation rate of 42.7 ± 2.1% in the HA medium containing 5 mM histamine within 10 days at 37 °C. Furthermore, 114 proteolytic and 19 lipolytic genes were detected which might contribute to its survival as well as the nutrient quality and flavor of shrimp paste. Of note, a putative gene vdo2592 was found as a possible novel lipase/esterase due to its unique Glycine-Aspartate-Serine-Leucine (GDSL) sequence motif. This is the first report to reveal the adaptative strategies and related biotechnological potential of Virgibacillus associated with femented foods. Our findings indicated that V. dokdonensis T4.6 is a promising starter culture for the production of fermented shrimp paste products.

Biosynthetic gene cluster profiling from North Java Sea Virgibacillus salarius reveals hidden potential metabolites.

Virgibacillus salarius 19.PP.SC1.6 is a coral symbiont isolated from Indonesia's North Java Sea; it has the ability to produce secondary metabolites that provide survival advantages and biological functions, such as ectoine, which is synthesized by an ectoine gene cluster. Apart from being an osmoprotectant for bacteria, ectoine is also known as a chemical chaperone with numerous biological activities such as maintaining protein stability, which makes ectoine in high demand in the market industry and makes it beneficial to investigate V. salarius ectoine. However, there has been no research on genome-based secondary metabolite and ectoine gene cluster characterization from Indonesian marine V. salarius. In this study, we performed a genomic analysis and ectoine identification of V. salarius. A high-quality draft genome with total size of 4.45 Mb and 4426 coding sequence (CDS) was characterized and then mapped into the Cluster of Orthologous Groups (COG) category. The genus Virgibacillus has an "open" pangenome type with total of 18 genomic islands inside the V. salarius 19.PP.SC1.6 genome. There were seven clusters of secondary metabolite-producing genes found, with a total of 80 genes classified as NRPS, PKS (type III), terpenes, and ectoine biosynthetic related genes. The ectoine gene cluster forms one operon consists of ectABC gene with 2190 bp gene cluster length, and is successfully characterized. The presence of ectoine in V. salarius was confirmed using UPLC-MS/MS operated in Multiple Reaction Monitoring (MRM) mode, which indicates that V. salarius has an intact ectoine gene clusters and is capable of producing ectoine as compatible solutes.

Distribution of class IId bacteriocin-producing Virgibacillus salexigens in various environments.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations.

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

Mining biosynthetic gene clusters in Virgibacillus genomes.

BACKGROUND: Biosynthetic gene clusters produce a wide range of metabolites with activities that are of interest to the pharmaceutical industry. Specific interest is shown towards those metabolites that exhibit antimicrobial activities against multidrug-resistant bacteria that have become a global health threat. Genera of the phylum Firmicutes are frequently identified as sources of such metabolites, but the biosynthetic potential of its Virgibacillus genus is not known. Here, we used comparative genomic analysis to determine whether Virgibacillus strains isolated from the Red Sea mangrove mud in Rabigh Harbor Lagoon, Saudi Arabia, may be an attractive source of such novel antimicrobial agents. RESULTS: A comparative genomics analysis based on Virgibacillus dokdonensis Bac330, Virgibacillus sp. Bac332 and Virgibacillus halodenitrificans Bac324 (isolated from the Red Sea) and six other previously reported Virgibacillus strains was performed. Orthology analysis was used to determine the core genomes as well as the accessory genome of the nine Virgibacillus strains. The analysis shows that the Red Sea strain Virgibacillus sp. Bac332 has the highest number of unique genes and genomic islands compared to other genomes included in this study. Focusing on biosynthetic gene clusters, we show how marine isolates, including those from the Red Sea, are more enriched with nonribosomal peptides compared to the other Virgibacillus species. We also found that most nonribosomal peptide synthases identified in the Virgibacillus strains are part of genomic regions that are potentially horizontally transferred. CONCLUSIONS: The Red Sea Virgibacillus strains have a large number of biosynthetic genes in clusters that are not assigned to known products, indicating significant potential for the discovery of novel bioactive compounds. Also, having more modular synthetase units suggests that these strains are good candidates for experimental characterization of previously identified bioactive compounds as well. Future efforts will be directed towards establishing the properties of the potentially novel compounds encoded by the Red Sea specific trans-AT PKS/NRPS cluster and the type III PKS/NRPS cluster.

Characterization of candidate genes involved in halotolerance using high-throughput omics in the halotolerant bacterium Virgibacillus chiguensis.

We previously used whole-genome sequencing and Tn5 transposon mutagenesis to identify 16 critical genes involved in the halotolerance of Halomonas beimenensis, a species in the phylum Proteobacteria. In this present study, we sought to determine if orthologous genes in another phylum are also critical for halotolerance. Virgibacillus spp. are halotolerant species that can survive in high-saline environments. Some Virgibacillus species are used in different aspects of food processing, compatible solute synthesis, proteinase production, and wastewater treatment. However, genomic information on Virgibacillus chiguensis is incomplete. We assembled a draft V. chiguensis strain NTU-102 genome based on high-throughput next-generation sequencing (NGS) and used transcriptomic profiling to examine the high-saline response in V. chiguensis. The V. chiguensis draft genome is approximately 4.09 Mbp long and contains 4,166 genes. The expression profiles of bacteria grown in 5% and 20% NaCl conditions and the corresponding Gene Ontology (GO) and clusters of orthologous groups (COG) categories were also analyzed in this study. We compared the expression levels of these 16 orthologs of halotolerance-related genes in V. chiguensis and H. beimenensis. Interestingly, the expression of 7 of the 16 genes, including trkA2, smpB, nadA, mtnN2, rfbP, lon, and atpC, was consistent with that in H. beimenensis, suggesting that these genes have conserved functions in different phyla. The omics data were helpful in exploring the mechanism of saline adaptation in V. chiguensis, and our results indicate that these 7 orthologs may serve as biomarkers for future screening of halotolerant species in the future.

Virgibacillus indicus sp. nov. and Virgibacillus profundi sp. nov, two moderately halophilic bacteria isolated from marine sediment by using microfluidic streak plates.

Three Gram-variable, moderately halophilic, motile, endospore-forming rods, designated P2-C2T, P3-H5T and P3-B8, were isolated from marine sediment of the Southwest Indian Ocean by using the microfluidic streak plate method. Phylogeny based on 16S rRNA gene sequences showed that strains P2-C2T and P3-H5T formed a robust cluster within the genus Virgibacillus and exhibited 16S rRNA gene similarity levels of 95.3-96.8 and 94.9-96.3 % to the type strains of Virgibacillus species, respectively. The 16S rRNA gene similarity between P2-C2T and P3-H5T was 97.6 %. Strain P3-B8 has an identical 16S rRNA gene sequence to strain P3-H5T. For all the novel strains, the predominant cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0, the main menaquinone was MK-7, and the polar lipid profiles contained diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G+C contents of strains P2-C2T, P3-H5T and P3-B8 were 38.3, 37.3 and 37.5 mol%, respectively. Combined data from phenotypic and genotypic studies demonstrated that strains P2-C2T and P3-H5T/P3-B8 are representatives of two different novel species of the genus Virgibacillus, for which the name Virgibacillus indicus sp. nov. and Virgibacillusprofundi are proposed. The type strains are P2-C2T (=CGMCC 1.16138T=NBRC 113014T) and P3-H5T (=CGMCC 1.16139T=NBRC 113015T).

Virgibacillus phasianinus sp. nov., a halophilic bacterium isolated from faeces of a Swinhoe's pheasant, Lophura swinhoii.

A rod-shaped, Gram-stain-positive, motile and aerobic bacterium, designated LM2416T, was isolated from faeces of Lophuras winhoii living in Seoul Grand Park, Gyeonggi-do, Republic of Korea. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain LM2416T belonged to the genus Virgibacillus, sharing high 16S rRNA gene sequence similarities to Virgibacillus necropolis LMG 19488T (99.0 %), Virgibacillus carmonensis LMG 20964T (98.4 %), Virgibacillus arcticus Hal 1T (98.3 %) and Virgibacillus flavescens S1-20T (97.9 %). The isolate grew at 10-30 °C, pH 6-7 and 0-20 % (w/v) NaCl. Optimal growth was observed at 30 °C, pH 6-7 and 10 % (w/v) NaCl. The major fatty acid was anteiso-C15 : 0. Polar lipids were composed of phosphatidylglycerol, diphosphatidylglycerol, three unknown phospholipids and two unknown aminophospholipids. The main menaquinone was MK-7. Strain LM2416T had alanine, lysine, glutamic acid, glycine and aspartic acid as cell-wall amino acids and ribose as a cell-wall sugar. The whole genome sequences of strain LM2416T and V. necropolis KCTC 3820T were sequenced by PacBio RS II sequencing. The genome sequence-based G+C content of strain LM2416T was 39.5 mol%. The orthologous average nucleotide identity value, showing genetic relatedness between strain LM2416T and V. necropolis KCTC 3820T, was 78.3 %. Based on the phylogenetic, biochemical, chemotaxonomic and genotypic data presented in this study, strain LM2416T is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus phasianinus is proposed. The type strain is LM2416T (=KCTC 33927T=JCM 32144T).

Identification, Characteristics and Mechanism of 1-Deoxy-N-acetylglucosamine from Deep-Sea Virgibacillus dokdonensis MCCC 1A00493.

Xanthomonas oryzae pv. oryzae, which causes rice bacterial blight, is one of the most destructive pathogenic bacteria. Biological control against plant pathogens has recently received increasing interest. 1-Deoxy-N-acetylglucosamine (1-DGlcNAc) was extracted from the supernatant of Virgibacillus dokdonensis MCCC 1A00493 fermentation through antibacterial bioassay-guided isolation. Its structure was elucidated by LC/MS, NMR, chemical synthesis and time-dependent density functional theory (TD-DFT) calculations. 1-DGlcNAc specifically suppressed X. oryzae pv. oryzae PXO99A (MIC was 23.90 µg/mL), but not other common pathogens including Xanthomonas campestris pv. campestris str.8004 and Xanthomonas oryzae pv. oryzicola RS105. However, its diastereomer (2-acetamido-1,5-anhydro-2-deoxy-d-mannitol) also has no activity to X. oryzae pv. oryzae. This result suggested that activity of 1-DGlcNAc was related to the difference in the spatial conformation of the 2-acetamido moiety, which might be attributed to their different interactions with a receptor. Eighty-four unique proteins were found in X. oryzae pv. oryzae PXO99A compared with the genome of strains8004 and RS105 by blastp. There may be unique interactions between 1-DGlcNAc and one or more of these unique proteins in X. oryzae pv. oryzae. Quantitative real-time PCR and the pharmMapper server indicated that proteins involved in cell division could be the targets in PXO99A. This research suggested that specificity of active substance was based on the active group and spatial conformation selection, and these unique proteins could help to reveal the specific mechanism of action of 1-DGlcNAc against PXO99A.

Virgibacillus kimchii sp. nov., a halophilic bacterium isolated from kimchi.

A Gram-stain-positive, halophilic, rod-shaped, non-motile, spore forming bacterium, strain NKC1-2T, was isolated from kimchi, a Korean fermented food. Comparative analysis based on 16S rRNA gene sequence demonstrated that the isolated strain was a species of the genus Virgibacillus. Strain NKC1-2T exhibited high level of 16S rRNA gene sequence similarity with the type strains of Virgibacillus xinjiangensis SL6-1T (96.9%), V. sediminis YIM kkny3T (96.8%), and V. salarius SA-Vb1T (96.7%). The isolate grew at pH 6.5-10.0 (optimum, pH 8.5-9.0), 0.0-25.0% (w/v) NaCl (optimum, 10-15% NaCl), and 15-50°C (optimum, 37°C). The major menaquinone in the strain was menaquinone-7, and the main peptidoglycan of the strain was meso-diaminopimelic acid. The predominant fatty acids of the strain were iso-C14:0, anteisio-C15:0, iso- C15:0, and iso-C16:0 (other components were < 10.0%). The polar lipids consisted of diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G + C content of NKC1-2T was 42.5 mol%. On the basis of these findings, strain NKC1-2T is proposed as a novel species in the genus Virgibacillus, for which the name Virgibacillus kimchii sp. nov. is proposed (=KACC 19404T =JCM 32284T). The type strain of Virgibacillus kimchii is NKC1-2T.

Virgibacillus ainsalahensis sp. nov., a Moderately Halophilic Bacterium Isolated from Sediment of a Saline Lake in South of Algeria.

A Gram-positive, moderately halophilic, endospore-forming bacterium, designated MerVT, was isolated from a sediment sample of a saline lake located in Ain Salah, south of Algeria. The cells were rod shaped and motile. Isolate MerVT grew at salinity interval of 0.5-25% NaCl (optimum, 5-10%), pH 6.0-12.0 (optimum, 8.0), and temperature between 10 and 40 °C (optimum, 30 °C).The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, a glycolipid, a phospholipid, and two lipids, and MK-7 is the predominant menaquinone. The predominant cellular fatty acids were anteiso C15:0 and anteiso C17:0. The DNA G+C content was 45.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain MerVT was most closely related to Virgibacillus halodenitrificans (gene sequence similarity of 97.0%). On the basis of phenotypic, chemotaxonomic properties, and phylogenetic analyses, strain MerVT (=DSM = 28944T) should be placed in the genus Virgibacillus as a novel species, for which the name Virgibacillus ainsalahensis is proposed.

Quantification of viable bacterial starter cultures of Virgibacillus sp. and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR.

Real-time quantitative polymerase chain reaction (qPCR) methods were developed for the quantification of Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33, which were added as starter cultures in fish sauce fermentation. The PCR assays were coupled with propidium monoazide (PMA) treatment of samples to selectively quantify viable cells and integrated with exogenous recombinant Escherichia coli cells to control variabilities in analysis procedures. The qPCR methods showed species-specificity for both Virgibacillus halodenitrificans and T. halophilus as evaluated using 6 reference strains and 28 strains of bacteria isolated from fish sauce fermentation. The qPCR efficiencies were 101.1% for V. halodenitrificans and 90.2% for T. halophilus. The quantification limits of the assays were 10(3) CFU/mL and 10(2) CFU/mL in fish sauce samples with linear correlations over 4 Logs for V. halodenitrificans and T. halophilus, respectively. The matrix effect was not observed when evaluated using fish sauce samples fermented for 1-6 months. The developed PMA-qPCR methods were successfully applied to monitor changes of Virgibacillus sp. SK37 and T. halophilus MS33 in a mackerel fish sauce fermentation model where culture-dependent techniques failed to quantify the starter cultures. The results demonstrated the usability of the methods as practical tools for monitoring the starter cultures in fish sauce fermentation.

Production of bacteriocin by Virgibacillus salexigens isolated from "terasi": a traditionally fermented shrimp paste in Indonesia.

A natural antibacterial-substance-producing gram-positive bacterium was isolated from terasi shrimp paste, a popular fermented product in Indonesia. This strain, a spore-forming and strictly aerobic bacterium, was identified as Virgibacillus salexigens by 16S rRNA gene sequence analysis. The antibacterial substance purified from the precipitated product in the culture supernatant of the strain using ammonium sulfate showed a broad inhibition spectrum against gram-positive bacteria, including a typical foodborne bacterium, namely, Listeria monocytogenes. The antibacterial activity of the substance was inactivated by treatments with various proteolytic enzymes. It was stable after heating or pH treatment, and approximately 60% of the initial activity remained even after heating at 121 °C for 15 min. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that its monoisotopic mass weight was 5318.4 Da (M+H)(+). On the basis of the results obtained by the automated Edman degradation technique and MALDI-TOF MS analysis, the substance can be classified as a member of Class IId bacteriocins, but it could not be identified as any of the previously purified substances except for the putative bacteriocin predicted from the draft genome sequence data of gram-positive bacteria such as Virgibacillus and Bacillus strains.

Virgibacillus kapii sp. nov., isolated from Thai shrimp paste (Ka-pi).

A Gram-reaction-positive and rod-shaped bacterium, designated KN3-8-4T, was isolated from shrimp paste collected from a market in Nakhon Sri Thammarat province, Thailand. Strain KN3-8-4T was a strictly aerobic, motile bacterium that produced ellipsoidal endospores at a terminal position. The isolate grew at pH 4.5-10 (optimum pH 7.5), at 11-42 °C (optimum 37 °C) and in the presence of 0-15% (w/v) NaCl (optimum 1-5%). On the basis of 16S rRNA gene sequences, strain KN3-8-4T belonged to the genus Virgibacillus and showed highest similarity with Virgibacillus olivae JCM 30551T (97.85% 16S rRNA gene sequence similarity). The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid and the major isoprenoid quinone was MK-7. Strain KN3-8-4T contained anteiso-C15:0 and anteiso-C17:0 as major cellular fatty acids and had phosphatidylglycerol, diphosphatidylglycerol, two unknown phospholipids and one glycolipid as polar lipids. The DNA G+C content was 43.5 mol%. Strain KN3-8-4T showed low DNA-DNA relatedness (20.44%) with V. olivae JCM 30551T. On the basis of phenotypic and chemotaxonomic data and phylogenetic analyses, strain KN3-8-4T represents a novel species of the genus Virgibacillus for which the name Virgibacillus kapii sp. nov. is proposed. The type strain is KN3-8-4T (=JCM 30071T=LMG 28282T=TISTR 2279T=PCU 345T).

Virgibacillus oceani sp. nov. isolated from ocean sediment.

A Gram-stain-positive, moderately halophilic, motile, strictly aerobic, endospore-forming, rod-shaped bacterium, strain MY11(T), was isolated from a sediment sample collected from the Western Pacific. This isolate grew in the presence of 0.5-18% (w/v) NaCl and at pH 6.0-10.0 and 15-45 °C; optimum growth was observed with 3.5% (w/v) NaCl and at pH 8.0-9.0 and 35-37 °C. Strain MY11(T) had menaquinone 7 (MK-7) as the predominant respiratory quinone and anteiso-C15:0 and anteiso-C17:0 as major fatty acids. Major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C content was 34.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain MY11(T) was a member of the genus Virgibacillus, exhibiting sequence similarities of 95.3-97.6% to the type strains of recognized Virgibacillus species. Strain MY11(T) could be differentiated from recognized species of the genus Virgibacillus based on phenotypic characteristics, chemotaxonomic differences, phylogenetic analysis and DNA-DNA hybridization data. On the basis of the data presented, strain MY11(T) is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus oceani sp. nov. is proposed. The type strain is MY11(T) ( =LMG 28105(T) =CGMCC 1.12754(T) =MCCC 1A09973(T)).

Aquibacillus halophilus gen. nov., sp. nov., a moderately halophilic bacterium from a hypersaline lake, and reclassification of Virgibacillus koreensis as Aquibacillus koreensis comb. nov. and Virgibacillus albus as Aquibacillus albus comb. nov.

A novel Gram-stain-positive, moderately halophilic bacterium, designated strain B6B(T), was isolated from the water of an Iranian hypersaline lake, Aran-Bidgol, and characterized taxonomically using a polyphasic approach. Cells of strain B6B(T) were rod-shaped, motile and produced ellipsoidal endospores in terminal positions in non-swollen sporangia. Strain B6B(T) was a strictly aerobic bacterium and catalase- and oxidase-positive. The strain was able to grow at NaCl concentrations of 0.5-20.0% (w/v), with optimum growth occurring at 10.0% (w/v) NaCl. The optimum temperature and pH for growth were 35 °C and pH 7.0. On the basis of 16S rRNA gene sequence analysis, strain B6B(T) was shown to belong to the phylum Firmicutes and its closest phylogenetic similarities were with the species Virgibacillus koreensis BH30097(T) (97.5%), Virgibacillus albus YIM 93624(T) (97.4%), Sediminibacillus halophilus EN8d(T) (96.8%), Sediminibacillus albus NHBX5(T) (96.6%), Virgibacillus carmonensis LMG 20964(T) (96.3%) and Paraliobacillus quinghaiensis YIM-C158(T) (96.0%), respectively. Phylogenetic analysis revealed that strain B6B(T), along with V. koreensis BH30097(T) and V. albus YIM 93624(T), clustered in a separate clade in the family Bacillaceae. The DNA G+C content of the novel isolate was 35.8 mol%. DNA-DNA hybridization experiments revealed low levels of relatedness between strain B6B(T)and V. koreensis BH30097(T) (13%) and V. albus YIM 93624(T) (33%). The major cellular fatty acid of strain B6B(T) was anteiso-C15 : 0 (75.1%) and its polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. The isoprenoid quinones were MK-7 (90%) and MK-6 (3%). The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. All of these features support the placement of isolate B6B(T) within the phylum Firmicutes. It is closely related to V. koreensis and V. albus, but with features that clearly distinguish it from species of the genus Virgibacillus or of other related genera. On the basis of the polyphasic evidence derived in this study, we propose that strain B6B(T) be placed within a new genus, as Aquibacillus halophilus gen. nov., sp. nov., with B6B(T) as the type strain ( =IBRC-M 10775(T) =KCTC 13828(T)). We also propose that V. koreensis and V. albus should be transferred to this new genus and be named Aquibacillus koreensis comb. nov. and Aquibacillus albus comb. nov., respectively. The type strain of Aquibacillus koreensis comb. nov. is BH30097(T) ( =KCTC 3823(T) =IBRC-M 10657(T) =JCM 12387(T)) and the type strain of Aquibacillus albus comb. nov. is YIM 93624(T) ( =DSM 23711(T) =IBRC-M 10798(T) =JCM 17364(T)).

Virgibacillus halotolerans sp. nov., isolated from a dairy product.

A Gram-stain-positive, strictly aerobic, rod-shaped and weakly motile bacterium, designated WS 4627(T), was isolated from a dairy product sample collected in southern Germany. Spherical to slightly ellipsoidal endospores were formed centrally or subterminally in sometimes slightly swollen sporangia. The isolate was able to grow at 8-35 °C, at pH 6.5-8.5 and with 0.5-16.5% (w/v) NaCl. The diamino acid of the cell wall was meso-diaminopimelic acid (peptidoglycan type A1γ) and the genomic DNA G+C content was 39.1 mol%. The major menaquinone was MK-7, the cellular fatty acid profile contained major amounts of anteiso-C(15:0) and anteiso-C(17:0) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Strain WS 4627(T) was most closely related to 'Virgibacillus natachei' FarD (96.5% 16S rRNA gene sequence similarity) and 'Virgibacillus zhanjiangensis' JSM 079157 (96.0%). Based on the data presented, strain WS 4627(T) represents a novel species of the genus Virgibacillus, for which the name Virgibacillus halotolerans sp. nov. is proposed. The type strain is WS 4627(T) ( =DSM 25060(T) =LMG 26644(T)).

Virgibacillus natechei sp. nov., a moderately halophilic bacterium isolated from sediment of a saline lake in southwest of Algeria.

A novel, Gram-positive, moderately halophilic bacterium, oxidase- and catalase-positive designated FarD(T) was isolated from sediments of a saline lake located in Taghit, 93 km from Bechar, southwest of Algeria. Cells were rod-shaped, endospore forming, and motile. Growth occurred at 15-40 °C (optimum, 35 °C), pH 6.0-12.0 (optimum, 7.0) and in the presence of 1-20 % NaCl (optimum, 10 %). Strain FarD(T) used glucose, mannitol, melibiose, D-mannose, and 5 ketogluconate. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, and three phospholipids; MK-7 is the predominant menaquinone. The predominant cellular fatty acids were anteiso C15:0, anteiso C17:0, C20:0, and anteiso C19:0. The DNA G+C content was 42.1 mol%. Phylogenetic analysis of the small-subunit ribosomal RNA gene sequence indicated that strain FarD(T) had as its closest relative Virgibacillus salinus (similarity of 96.3 %). Based on phenotypic, phylogenetic, and taxonomic characteristics, strain FarD(T) is proposed as a novel species of the genus Virgibacillus within the order Clostridiales, for which the name V. natechei is proposed. The type strain is FarD(T) (=DSM 25609(T) = CCUG 62224(T)).

Draft genome sequence of Virgibacillus halodenitrificans 1806.

Virgibacillus halodenitrificans 1806 is an endospore-forming halophilic bacterium isolated from salterns in Korea. Here, we report the draft genome sequence of V. halodenitrificans 1806, which may reveal the molecular basis of osmoadaptation and insights into carbon and anaerobic metabolism in moderate halophiles.

Optimization and characterization of chromium(VI) reduction in saline condition by moderately halophilic Vigribacillus sp. isolated from mangrove soil of Bhitarkanika, India.

A Gram-positive moderately halophilic Cr(VI) tolerant bacterial strain H4, isolated from saline mangrove soil, was identified as Vigribacillus sp. by biochemical characterization and 16S rRNA analysis. In LB medium, the strain could tolerate up to 1000 mg L(-1) Cr(VI) concentration and reduced 90.2 and 99.2% of 100 mg L(-1) Cr(VI) under optimized set of condition within 70 h in absence and presence of 6 wt.% NaCl, respectively. The fitting of time course reduction data to an exponential rate equation yielded the Cr(VI) reduction rate constants in the range (0.69-5.56)×10(-2)h(-1). Analyses of total chromium and bacterial cell associated with reduced product by AAS, SEM/EDS, TEM/SAED, FT-IR and UV-vis-DRS indicated the formation of about 35% of insoluble Cr(III) either as Cr(OH)(3) precipitate in nanometric size or immobilized on the bacterial cell surface while the remaining 65% of reduced chromium was present as soluble Cr(III) in the growth medium. Powder XRD analysis revealed the amorphous nature of the precipitated Cr(OH)(3). The high Cr(VI) reducing ability of the strain under saline condition suggests the Vigribacillus sp. as a new and efficient strain capable of remediating highly saline Cr(VI) polluted industrial effluents.