ApoFnr binds as a monomer to promoters regulating expression of enterotoxin genes of Bacillus cereus.

International audienceBacillus cereus Fnr is a member of the Crp/Fnr (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of Hbl and Nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occured as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulphide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed

The Fnr protein and the two component system ResDE, two major regulators of enterotoxin gene expression in Bacillus cereus

Bacillus cereus is an opportunistic pathogen responsible of two types of food-borne diseases, classified as emetic and diarrhoeal syndromes. The diarrhoeal syndrome results from the production of enterotoxins (Hbl, Nhe and CytK) in the host small intestine, which constitutes a high reducing anoxic environment. The ability of B. cereus to produce enterotoxins and grow well in such environment is controlled by two global regulators that may function independently of the pleiotropic virulence regulator PlcR (Phospholipase C Regulator). These two regulators are the two-component system ResDE and the redox regulator Fnr (Fumarate Nitrate Reductase). The aim of this study was to establish the role of Fnr and ResDE in the virulence regulatory pathway of B. cereus. The results showed that transcriptional regulation of hbl and nhe was directly and indirectly controlled by Fnr and ResD. In aerobiosis, Fnr interaction (apo form) with the promoter regions of the enterotoxin structural genes (pnhe and phbl) and the enterotoxin regulator genes (presDE, pfnr and pplcR) depends on its oligomeric state. DNA binding affinity of ResD for pnhe, phbl, presDE, pfnr and pplcR depends on the promoter sequences and affinity for presDE and pfnr depends on its phosphorylation state. ResD and Fnr were found to physically interact and simultaneously bind their target DNAs. We proposed a model for regulation of enterotoxin genes expression in which ResD and Fnr could act synergically. Finally, yeast two-hybride experiments showed that PlcR could physically interact in vivo with Fnr and ResD. Enterotoxin genes expression of B. cereus could thus be controlled through a mechanism including a ternary complexBacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculair

La protéine Fnr et le système à deux composants ResDE, des régulateurs majeurs de la synthèse des entérotoxines de Bacillus cereus

Bacillus cereus is an opportunistic pathogen responsible of two types of food-borne diseases, classified as emetic and diarrhoeal syndromes. The diarrhoeal syndrome results from the production of enterotoxins (Hbl, Nhe and CytK) in the host small intestine, which constitutes a high reducing anoxic environment. The ability of B. cereus to produce enterotoxins and grow well in such environment is controlled by two global regulators that may function independently of the pleiotropic virulence regulator PlcR (Phospholipase C Regulator). These two regulators are the two-component system ResDE and the redox regulator Fnr (Fumarate Nitrate Reductase). The aim of this study was to establish the role of Fnr and ResDE in the virulence regulatory pathway of B. cereus. The results showed that transcriptional regulation of hbl and nhe was directly and indirectly controlled by Fnr and ResD. In aerobiosis, Fnr interaction (apo form) with the promoter regions of the enterotoxin structural genes (pnhe and phbl) and the enterotoxin regulator genes (presDE, pfnr and pplcR) depends on its oligomeric state. DNA binding affinity of ResD for pnhe, phbl, presDE, pfnr and pplcR depends on the promoter sequences and affinity for presDE and pfnr depends on its phosphorylation state. ResD and Fnr were found to physically interact and simultaneously bind their target DNAs. We proposed a model for regulation of enterotoxin genes expression in which ResD and Fnr could act synergically. Finally, yeast two-hybride experiments showed that PlcR could physically interact in vivo with Fnr and ResD. Enterotoxin genes expression of B. cereus could thus be controlled through a mechanism including a ternary complexBacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculair

La protéine Fnr et le système à deux composants ResDE, des régulateurs majeurs de la synthèse des entérotoxines de Bacillus cereus

Bacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculaireBacillus cereus is an opportunistic pathogen responsible of two types of food-borne diseases, classified as emetic and diarrhoeal syndromes. The diarrhoeal syndrome results from the production of enterotoxins (Hbl, Nhe and CytK) in the host small intestine, which constitutes a high reducing anoxic environment. The ability of B. cereus to produce enterotoxins and grow well in such environment is controlled by two global regulators that may function independently of the pleiotropic virulence regulator PlcR (Phospholipase C Regulator). These two regulators are the two-component system ResDE and the redox regulator Fnr (Fumarate Nitrate Reductase). The aim of this study was to establish the role of Fnr and ResDE in the virulence regulatory pathway of B. cereus. The results showed that transcriptional regulation of hbl and nhe was directly and indirectly controlled by Fnr and ResD. In aerobiosis, Fnr interaction (apo form) with the promoter regions of the enterotoxin structural genes (pnhe and phbl) and the enterotoxin regulator genes (presDE, pfnr and pplcR) depends on its oligomeric state. DNA binding affinity of ResD for pnhe, phbl, presDE, pfnr and pplcR depends on the promoter sequences and affinity for presDE and pfnr depends on its phosphorylation state. ResD and Fnr were found to physically interact and simultaneously bind their target DNAs. We proposed a model for regulation of enterotoxin genes expression in which ResD and Fnr could act synergically. Finally, yeast two-hybride experiments showed that PlcR could physically interact in vivo with Fnr and ResD. Enterotoxin genes expression of B. cereus could thus be controlled through a mechanism including a ternary complexAVIGNON-Bib. numérique (840079901) / SudocSudocFranceF

Desiccation: An environmental and food industry stress that bacteria commonly face

International audienceWater is essential for all living organisms, for animals as well as for plants and micro-organisms. For these latter, the presence of water or a humid environment with a high air relative humidity (RH) is necessary for their survival and growth. Thus, variations in the availability of water or in the air relative humidity constitute widespread environmental stresses which challenge microorganisms, and especially bacteria. Indeed, in their direct environment, bacteria are often faced with conditions that remove cellbound water through air-drying of the atmosphere. Bacterial cells are subject to daily or seasonal environmental variations, sometimes going through periods of severe desiccation. This is also the case in the food industry, where air dehumidification treatments are applied after the daily cleaning-disinfection procedures. In plants producing low-water activity products, it is also usual to significantly reduce or eliminate water usage. Periodic desiccation exposure affects bacteria viability and so they require strategies to persist. Negative effects of desiccation are wide ranging and include direct cellular damage but also changes in the biochemical and biophysical properties of cells for which planktonic cells are more exposed than cells in biofilm. Understanding the mechanisms of desiccation adaptation and tolerance has a biological and biotechnological interest. This review gives an overview of the factors influencing desiccation tolerance and the biological mechanisms involved in this stress response

ResDE-dependent regulation of enterotoxin gene expression in <em>Bacillus cereus</em>: evidences for multiple modes of binding for ResD and interaction with Fnr

International audienceIn the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe and phbl was not dependent on ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an anti-activator of Fnr while phosphorylated ResD acts as a co-activator of Fnr. Finally, our findings represent the first molecular evidence for the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulenc

<it>Bacillus cereus</it> Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR

<p>Abstract</p> <p>Background</p> <p>Bacillus <it>cereus</it> is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. <it>B. cereus</it> Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr.</p> <p>Results</p> <p>Fnr has been subjected to <it>in vitro</it> Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]²⁺ cluster per monomer. Atmospheric O₂ causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the <it>nhe</it> and <it>hbl</it> promoter regions, while holoFnr has a higher affinity for <it>fnr</it> promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex.</p> <p>Conclusions</p> <p>Overall, this work shows that incorporation of the [4Fe-4S]²⁺ cluster is not required for DNA binding of Fnr to promoter regions of <it>hbl</it> and <it>nhe</it> enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.</p

ApoFnr Binds as a Monomer to Promoters Regulating the Expression of Enterotoxin Genes of Bacillus cereus▿ †

Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed

Adaptive response of Listeria monocytogenes biofilms to a dehumidification stress

International audienc