ABSTRACT
The mechanism of pathogenicity in Shigella and enteroinvasive Escherichia coli (EIEC) requires the co-ordinated expression of several genes located on both the virulence plasmid and the chromosome. We found that cells lacking a functional FIS protein (factor for inversion stimulation) are partially impaired in expressing the virulence genes and that full expression is totally restored when Shigella wild-type fis gene is offered in trans. We also identified virF, among the virulence genes, as a target of FIS-mediated activation and showed that FIS binds to four specific sites in the promoter region of virF. Previous studies have demonstrated that the expression of VirF, the first positive activator of a multistep regulatory cascade, is subject to temperature-dependent regulation by H-NS, one of the main nucleoid-associated proteins. We now demonstrate that two of the four FIS sites overlap one of the two H-NS sites responsible for thermoregulation (H-NS site I). FIS was found to exercise a direct positive transcriptional control at permissive temperature (37 degrees C), when H-NS fails to repress virF, as well as an indirect effect by partially counteracting H-NS inhibition at the transition temperature (32 degrees C). Our data indicate that FIS may be relevant for the rapid increase in virF expression after penetration of bacteria into the host.
Subject(s)
Bacterial Proteins/genetics , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Escherichia coli/pathogenicity , Gene Expression Regulation, Bacterial , Shigella/genetics , Transcription Factors/metabolism , Virulence Factors , Base Sequence , Binding Sites , Carrier Proteins/genetics , DNA Footprinting , DNA-Binding Proteins/genetics , Factor For Inversion Stimulation Protein , Integration Host Factors , Molecular Sequence Data , Plasmids/genetics , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/metabolism , Response Elements/genetics , Shigella/physiology , Temperature , Transcription Factors/genetics , Transcription, Genetic , Virulence/geneticsABSTRACT
Two Csp proteins (CspA and CspD) were fused to the green fluorescent protein GFP and expressed from their natural promoters or from an inducible promoter. Fluorescence microscopy and computerized image analysis indicate that in Escherichia coli growing at 37 degrees C CspD localizes in the nucleoid like the control H-NS while CspA occupies a polar position away from the nucleoid. Following cold shock CspA maintains its location, while CspD is not sufficiently expressed to permit its localization. The different localization of CspA and CspD indicates that these proteins play different roles in the cell in spite of their extensive structural similarity.
Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/metabolism , Transcription Factors/biosynthesis , Bacterial Proteins/analysis , Blotting, Western , Cell Nucleolus , Cold Temperature , Green Fluorescent Proteins , Luminescent Proteins , Microscopy, Fluorescence , Transcription Factors/analysisABSTRACT
The promoter of hns, the structural gene for the abundant nucleoid-associated protein H-NS of Escherichia coli, contains, downstream of the initiation site, two four bp-long 'CG clamps', one of which overlaps the potential target sequence (CCAAT) of CspA, the cold-shock transcriptional enhancer of this gene. To establish the role of these potential regulatory signals during the cold-shock activation of hns, the CCCCAAT sequence has been subjected to mutagenesis, weakening the strength of the CG clamp and scrambling or inverting the CCAAT sequence. The resulting mutated hns promoters were placed in front of a reporter gene (cat) and their activity was studied in cells subjected to cold-shock under conditions where the increase in the concentration of CspA is either large or small. Our results allow us to conclude that although not essential, the CCCCAAT sequence, mainly due to the presence of the CG clamp, may play an important role in the CspA-mediated regulation of hns expression at both transcriptional and translational levels.
