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 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.

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)).

Ectoine and hydroxyectoine as protectants against osmotic and cold stress: uptake through the SigB-controlled betaine-choline- carnitine transporter-type carrier EctT from Virgibacillus pantothenticus.

Virgibacillus pantothenticus has been shown to synthesize the compatible solute ectoine in response to high salinity or low growth temperature. We found that exogenously provided ectoine and hydroxyectoine also serve as protectants against these challenges. Transport studies with [(14)C]ectoine revealed that both types of stress induced a high-affinity ectoine uptake activity in V. pantothenticus. By using an Escherichia coli mutant defective in osmoprotectant uptake systems, a functional complementation approach for osmostress resistance in the presence of ectoine was employed to retrieve a gene encoding an ectoine transporter from V. pantothenticus. The cloned gene (ectT) encodes a protein (EctT) that is a member of the BCCT (betaine-choline-carnitine-transporter) family of carriers. Osmoprotection assays demonstrated that the EctT carrier mediates the preferential import of ectoine and hydroxyectoine but also possesses minor uptake activities for the compatible solutes proline and glycine betaine. Northern blot analysis with RNA isolated from V. pantothenticus revealed that a rise in the external osmolality or a reduction in growth temperature strongly increased the transcription of the ectT gene. Primer extension analysis demonstrated that ectT was transcribed under these conditions from a SigB-type promoter. SigB is the master regulator of the general stress regulon of bacilli and provides protection to cells against various challenges, including high salinity and low temperature. Both the synthesis of ectoine and the EctT-mediated uptake of ectoine and hydroxyectoine are triggered by the same environmental cues, high salinity and cold stress, and thereby provide, in a concerted fashion, the protection of V. pantothenticus against these challenges.

Enhanced protease production in a polymethylmethacrylate conico-cylindrical flask by two biofilm-forming bacteria.

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment was employed for protease production by two biofilm-forming bacteria, an intertidal gamma-Proteobacterium (DGII) and a chicken meat isolate, Virgibacillus pantothenticus. The flask design allowed comparison of protease production during cultivation with a hydrophilic (glass) or hydrophobic (PMMA) surface. Compared to the Erlenmeyer flask, the CCF allowed protease production that was 30% and 35% higher and growth that was 20% and 345% higher for DGII and V. pantothenticus, respectively. Protease production increased by 202% and 22% and growth by 19,275% and 940% for DGII and V. pantothenticus, respectively, in the presence of a hydrophobic as compared to a hydrophilic surface. This investigation pioneers the application of a vessel beyond the traditional shake-flask for enhancing protease production by biofilm-formers.