The PlcR Virulence Regulon of Bacillus cereus

PlcR is a Bacillus cereus transcriptional regulator, which activates gene expression by binding to a nucleotidic sequence called the ‘PlcR box’. To build a list of all genes included in the PlcR regulon, a consensus sequence was identified by directed mutagenesis. The reference strain ATCC14579 sequenced genome was searched for occurrences of this consensus sequence to produce a virtual regulon. PlcR control of these genes was confirmed by comparing gene expression in the reference strain and its isogenic Δ-plcR strain using DNA microarrays, lacZ fusions and proteomics methods. The resulting list included 45 genes controlled by 28 PlcR boxes. Forty of the PlcR controlled proteins were exported, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). The functions of these proteins were related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (two-component sensors, chemotaxis proteins, GGDEF family regulators). Four genes coded for cytoplasmic regulators. The PlcR regulon appears to integrate a large range of environmental signals, including food deprivation and self cell-density, and regulate the transcription of genes designed to overcome obstacles that hinder B. cereus growth within the host: food supply, host barriers, host immune defenses, and competition with other bacterial species. PlcR appears to be a key component in the efficient adaptation of B. cereus to its host environment

Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides

The transcriptional regulator PlcR and its cognate cell–cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR–PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli

Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading

Relationship of plcR-Regulated Factors to BacillusEndophthalmitis Virulence

The explosive, destructive course of Bacillus endophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator plcR to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic plcR-deficient mutants of Bacillus cereus and Bacillus thuringiensis were constructed by insertional inactivation of plcR by the kanamycin resistance cassette, aphA3. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B. cereus or B. thuringiensis or a plcR-deficient mutant. The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with plcR mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of plcR-regulated factors significantly attenuated the severity of Bacillus endophthalmitis. The results therefore suggest that plcR may represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillus endophthalmitis

Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis

Bacteria of the Bacillus genus are able to synthesize several families of lipopeptides. These small molecules are the product of non-ribosomal peptide synthetases. In 2000, it was found that Bacillus thuringiensis, an entomopathogenic bacterium of the Bacillus cereus group, produced a previously unknown lipopeptide: kurstakin. Genomic analyses reveal that the krs locus, encoding the kurstakin synthetases, is specific to the B. cereus group, but is unevenly distributed within this group. Previous work showed that krs transcription requires the necrotrophism quorum-sensor NprR. Here, we demonstrated that the genes of the krs locus form an operon and we defined its transcription start site. Following krs transcription at the population and single-cell levels in multiple culture conditions, we depicted a condition-dependent transcription pattern, indicating that production of kurstakin is subject to environmental regulation. Consistent with this idea, we found krs transcription to be regulated by another master regulator, Spo0A, suggesting that krs expression is fine-tuned by integrating multiple signals. We also reported an unknown DNA palindrome in the krs promoter region that modulates krs expression. Due to their surfactant properties, lipopeptides could play several physiological roles. We showed that the krs locus was required for proper biofilm structuration