Fast and accurate identification by MALDI-TOF of the zoonotic serovar E of Vibrio vulnificus linked to eel culture.

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Analyses of competent and non-competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence.

Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.

Fluorescently Labeled DNA Interacts with Competence and Recombination Proteins and Is Integrated and Expressed Following Natural Transformation of Bacillus subtilis.

During competence, Bacillus subtilis is able to take up DNA from its environment through the process of transformation. We investigated the ability of B. subtilis to take up fluorescently labeled DNA and found that it is able to take up fluorescein-dUTP-, DyLight 550-dUTP-, and DyLight 650-dUTP-labeled DNA. Transformation with labeled DNA containing an antibiotic cassette resulted in uptake of the labeled DNA and also generated antibiotic-resistant colonies. DNA is primarily taken up at the pole, as it can be seen to colocalize with ComFC, which is a component of the competence machinery. The DNA is taken up rapidly and can be seen to localize with (the actively searching form of) RecA. Colocalization with a homologous locus on the chromosome increases over time. Using microfluidics, we observed replacement of the homologous locus and subsequent expression of the integrated labeled and unlabeled DNA, although whether the integrated DNA contains labeled nucleotides needs to be determined conclusively. Integrated DNA in cells with a doubling time of 60 min is expressed on average 6 h 45 min after the addition of DNA and 4 h 45 min after the addition of fresh medium. We also found that the expression of the incoming DNA under these conditions can occur before cell division and, thus, before complete exit from the competence state. Because the competence machinery is conserved among naturally competent bacteria, this method of labeling is also suitable for studying transformation of other naturally competent bacteria.IMPORTANCE We used DNA that was covalently labeled with fluorescent nucleotides to investigate the transformation process of Bacillus subtilis at the molecular level. We show that the labeled DNA colocalizes with components of the competence machinery, the chromosome, and the recombination protein RecA. Using time-lapse microscopy and microfluidics, we visualized, in real-time, the uptake of fluorescently labeled DNA. We found that under these conditions, cell division is not required for the expression of integrated DNA. Because the competence machinery is conserved in naturally competent bacteria, this method can also be used to investigate the transformation process in many other bacterial species.

Bacillus subtilis biosensor engineered to assess meat spoilage.

Here, we developed a cell-based biosensor that can assess meat freshness using the Gram-positive model bacterium Bacillus subtilis as a chassis. Using transcriptome analysis, we identified promoters that are specifically activated by volatiles released from spoiled meat. The most strongly activated promoter was PsboA, which drives expression of the genes required for the bacteriocin subtilosin. Next, we created a novel BioBrick compatible integration plasmid for B. subtilis and cloned PsboA as a BioBrick in front of the gene encoding the chromoprotein amilGFP inside this vector. We show that the newly identified promoter could efficiently drive fluorescent protein production in B. subtilis in response to spoiled meat and thus can be used as a biosensor to detect meat spoilage.

The YmdB phosphodiesterase is a global regulator of late adaptive responses in Bacillus subtilis.

Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2',3'- and 3',5'-cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the σ(D)-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.

Spo0A regulates chromosome copy number during sporulation by directly binding to the origin of replication in Bacillus subtilis.

When starved, Bacillus subtilis cells can enter the developmental programme of endospore formation by activation of the master transcriptional regulator Spo0A. Correct chromosome copy number is crucial for the production of mature and fully resistant spores. The production and maintenance of one chromosome for the mother cell and one copy for the forespore requires accurate co-ordination between DNA replication and initiation of sporulation. Here, we show that Spo0A regulates chromosome copy number by directly binding to a number of Spo0A binding sites that are present near the origin of replication (oriC). We demonstrate that cells lacking three specific Spo0A binding sites at oriC display increased chromosome copy numbers when sporulation is induced. Our data support the hypothesis that Spo0A directly controls DNA replication during sporulation by binding to oriC.

Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily.

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.