Characterization of anthranilic acid produced by Virgibacillus salarius MML1918 and its bio-imaging application.

Anthranilic acid (AA) holds significant importance in the chemical industry. It serves as a crucial building block for the amino acid tryptophan by manipulating the tryptophan biosynthesis pathway, it is possible to increase the production of anthranilic acid. In this study, we utilized metabolic engineering approaches to produce anthranilic acid from the halophilic bacterium Virgibacillus salarius MML1918. The halophilic bacteria were grown in an optimized production medium, and mass production of secondary metabolites was made in ATCC medium 1097 Proteose peptone-for halophilic bacteria and subjected to column chromatography followed by sub-column chromatography the single band for the purified compound was confirmed. Further, various spectral analyses were made for the partially purified compounds, and fluorescence microscopy for fungal cell observation was performed. The purified compound was confirmed by single crystal X-ray diffraction (XRD) analysis, and it was identified as 2-amino benzoic acid. The Fourier transform infrared Spectroscopy (FT-IR) spectrum and nuclear magnetic resonance (NMR) spectrum also confirm the structural characteristic of 2-amino benzoic acid. The UV-Vis absorption spectrum of AA shows the maximum absorption at 337.86 nm. The emission spectrum of 2-amino benzoic acid showed the maximum emission at 453 nm. The bio-imaging application of 2-amino benzoic acid was examined with fungal mycelium of Rhizoctonia solani. It was effectively bound and emitted the blue color at the concentration of 200 and 300 µg/mL. The halophilic bacterium (V. salarius), may have unique metabolic pathways and requirements compared to non-halophilic organisms, to produce AA effectively. This could have implications for industrial biotechnology, particularly in manufacturing environments where high salt concentrations are present and also it can be used as bio-imaging agent.

Antimicrobial potential of secondary metabolites and DNA gyrase B blocking molecules produced by a halophilic bacterium Virgibacillus salarius (MML1918).

AIM: The present study aims to determine the antimicrobial potential of Virgibacillus salairus (MML1918) against human pathogens and its in-vitro and in-silico properties. METHODS AND RESULTS: In this present study, totally 63 halophilic bacterial cultures were obtained and cultivated in nutrient broth medium containing 8% NaCl and the metabolites, were extracted using ethyl acetate and screened for their antimicrobial property by cell viability assay against 12 pathogenic bacteria and fungi, among 63 halophilic bacteria the Vir. salaries (MML1918) found to be the best producer for secondary metabolites production against clinical pathogens. The optimization of growth for important physiochemical parameters was characterized and applied for different production media and based on its highest activity as 17.5 ± .07 mm zone of inhibition (ZOI) for Bacillus cereus followed by 17.5 ± 00 mm ZOI for Staphylococcus aureus, the production medium ATCC1097 was chosen for mass production. The mass production of secondary metabolites from Vir. salaries MML1918 was carried out in a fermenter under controlled conditions and crude metabolites was extracted and condensed. The antimicrobial activity of crude metabolites showed B. cereus (19.3 ± 0.5 mm ZOI), Staph. aureus, and Candida albicans (18.3 ± 0.5 mm ZOI) as the highest ZOI in production media for halophilic bacteria ATCC1097. Further, the gas chromatography-mass spectrometry analysis showed 24 compounds present in crude metabolites. Among the 24 compounds, four molecules were found to be important based on molecule percentage in crude and structural similarity. The molecular docking studies show that the selected four molecules effectively bind with the active region DNA gyrase B. CONCLUSION: Virgibacillus salarius (MML1918) effectively showed antimicrobial activity against several pathogenic organisms and can be employed as a suitable candidate for producing novel antimicrobial agents.

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.

Histamine-degrading halophilic bacteria from traditional fish sauce: Characterization of Virgibacillus campisalis TT8.5 for histamine reduction.

Traditionally produced fish sauce can contain significant amounts of histamine. In some instances, the histamine concentration may be well above the limit recommended by the Codex Alimentarius Commission. The aim of this study was to discover new bacterial strains capable of growing under the stressful environmental conditions of fish sauce fermentation and metabolizing histamine. In this study, 28 bacterial strains were isolated from Vietnamese fish sauce products based on their ability to grow at high salt concentrations (23% NaCl) and tested for their ability to degrade histamine. Strain TT8.5 showed the highest histamine-degradation (45.1 ± 0.2% of initially 5 mM histamine within 7 days) and was identified as Virgibacillus campisalis TT8.5. Its histamine-degrading activity was shown to be localized intracellularly and the enzyme is a putative histamine dehydrogenase. The strain exhibited optimal growth and histamine-degrading activity at 37°C, pH 7%, and 5% NaCl in halophilic archaea (HA) histamine broth. It also showed pronounced histamine-degrading activity in HA histamine broth when cultivated at temperatures of up to 40 °C as well as in the presence of up to 23% NaCl. After treatment with immobilized cells, 17.6-26.9% of the initial histamine in various fish sauce products were reduced within 24 h of incubation, while no significant changes in other parameters of fish sauce quality were observed after this treatment. Our results indicate that V. campisalis TT8.5 is of potential interest to be applied in histamine degradation of traditional fish sauce.

Anaerobic phenanthrene biodegradation by a new salt-tolerant/halophilic and nitrate-reducing Virgibacillus halodenitrificans strain PheN4 and metabolic processes exploration.

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) under hypersaline environments has received increasing attention, whereas the study of anaerobic PAH biodegradation under hypersaline environments is still lacking. Here, we found a pure culture designated PheN4, which was affiliated with Virgibacillus halodenitrificans and could degrade phenanthrene with nitrate as the terminal electron acceptor and a wide range of salinities (from 0.3% to 20%) under anaerobic environments. The optimal salinity for biodegradation of phenanthrene by PheN4 was 5%, which could degrade 93.5% of 0.62 ± 0.04 mM phenanthrene within 10 days with the initial inoculum of 0.01 gVSS/L. Meanwhile, an increased microbial amount could efficiently promote the phenanthrene biodegradation rate. The metabolic processes of anaerobic phenanthrene biodegradation under hypersaline conditions by PheN4 were proposed based on intermediates and genome analyses. Phenanthrene was initially activated via methylation to form 2-methylphenanthrene. Next, fumarate addition and ß-oxidation or direct oxidation of the methyl group, ring reduction and ring cleavage were identified as the midstream and downstream steps. In addition, PheN4 could utilize benzene, naphthalene, and anthracene as carbon sources, but Benz[a]anthracene, pyrene, and Benzo[a]pyrene could not be biodegraded by PheN4. This study could provide some guidance for the bioremediation of PAH pollutants in anaerobic and hypersaline zones.

Virgibacillus dokdonensis VITP14 produces α-amylase and protease with a broader operational range but with differential thermodynamic stability.

Extracellular α-amylase and protease were coproduced from halo tolerant Virgibacillus dokdonensis VITP14 with banana peels (2% w/v) as substrate. The pH optima for α-amylase and protease were 6.5 and 7.0, respectively. The temperature optima of α-amylase and protease were 30 and 50 °C, respectively. Both the enzymes were active in the presence of various metal ions (1 mM of Ni2+ , Ca2+ , Ba2+ , Sr2+ , and Mg2+ ), detergents (Tween 20, Tween 80, Triton X-100), and other additives (2-mercaptoethanol and urea). Both the enzymes followed Michaelis-Menten type enzyme kinetics with Vmax of 121.40 and 4.17 µmol Min-1 mL-1 and Km of 0.59 and 0.28 mg mL-1 for amylase and protease, respectively. Amylase showed higher activation energy for inactivation (75.55 kJ mol-1 compared to 59.70 kJ mol-1 for protease) and higher thermal stability (reflected by longer half-life 53.23 Min compared to 0.11 Min for protease) at 60 °C. The coexistence of amylase and protease could be attributed to the difference in the optimum temperatures of activity and thermal stability of the two enzymes.

Aquibacillus kalidii sp. nov., an indole acetic acid-producing endophyte from a shoot of Kalidium cuspidatum, and reclassification of Virgibacillus campisalis Lee et al. 2012 as a later heterotypic synonym of Virgibacillus alimentarius Kim et al. 2011.

A Gram-stain-positive, strictly aerobic, motile, endospore-forming, milk-white, indole acetic acid-producing, rod-shaped bacterial strain, designated as HU2P27T, was isolated from a shoot of Kalidium cuspidatum collected in Tumd Right Banner, Inner Mongolia, PR China. Strain grew at 10-40 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0) and with 0-14.0 % NaCl (optimum, 5.0-8.0 %). The strain tested positive for oxidase, catalase and nitrate reductase. The phylogenetic trees based on the 16S rRNA gene sequence and the core genome both showed that strain HU2P27T clustered with Aquibacillus koreensis BH30097T, sharing 97.7 % and <97.0 % of 16S rRNA gene similarity with A. koreensis BH30097T and any other type strain. Strain HU2P27T contained MK-7 as the major respiratory quinone. Its major fatty acids were anteiso-C15 : 0 and iso-C15 : 0, and the major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and four unidentified phospholipids. The genomic DNA G+C content was 36.0 mol%. The average nucleotide identity, amino acid identity and digital DNA-DNA hybridization values of strain HU2P27T with A. koreensis BH30097T were 71.7, 69.2 and 19.4%, respectively. The phylogenetic, physiological and phenotypic results allowed the discrimination of strain HU2P27T from its phylogenetic relatives. The name Aquibacillus kalidii sp. nov. is therefore proposed. The type strain is strain HU2P27T (=CGMCC 1.18646T=KCTC 43248T). Based on the results of 16S rRNA gene and genome analyses, we propose the reclassification of Virgibacillus campisalis Lee et al. 2012 as a later heterotypic synonym of Virgibacillus alimentarius Kim et al. 2011.

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 and Characterization of Nematicidal Volatile Organic Compounds from Deep-Sea Virgibacillus dokdonensis MCCC 1A00493.

Root-knot nematode diseases cause severe yield and economic losses each year in global agricultural production. Virgibacillus dokdonensis MCCC 1A00493, a deep-sea bacterium, shows a significant nematicidal activity against Meloidogyne incognita in vitro. However, information about the active substances of V. dokdonensis MCCC 1A00493 is limited. In this study, volatile organic compounds (VOCs) from V. dokdonensis MCCC 1A00493 were isolated and analyzed through solid-phase microextraction and gas chromatography-mass spectrometry. Four VOCs, namely, acetaldehyde, dimethyl disulfide, ethylbenzene, and 2-butanone, were identified, and their nematicidal activities were evaluated. The four VOCs had a variety of active modes on M. incognita juveniles. Acetaldehyde had direct contact killing, fumigation, and attraction activities; dimethyl disulfide had direct contact killing and attraction activities; ethylbenzene had an attraction activity; and 2-butanone had a repellent activity. Only acetaldehyde had a fumigant activity to inhibit egg hatching. Combining this fumigant activity against eggs and juveniles could be an effective strategy to control the different developmental stages of M. incognita. The combination of direct contact and attraction activities could also establish trapping and killing strategies against root-knot nematodes. Considering all nematicidal modes or strategies, we could use V. dokdonensis MCCC 1A00493 to set up an integrated strategy to control root-knot nematodes.

Optimization of production and intrinsic viscosity of an exopolysaccharide from a high yielding Virgibacillus salarius BM02: Study of its potential antioxidant, emulsifying properties and application in the mixotrophic cultivation of Spirulina platensis.

Virgibacillus salarius BM02 was identified as a highly exopolysaccharide (EPS) producing bacterium. The EPS production and its physico-chemical properties (intrinsic viscosity and total sugars/protein (TS/P) ratio) were optimized using Box-Behnken experimental design. Maximum EPS production of 5.87 g L-1 with TS/P ratio of 12.56 and intrinsic viscosity of 0.13 dL g-1 was obtained at optimal conditions of sucrose (4.0% w/v), peptone (0.75% w/v) and incubation period of 4.69 day. The monosaccharide composition of EPS was mannose, arabinose and glucose at a molar ratio of 1.0:0.26:0.08. The EPS showed high water solubility (38.5%), water holding capacity (514.46%) and foaming capacity (55.55%). The EPS showed moderate antioxidant activity in vitro and good emulsion stabilizing properties against several hydrophobic compounds. The emulsifying activity was stable at different temperatures, pH and ionic strength. Additionally, the acid hydrolysate of the EPS was evaluated as a carbon source for the mixotrophic cultivation of industrially important Spirulina platensis. It induced an enhancement of not only biomass production of S. platensis, but also cellular contents (pigments, proteins and lipids) leading to higher nutritional value.

Isolation and characterization of the novel Virgibacillus-infecting bacteriophage Mimir87.

The novel bacterial virus Mimir87, infecting the salt-tolerant bacterium Virgibacillus halotolerans, was isolated from worker honey bees. Mimir87 has an elongated head and a long non-contractile tail consistent with members of the Siphoviridae phage family. The phage genome comprises 48,016 base pairs and encodes 68 predicted proteins, to 34 of which a function could be assigned from homology analysis. The phage encodes two metabolism-related transporter proteins previously not observed in bacteriophage genomes. Mimir87 displays some relatedness to several Bacillus and Paenibacillus viruses; however, the overall sequence dissimilarity suggests Mimir87 to be a representative of a new phage genus.

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.

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.

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.

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form and characterised. VbTA T16F showed high stability towards polar organic solvents and salt exposure, accepting mainly hydrophobic aromatic amine and carbonyl substrates. The 2.0 Å resolution crystal structure of VbTA T16F is here reported, and together with computational calculations, revealed that this mutation is crucial for correct dimerisation and thus correct folding, leading to soluble protein expression.

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.

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).

Developing a colorimetric assay for Fe(II)/2-oxoglutarate-dependent dioxygenase.

The Fe(II)/2-oxoglutarate-dependent dioxygenases (2-OGDs) catalyze the oxidation of substrates ranging from small molecules to large biomolecules with concomitant oxidation of co-substrate (2-oxoglutarate) into succinate. In the present study, we reported a coupled colorimetric assay that can be generally applied to measure the activities of all members of 2-OGDs family. Succinyl-CoA synthetase is employed as the coupling enzyme to transform succinate produced from 2-OGDs catalysis to form succinyl-CoA with concomitant hydrolysis of ATP to form ADP and orthophosphate. Orthophosphate can be quantitated by reacting it with molybdic acid forming a blue pigment. As a proof of concept, kinetic parameters of ectoine hydroxylase obtained using this method are compared to a traditional time- and labor-consuming HPLC based method. As 2-OGDs family enzymes are important drug targets due to their impressive versatility in catalyzing numerous oxidative reactions that are still very challenging using synthetic chemistry, colorimetric method detailed in the manuscript has the potential to enable the practice of high throughput drug screening for 2-OGDs.