Gram-positive bacteria use a wealth of extracellular signaling peptides, so-called autoinducers, to regulate gene expression according to population densities. These “quorum sensing” systems control vital processes such as virulence, sporulation, and gene transfer. Using x-ray analysis, we determined the structure of PlcR, the major virulence regulator of the Bacillus cereus group, and obtained mechanistic insights into the effects of autoinducer binding. Our structural and phylogenetic analysis further suggests that all of those quorum sensors that bind directly to their autoinducer peptide derive from a common ancestor and form a single family (the RNPP family, for Rap/NprR/PlcR/PrgX) with conserved features. As a consequence, fundamentally different processes in different bacterial genera appear regulated by essentially the same autoinducer recognition mechanism. Our results shed light on virulence control by PlcR and elucidate origin and evolution of multicellular behavior in bacteria.

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.

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 .

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. Bacillus thuringiensis (Bt) is a well known entomopathogenic bacterium successfully used as a biopesticide for fifty years. The insecticidal properties of Bt are mainly due to specific toxins forming a crystal inclusion associated with the spore. After ingestion by susceptible insect larvae, toxins could induce favorable conditions for spore germination. The bacteria multiply in the insect and coordinate their behavior using signaling molecules involved in quorum sensing. The activation of the quorum sensor PlcR leads to the production of virulence factors allowing the bacteria to kill the insect host. Here we show that, in the cadaver, Bt shifts from a virulent to a necrotrophic lifestyle during which a second quorum sensor (NprR) becomes functional. NprR activates genes encoding degradative enzymes (proteases, lipases and chitinases) and a lipopeptide (kurstakin) involved in swarming and biofilm formation. The kurstakin is also essential for the survival of Bt after insect death. This suggests that NprR allows the bacteria to survive and eventually to sporulate in the host cadaver, thus improving their ability to disseminate in the environment. Altogether these results show that the pathogenic and necrotrophic lifestyles of Bt are tightly controlled by two quorum-sensing systems acting sequentially during the infection process.

Bacterial cell-cell communication or quorum sensing (QS) is a biological process commonly described as allowing bacteria belonging to a same pherotype to coordinate gene expression to cell density. In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors regulated by secreted and re-imported signaling peptides. The Bacillus quorum sensors Rap, NprR, and PlcR were previously identified as the first members of a new protein family called RNPP. Except for the Rap proteins, these RNPP regulators are transcription factors that directly regulate gene expression. QS regulates important biological functions in bacteria of the Bacillus cereus group. PlcR was first characterized as the main regulator of virulence in B. thuringiensis and B. cereus. More recently, the PlcR-like regulator PlcRa was characterized for its role in cysteine metabolism and in resistance to oxidative stress. The NprR regulator controls the necrotrophic properties allowing the bacteria to survive in the infected host. The Rap proteins negatively affect sporulation via their interaction with a phosphorelay protein involved in the activation of Spo0A, the master regulator of this differentiation pathway. In this review we aim at providing a complete picture of the QS systems that are sequentially activated during the lifecycle of B. cereus and B. thuringiensis in an insect model of infection.

Summary: Bacillus cereus is an opportunistic bacterium frequently associated with food‐borne infections causing gastroenteritis. We developed an in vivo expression technology (IVET), with an insect host, for identification of the B. cereus genes specifically expressed during infection. This IVET‐based approach uses site‐specific recombinase TnpI to identify transient promoter activation. We constructed a genomic library of B. cereus ATCC14579 by cloning DNA fragments upstream from tnpI. The library was screened in vivo by oral infection of the insect Galleria mellonella. We selected 100 clones from dead larvae. Sequencing of the inserts followed by a second screen for specific in vivo induction led to the identification of 20 in vivo‐induced genes (ivi genes). They belonged to several different functional classes: regulation, metabolism, DNA repair and replication, cell division, transport, virulence and adaptation. A strongly induced gene, ivi29, was further analysed. It encodes an internalin‐like protein with four distinct domains: an N‐terminal signal peptide for export, a NEAT domain thought to be involved in iron transport, a leucine‐rich repeat domain that may interact with host cells, and a C‐terminal SLH domain presumably binding the protein to the peptidoglycan. As suggested by a Fur box in the promoter, transcriptional analysis showed ivi29 expression to be repressed by iron, suggesting that expression was induced in vivo due to iron deprivation in the host. This iron‐regulated, leucine‐rich surface protein was designated IlsA. Disruption of ilsA reduced the virulence of the bacteria to the insect larvae indicating its role in the overall pathogenesis of B. cereus.