We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the exgression of the virF gene present on the large virulence plasmid of Shigeila and enteroinvasive Escherichia coil in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the virG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of HNS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30°C. The expression of a virF-lacZ translational fusion in E.cofi wild type and in an hns-defective derivative grown at 30 ° or 370C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virFexpression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controis virFexpression by binding to the virFpromoter and by repressing its expression at low temperature and at low pH.
A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.
NICOLETTI, Mauro
1998-01-01
Abstract
We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the exgression of the virF gene present on the large virulence plasmid of Shigeila and enteroinvasive Escherichia coil in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the virG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of HNS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30°C. The expression of a virF-lacZ translational fusion in E.cofi wild type and in an hns-defective derivative grown at 30 ° or 370C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virFexpression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controis virFexpression by binding to the virFpromoter and by repressing its expression at low temperature and at low pH.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
