title: Silencing and activation of ClyA cytotoxin expression in Escherichia coli.
abstract: Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene of Escherichia coli K-12. Genetic analysis suggested that clyA is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to clyA sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal clyA::luxAB operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in hns(+) E. coli derepresses clyA transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the clyA upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the clyA gene could be activated by overexpression of FNR. A major clyA transcript having its 5' end (+1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The clyA promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the clyA promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.
authors.affiliation: Department of Microbiology, Umeå University, S-90187 Umeå, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Westermark
authors.forename: J
authors.initials: J
authors.lastname: Oscarsson
authors.forename: Y
authors.initials: Y
authors.lastname: Mizunoe
authors.forename: J
authors.initials: J
authors.lastname: Urbonaviciene
authors.forename: B E
authors.initials: BE
authors.lastname: Uhlin
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D001427:Bacterial Toxins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; C031908:H-NS protein, bacteria; D006460:Hemolysin Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C119944:hlyE protein, E coli; C056785:salmolysin