Influence of temperature, salinity and Mg2+:Ca2+ ratio on microbially-mediated formation of Mg-rich carbonates by Virgibacillus strains isolated from a sabkha environment.

Studies have demonstrated that microbes facilitate the incorporation of Mg2+ into carbonate minerals, leading to the formation of potential dolomite precursors. Most microbes that are capable of mediating Mg-rich carbonates have been isolated from evaporitic environments in which temperature and salinity are higher than those of average marine environments. However, how such physicochemical factors affect and concur with microbial activity influencing mineral precipitation remains poorly constrained. Here, we report the results of laboratory precipitation experiments using two mineral-forming Virgibacillus strains and one non-mineral-forming strain of Bacillus licheniformis, all isolated from the Dohat Faishakh sabkha in Qatar. They were grown under different combinations of temperature (20°, 30°, 40 °C), salinity (3.5, 7.5, 10 NaCl %w/v), and Mg2+:Ca2+ ratios (1:1, 6:1 and 12:1). Our results show that the incorporation of Mg2+ into the carbonate minerals is significantly affected by all of the three tested factors. With a Mg2+:Ca2+ ratio of 1, no Mg-rich carbonates formed during the experiments. With a Mg2+:Ca2+ ratios of 6 and 12, multivariate analysis indicates that temperature has the highest impact followed by salinity and Mg2+:Ca2+ ratio. The outcome of this study suggests that warm and saline environments are particularly favourable for microbially mediated formation of Mg-rich carbonates and provides new insight for interpreting ancient dolomite formations.

Virgicin, a novel lanthipeptide from Virgibacillus sp. strain AK90 exhibits inhibitory activity against Gram-positive bacteria.

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.

Bacillus and Virgibacillus strains isolated from three Mexican coasts antagonize Staphylococcus aureus and Vibrio parahaemolyticus.

This study identified marine microorganisms from Mexican coasts that had antimicrobial activity against Staphylococcus aureus and Vibrio parahaemolyticus, which are known worldwide to be food-poisoning agents. Representative specimens of algae, saline sediment, crustaceans and mollusks were collected. Of the 42 tested strains, 15 inhibited these pathogens. Bacillus and Virgibacillus strains were identified by 16S rRNA gene sequencing. The strains with the highest inhibitory activity against S. aureus were PCRS1-07 (B. aerius), BLCG-05 and GUO-01 (B. pumilus). The strains GUHC-04, BLCG-05, GUHC-03 (B. altitudinis) and BLBSe-05 (B. oryzicola) showed higher antimicrobial activity against V. parahaemolyticus. Biofilm production by all strains was moderate, but B. altitudinis produced a stronger biofilm. This is the first study to isolate B. aerius, B. oryzicola, B. safensis, B. boroniphilus, B. altitudinis and V. senegalensis from marine ecosystems in Mexico as well as the first study to report their inhibitory effects against both S. aureus and V. parahaemolyticus. Bioactivity of spent media from the antagonistic strains cultured as biofilm also demonstrated high antimicrobial activity. The active compounds of the antagonists are currently being studied and tested. Marine ecosystems have the highest bacterial diversity associated with invertebrates and seaweed; however, this bacterial diversity has not been well-studied on Mexican coasts.

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).

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.

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)).

[Screening of epoxy-degrading halophiles and their application in high-salt wastewater treatment].

In this study, two halophilic bacteria were isolated from activated sludge in the epoxy wastewater treatment system. The strains were identified, and the growth and degradation characteristics were investigated. Strain J1 and J2 was identified respectively by morphological observation and 16S rDNA sequence alignment analysis. It was found that both strains belong to the Bacillus genus (Bacillus sp.) and branch Bacillus (Virgibacillus sp.). The optimized growth condition of strain J1 and J2 in the high salt CM culture medium was as follows: solution temperature 30 degrees C, pH 7.0 and 5-50 g x L(-1) of NaCl. Furthermore, the best degradation condition of the organic epoxy wastewater was: temperature 30 degrees C, pH 7.0 and NaCl concentration 30 g x L(-1). When the volume ratio of bacterial suspension mixture of J1 and J2 was 2:1 and the inoculum size of the composite strains was 10%, the highest COD removal efficiency was achieved in the epoxy wastewater treatment.

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)).

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.

Virgibacillus albus sp. nov., a novel moderately halophilic bacterium isolated from Lop Nur salt lake in Xinjiang province, China.

A Gram-positive, moderately halophilic, strictly aerobic bacterium, designated YIM 93624(T), was isolated from a salt lake in Xinjiang province of China and subjected to a polyphasic taxonomic study. Strain YIM 93624(T) grew at 15-45 °C (optimum 25-30 °C), 1-17% (w/v) NaCl (optimum 5-10 %, w/v) and pH 4.0-9.0 (optimum pH 7.0). The predominant menaquinone was found to be MK-7. The major fatty acids were anteiso-C(15:0) and C(16:0). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, a glycolipid and two unidentified phospholipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The G+C content of the genomic DNA was 37.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 93624(T) was a member of the genus Virgibacillus and exhibited the highest similarity of 97.0 % to Virgibacillus koreensis KCTC 3823(T). However, the level of DNA-DNA relatedness between strain YIM 93624(T) and V. koreensis KCTC 3823(T) was 32.5 %. On the basis of phylogenetic, physiological and chemotaxonomic analysis data, the isolate is concluded to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus albus sp. nov., is proposed, with type strain of YIM 93624(T) (=DSM 23711(T) = JCM 17364(T)).

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation.

UNLABELLED: Cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris-maleate (pH 7) at 37 °C for 2 h. Major cell-associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32-kDa proteinase showed strong amidolytic activity toward Suc-Ala-Ala-Pro-Phe-AMC. Activity of all cell-associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65-kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal-dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell-associated proteinases from a moderate halophile, Virgibacillus sp. PRACTICAL APPLICATION: The cell-associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell-associated proteinases are key factors contributing to protein-degrading ability at high salt environment of Virgibacillus sp. SK 33.

Bioflocculant production by Virgibacillus sp. Rob isolated from the bottom sediment of Algoa Bay in the Eastern Cape, South Africa.

A bioflocculant-producing marine bacterium previously isolated from marine sediment of Algoa Bay was screened for flocculant production. Comparative analysis of 16S rDNA sequence identified the isolate to have 99% similarity to Virgibacillus sp. XQ-1 and it was deposited in the GenBank as Virgibacillus sp. Rob with accession number HQ537127. The bacterium produced biflocculants optimally in glucose (70.4%) and peptone (70.4%) as sole sources of carbon and nitrogen, alkaline pH (12) (74%); and the presence of Fe2+ (74%). Chemical analysis of the bioflocculant revealed it to be a polysaccharide.

Virgibacillus alimentarius sp. nov., isolated from a traditional Korean food.

A novel, Gram-positive, rod-shaped, motile, endospore-forming, halophilic bacterial strain, J18(T), was isolated from a traditional salt-fermented seafood made of gizzard shad in Korea. Colonies were convex, cream-coloured and 1.0-2.0 mm in diameter after incubation for 3 days on marine agar. Growth occurred at pH 7.0-11.0 (optimum, pH 10.0), at 4-40 °C (optimum, 37 °C) and in the presence of 0-30% NaCl (optimum, 9-10%). On the basis of 16S rRNA gene sequence analysis, strain J18(T) was related most closely to Virgibacillus byunsanensis ISL-24(T) (96.3% similarity), Virgibacillus carmonensis LMG 20964(T) (96.2%), Virgibacillus halodenitrificans DSM 10037(T) (96.0%), Virgibacillus arcticus Hal 1(T) (95.5%) and Virgibacillus necropolis LMG 19488(T) (95.5%). The major fatty acids were anteiso-C(15:0) and anteiso-C(17:0). The DNA G+C content of strain J18(T) was 37.0 mol%. The cell-wall peptidoglycan was of the meso-diaminopimelic acid type. The major quinone was menaquinone 7 (MK-7). Based on phenotypic, chemotaxonomic and phylogenetic data, strain J18(T) is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus alimentarius sp. nov. is proposed. The type strain is J18(T) (=KACC 14624(T) =JCM 16994(T)).

Virgibacillus soli sp. nov., isolated from mountain soil.

A Gram-positive bacterium (strain CC-YMP-6(T)) was isolated from soil samples collected from Yang-Ming Mountain, Taiwan. On the basis of 16S rRNA gene sequence analysis, strain CC-YMP-6(T) clearly belonged to the genus Virgibacillus and was most closely related to the type strains of Virgibacillus halophilus (96.2 % similarity) and Virgibacillus kekensis (96.3 %). The predominant isoprenoid quinone was menaquinone MK-7 and the polar lipid profile was composed of the major components diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified phospholipid plus moderate amounts of two unidentified aminophospholipids and a phospholipid. The polyamine pattern comprised spermidine as the single major component with spermine and putrescine present in minor amounts. The major fatty acids of strain CC-YMP-6(T) were iso-C(15 : 0) and anteiso-C(15 : 0). The results of physiological and biochemical tests allowed the clear phenotypic differentiation of strain CC-YMP-6(T) from all recognized species of the genus Virgibacillus. Strain CC-YMP-6(T) is therefore considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus soli sp. nov. is proposed. The type strain is CC-YMP-6(T) (=DSM 22952(T)=CCM 7714(T)).