NprR, a moonlighting quorum sensor shifting from a phosphatase activity to a transcriptional activator

Regulation of biological functions requires factors (proteins, peptides or chemicals) able to sense and translate environmental conditions or any circumstances in order to modulate the transcription of a gene, the stability of a transcript or the activity of a protein. Quorum sensing is a regulation mechanism connecting cell density to the physiological state of a single cell. In bacteria, quorum sensing coordinates virulence, cell fate and commitment to sporulation and other adaptation properties. The critical role of such regulatory systems was demonstrated in pathogenicity and adaptation of bacteria from the Bacillus cereus group (i.e. B. cereus and Bacillus thuringiensis). Furthermore, using insects as a model of infection, it was shown that sequential activation of several quorum sensing systems allowed bacteria to switch from a virulence state to a necrotrophic lifestyle, allowing their survival in the host cadaver, and ultimately to the commitment into sporulation. The chronological development of these physiological states is directed by quorum sensors forming the RNPP family. Among them, NprR combines two distinct functions connecting sporulation to necrotrophism in B. thuringiensis. In the absence of its cognate signaling peptide (NprX), NprR negatively controls sporulation by acting as a phosphatase. In the presence of NprX, it acts as a transcription factor regulating a set of genes involved in the survival of the bacteria in the insect cadaver

Structural analysis of the bacterial HPr kinase/phosphorylase V267F mutant gives insights into the allosteric regulation mechanism of this bifunctional enzyme.

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phosphocarrier protein HPr, which regulates the utilization of carbon sources in gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of P-Ser46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of P-loop proteins. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the X-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6 A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide-binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P

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

The phosphocarrier protein HPr of Neisseria meningitidis interacts with the transcription regulator CrgA and its deletion affects capsule production, cell adhesion, and virulence

The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars, but also carries out numerous regulatory functions. The β-proteobacterium Neisseria meningitidis possesses an incomplete PTS unable to transport carbon sources because it lacks a membrane component. Nevertheless, the residual phosphorylation cascade is functional and the meningococcal PTS was therefore expected to carry out regulatory roles. Interestingly, a ΔptsH mutant (lacks the PTS protein HPr) exhibited reduced virulence in mice and after intraperitoneal challenge it was rapidly cleared from the bloodstream of BALB/c mice. The rapid clearance correlates with lower capsular polysaccharide production by the ΔptsH mutant, which is probably also responsible for its increased adhesion to Hec-1-B epithelial cells. In addition, compared to the wild-type strain more apoptotic cells were detected when Hec-1-B cells were infected with the ΔptsH strain. Coimmunoprecipitation revealed an interaction of HPr and P-Ser-HPr with the LysR type transcription regulator CrgA, which among others controls its own expression. Moreover, ptsH deletion caused increased expression of a ΦcrgA-lacZ fusion. Finally, the presence of HPr or phospho-HPr's during electrophoretic mobility shift assays enhanced the affinity of CrgA for its target sites preceding crgA and pilE, but HPr did not promote CrgA binding to the sia and pilC1 promoter regions