ResDE-dependent regulation of enterotoxin gene expression in Bacillus cereus: evidence for multiple modes of binding for ResD and interaction with Fnr.
In the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic 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 the 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 antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulence.
Esbelin
Julia
J
UMR408, Sécurité et Qualité des Produits d'Origine Végétale INRA, Université d'Avignon et des Pays de Vaucluse, Avignon, France.
Armengaud
Jean
J
Zigha
Assia
A
Duport
Catherine
C
D001409:Bacillus cereus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D026481:Blotting, Far-Western; D015153:Blotting, Western; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization
D001426:Bacterial Proteins