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Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry., The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.

The transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR

The transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR7, and the oligopeptide permease OppABCDF, required for PapR7 import, form a quorum-sensing system that controls the expression of virulence factors in Bacillus cereus and Bacillus thuringiensis species. In B. cereus strain ATCC 14579, the transcriptional regulator PlcRa activates the expression of abrB2 gene, which encodes an AbrB-like transcriptional regulator involved in cysteine biosynthesis. PlcRa is a structural homolog of PlcR: in particular, its C-terminal TPR peptide-binding domain could be similarly arranged as in PlcR. The signaling peptide of PlcRa is not known. As PlcRa is a PlcR-like protein, the cognate PapR7 peptide (ADLPFEF) is a relevant candidate to act as a signaling peptide for PlcRa activation. Also, the putative PapRa7 peptide (CSIPYEY), encoded by the papRa gene adjacent to the plcRa gene, is a relevant candidate as addition of synthetic PapRa7 induces a dose-dependent increase of abrB2 expression. To address the issue of peptide selectivity of PlcRa, the role of PapR and PapRa peptides in PlcRa activity was investigated in B. thuringiensis 407 strain, by genetic and functional complementation analyses. A transcriptional fusion between the promoter of abrB2 and lacZ was used to monitor the PlcRa activity in various genetic backgrounds. We demonstrated that PapR was necessary and sufficient for PlcRa activity. We showed that synthetic PapRs from pherogroups II, III and IV and synthetic PapRa7 were able to trigger abrB2 expression, suggesting that PlcRa is less selective than PlcR. Lastly, the mode of binding of PlcRa was addressed using an in silico approach. Overall, we report a new role for PapR as a signaling peptide for PlcRa activity and show a functional link between PlcR and PlcRa regulons in B. 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.

International audience; The current experimental study investigates the influence of latex microsphere particles' size on the assessment of their hydrophilic/hydrophobic character, using the method known as “Microbial Adhesion to Hydrocarbons” (MATH). Since bacteria surfaces often change according to the environment in which they find themselves, most of the experiments here were carried out using the calibrated latex microspheres Polybeads® and Yellow-green Fluoresbrite® (Polyscience) microspheres with diameters between 0.2 μm and 4.5 μm. All the beads had a density of ˜1.05 g/cm3. The first set of experiments was performed to adapt the procedure for measurements of water contact angles to microsphere lawns. It was found that all the microspheres tested were hydrophobic, when using a water contact angle of around 110-118°. However, wide differences were observed using the MATH method. The smaller microspheres (0.2 μm, 0.5 μm +/- 0.75 μm) exhibited a poor affinity to hexadecane, even after long contact times, suggesting a hydrophilic character. In contrast, larger microspheres quickly adhered to hexadecane, which is consistent with the values obtained for the water contact angles observed. These results suggest that, at least where hydrophobic particles are concerned, the MATH method is not suitable for the assessment of the hydrophobic character of particles with diameters of less than 1.0 μm. We lastly investigated whether the data obtained for Bacillus spores could also be affected by spore size. The hydrophobicity of spores of eight Bacillus strains was analysed by both MATH and contact angle. Some discrepancies were observed between both methods but could not be related their size (length or width).

International audience; The infectious cycle of Bacillus thuringiensis in the insect host is regulated by quorum sensors of the RNPP family. The activity of these regulators is modulated by their cognate signaling peptides translocated into the bacterial cells by oligopeptide permeases (Opp systems). In B. thuringiensis, the quorum sensor NprR is a bi-functional regulator that connects sporulation to necrotrophism. The binding of the signaling peptide NprX switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. Here, we report that NprX is imported into the bacterial cells by two different oligopeptide permease systems. The first one is Opp, the system known to be involved in the import of the signaling peptide PapR in B. thuringiensis and Bacillus cereus. The second, designated as Npp (NprX peptide permease), was not previously described. We show that at least two substrate binding proteins (SBPs) are able to translocate NprX through OppBCDF. In contrast, we demonstrate that a unique SBP (NppA) can translocate NprX through NppDFBC. We identified the promoter of the npp operon, and we showed that transcription starts at the onset of stationary phase and is repressed by the nutritional regulator CodY during the exponential growth phase.

International audience; Cell differentiation within an isogenic population allows the specialisation of subpopulations and a division of labour. Bacillus thuringiensis is a spore‐forming bacterium that produces insecticidal crystal proteins (Cry proteins) in sporulating cells. We recently reported that strain B. thuringiensis LM1212 presents the unique ability to differentiate into two subpopulations during the stationary phase: spore‐formers and crystal‐producers. Here, we characterised the transcriptional regulator CpcR responsible for this differentiation and the expression of the cry genes. cpcR is located on a plasmid that also harbours cry genes. The alignment of LM1212 cry gene promoters revealed the presence of a conserved DNA sequence upstream from the −35 region. This presumed CpcR box was also found in the promoter of cpcR and we showed that cpcR transcription is positively autoregulated. Electrophoretic mobility shift assays suggested that CpcR directly controls the transcription of its target genes by binding to the CpcR box. We showed that CpcR was able to direct the production of a crystal consisting of a heterologous insecticidal Cry protein in non‐sporulating cells of a typical B. thuringiensis kurstaki strain. Moreover, the expression of cpcR induced a reduction in the sporulation of this B. thuringiensis strain, suggesting an interaction between CpcR and the sporulation regulatory networks.

5 CNRS, UPR 3082, Laboratoire d'Enzymologie et Biochimie Structurales, Centre de recherche de Gif, F-91198 Gif-sur-Yvette, France. Summary NprR is a quorum sensor of the RNPP family found in bacteria of the Bacillus cereus group. In association with its cognate peptide NprX, NprR controls the expression of genes essential for survival and sporu- lation of Bacillus thuringiensis during its necrotrophic development in insects. Here, we report that the nprR-nprX genes are not autoregulated and are co-transcribed from a s A -dependent promoter (PA) located upstream fromnprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY represses nprR- nprX transcription during the exponential growth phase and the quorum sensor PlcR activates nprR- nprX transcription at the onset of stationary phase. We show that nprX is also transcribed independently of nprR from two promoters, PH and PE, dependent on the sporulation-specific sigma factors, s H and s E respec- tively. Both promoters ensure nprX transcription during late stationary phase while transcription from PA has decreased. These results show that the activity of the NprR-NprX quorum sensing system is tightly co-ordinated to the physiological stage throughout the developmental process of the Bacillus.

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.

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.

A major challenge in bacterial developmental biology has been to understand the mechanisms underlying cell fate decisions. Some differentiated cell types display cooperative behaviour. Cooperation is one of the greatest mysteries of evolutionary biology and microbes have been considered as an excellent system for experimentally testing evolution theories. Bacillus thuringiensis (Bt) is a spore-forming bacterium, which is genetically closely related to B. anthracis, the agent of anthrax, and to B. cereus, an opportunistic human pathogen. The defining feature that distinguishes Bt from its relatives is its ability to produce crystal inclusions in the sporulating cells. These toxins are solubilized after ingestion and are cooperative public goods in insect hosts. In this study, we describe a Bt strain LM1212 that presents the unique ability to terminally differentiate into crystal producers and spore formers. Transcriptional analysis based on lacZ and gfp reporter genes suggested that this phenotype is the consequence of a new type of cell differentiation associated with a novel regulation mode of cry gene expression. The differentiating crystal-producer phenotype has higher spore productivity than a typical Bt strain and is better able to compete with Cry toxin null ‘cheaters'. Potentially, this division of labour provides additional fitness benefits in terms of spore viability or durability of Cry toxin.

NprR is a quorum sensor of the RNPP family found in bacteria of the Bacillus cereus group. In association with its cognate peptide NprX, NprR controls the expression of genes essential for survival and sporulation of Bacillus thuringiensis during its necrotrophic development in insects. Here, we report that the nprR-nprX genes are not autoregulated and are co-transcribed from a σ(A) -dependent promoter (PA ) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY represses nprR-nprX transcription during the exponential growth phase and the quorum sensor PlcR activates nprR-nprX transcription at the onset of stationary phase. We show that nprX is also transcribed independently of nprR from two promoters, PH and PE , dependent on the sporulation-specific sigma factors, σ(H) and σ(E) respectively. Both promoters ensure nprX transcription during late stationary phase while transcription from PA has decreased. These results show that the activity of the NprR-NprX quorum sensing system is tightly co-ordinated to the physiological stage throughout the developmental process of the Bacillus.

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., Author Summary 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.

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.