The megaplasmid pCER270 of Bacillus cereus emetic strain affects the timing of the sporulation process, spore resistance properties, and germination.

The Bacillus cereus group includes closely related spore-forming Gram-positive bacteria. In this group, plasmids play a crucial role in species differentiation and are essential for pathogenesis and adaptation to ecological niches. The B. cereus emetic strains are characterized by the presence of the pCER270 megaplasmid, which encodes the non-ribosomal peptide synthetase for the production of cereulide, the emetic toxin. This plasmid carries several genes that may be involved in the sporulation process. Furthermore, a transcriptomic analysis has revealed that pCER270 influences the expression of chromosome genes, particularly under sporulation conditions. In this study, we investigated the role of pCER270 on spore properties in different species of the B. cereus group. We showed that pCER270 plays a role in spore wet heat resistance and germination, with varying degrees of impact depending on the genetic background. In addition, pCER270 ensures that sporulation occurs at the appropriate time by delaying the expression of sporulation genes. This regulation of sporulation timing is controlled by the pCER270-borne Rap-Phr system, which likely regulates the phosphorylation state of Spo0A. Acquisition of the pCER270 plasmid by new strains could give them an advantage in adapting to new environments and lead to the emergence of new pathogenic strains. IMPORTANCE: The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the Bacillus cereus group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic B. cereus strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the B. cereus group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.

CalY is a major virulence factor and a biofilm matrix protein.

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.

How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.

Correction: How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.

[This corrects the article DOI: 10.1371/journal.ppat.1005779.].

Correction: Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis.

[This corrects the article DOI: 10.1371/journal.ppat.1002629.].

Bacterial swimmers that infiltrate and take over the biofilm matrix.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

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.

Pathogenic potential of Bacillus cereus strains as revealed by phenotypic analysis.

The Bacillus cereus pathogenic spectrum ranges from strains used as probiotics to human-lethal strains. However, prediction of the pathogenic potential of a strain remains difficult. Here, we show that food poisoning and clinical strains can be differentiated from harmless strains on the basis of host colonization phenotypes.

Plasmid - Chromosome interplay in natural and non-natural hosts: global transcription study of three Bacillus cereus group strains carrying pCER270 plasmid.

The Bacillus cereus group comprises genetically related Gram-positive spore-forming bacteria that colonize a wide range of ecological niches and hosts. Despite their high degree of genome conservation, extrachromosomal genetic material diverges between these species. The discriminating properties of the B. cereus group strains are mainly due to plasmid-borne toxins, reflecting the importance of horizontal gene transfers in bacterial evolution and species definition. To investigate how a newly acquired megaplasmid can impact the transcriptome of its host, we transferred the pCER270 from the emetic B. cereus strains to phylogenetically distant B. cereus group strains. RNA-sequencing experiments allowed us to determine the transcriptional influence of the plasmid on host gene expression and the impact of the host genomic background on the pCER270 gene expression. Our results show a transcriptional cross-regulation between the megaplasmid and the host genome. pCER270 impacted carbohydrate metabolism and sporulation genes expression, with a higher effect in the natural host of the plasmid, suggesting a role of the plasmid in the adaptation of the carrying strain to its environment. In addition, the host genomes also modulated the expression of pCER270 genes. Altogether, these results provide an example of the involvement of megaplasmids in the emergence of new pathogenic strains.

Environmental and biofilm-dependent changes in a Bacillus cereus secondary cell wall polysaccharide.

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(ß1-3)]GlcNAc(ß1-6)[Glc(ß1-3)][ManNAc(α1-4)]GalNAc(α1-4)ManNAc(ß1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(ß1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.

A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group.

In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signalling peptides and regulators of the RNPP family. We investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. We show that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. The transcription start site of the nprA gene has been identified and the minimal region necessary for full activation has been characterized by promoter mutagenesis. We demonstrate that the NprX peptide is secreted, processed and then reimported within the bacterial cell. Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. Alignment of available NprR sequences from different species of the B. cereus group defines seven NprR clusters associated with seven NprX heptapeptide classes. This cell-cell communication system was found to be strain-specific with a possible cross-talk between some pherotypes. The phylogenic relationship between NprR and NprX suggests a coevolution of the regulatory protein and its signalling peptide.

Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

IlsA, a unique surface protein of Bacillus cereus required for iron acquisition from heme, hemoglobin and ferritin.

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts.

Massive Integration of Planktonic Cells within a Developing Biofilm.

During biofilm growth, the coexistence of planktonic and sessile cells can lead to dynamic exchanges between the two populations. We have monitored the fate of these populations in glass tube assays, where the Bacillus thuringiensis 407 strain produces a floating pellicle. Time-lapse spectrophotometric measurement methods revealed that the planktonic population grew until the pellicle started to be produced. Thereafter, the planktonic population decreased rapidly down to a value close to zero while the biofilm was in continuous growth, showing no dispersal until 120 h of culture. We found that this decrease was induced by the presence of the pellicle, but did not occur when oxygen availability was limited, suggesting that it was independent of cell death or cell sedimentation and that the entire planktonic population has integrated the biofilm. To follow the distribution of recruited planktonic cells within the pellicle, we tagged planktonic cells with GFP and sessile cells with mCherry. Fluorescence binocular microscopy observations revealed that planktonic cells, injected through a 24-h-aged pellicle, were found only in specific areas of the biofilm, where the density of sessile cells was low, showing that spatial heterogeneity can occur between recruited cells and sessile cells in a monospecies biofilm.

CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation, and virulence.

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM.

Long inverted repeats around the chromosome replication terminus in the model strain Bacillus thuringiensis serovar israelensis BGSC 4Q7.

Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain, B. thuringiensis serovar israelensis BGSC 4Q7 (4Q7), has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element, pBtic235, which has been shown to be an inducible prophage, although three putative chromosomal prophages have been lost. Moreover, a 492 kb region, potentially including the standard replication terminus, has also been deleted in the 4Q7 strain, indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain, and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area, close to the 492 kb deletion, was found to be duplicated. This duplication presumably restored the equal sizes of the replichores, and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome, and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins, potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235, but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains, suggesting a duplication or over-replication in 4Q7. Thus, the 4Q7 strain is not a pure plasmid-less offshoot, but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence, genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model, but they also raise interesting fundamental questions about the functioning of the Bacillus genome.

Biofilm formation and cell surface properties among pathogenic and nonpathogenic strains of the Bacillus cereus group.

Biofilm formation by 102 Bacillus cereus and B. thuringiensis strains was determined. Strains isolated from soil or involved in digestive tract infections were efficient biofilm formers, whereas strains isolated from other diseases were poor biofilm formers. Cell surface hydrophobicity, the presence of an S layer, and adhesion to epithelial cells were also examined.

Bacillus thuringiensis Spores and Vegetative Bacteria: Infection Capacity and Role of the Virulence Regulon PlcR Following Intrahaemocoel Injection of Galleria mellonella.

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 °C using spores or vegetative cell inocula, and the 50% lethal dose (LD50) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD50 is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion.

The YvfTU two-component system is involved in plcR expression in Bacillus cereus.

BACKGROUND: Most extracellular virulence factors produced by Bacillus cereus are regulated by the pleiotropic transcriptional activator PlcR. Among strains belonging to the B. cereus group, the plcR gene is always located in the vicinity of genes encoding the YvfTU two-component system. The putative role of YvfTU in the expression of the PlcR regulon was therefore investigated. RESULTS: Expression of the plcR gene was monitored using a transcriptional fusion with a lacZ reporter gene in a yvfTU mutant and in its B. cereus ATCC 14579 parental strain. Two hours after the onset of the stationary phase, a stage at which the PlcR regulon is highly expressed, the plcR expression in the yvfTU mutant was only 50% of that of its parental strain. In addition to the reduced plcR expression in the yvfTU mutant, a few members of the PlcR regulon showed a differential expression, as revealed by transcriptomic and proteomic analyses. The virulence of the yvfTU mutant in a Galleria mellonella insect model was slightly lower than that of the parental strain. CONCLUSION: The YvfTU two-component system is not required for the expression of most of the virulence factors belonging to the PlcR regulon. However, YvfTU is involved in expression of plcR, a major regulator of virulence in B. cereus.

Two distinct pathways lead Bacillus thuringiensis to commit to sporulation in biofilm.

The spore-forming bacterium Bacillus thuringiensis is an efficient biofilm producer, responsible for persistent contamination of industrial food processing systems. B. thuringiensis biofilms are highly heterogeneous bacterial structures in which three distinct cell types controlled by quorum sensing regulators were identified: PlcR-controlled virulent cells, NprR-dependent necrotrophic cells and cells committed to sporulation, a differentiation process controlled by Rap phosphatases and Spo0A-P. Interestingly, a cell lineage study revealed that, in LB medium or in insect larvae, only necrotrophic cells became spores. Here we analyzed cellular differentiation undertaken by cells growing in biofilm in a medium optimized for sporulation. No virulent cells were identified; surprisingly, two distinct routes could lead to differentiation as a spore in this growth condition: the NprR-dependent route, followed by the majority of cells, and the newly identified NprR-independent route, which is followed by 20% of sporulating cells.