Silencing and activation of ClyA cytotoxin expression in Escherichia coli

Cytolysin A (CIS-A) is a pore-forming cytotoxic protein encoded by the clyA gene of Escherichia coli K-12. Genetic analysis suggested that clyA is sil enced by the nucleoid protein H-NS. Purified H-NS protein showed preferenti al 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 c onditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS -deficient bacteria neither the absence nor the overproduction of SlyA affe cted the derepressed ClyA expression any further. Therefore, we suggest tha t overproduction of SlyA in hns(+) E. coli derepresses clyA transcription b y counteracting H-NS. The cyclic AMP receptor protein (CRP) was required fo r ClyA expression, and it interacted with a predicted, albeit suboptimal, C RP binding site in the clyA upstream region. Site-specific alterations of t he 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, an d the clyA gene could be activated by overexpression of FNR. A major clyA t ranscript having its 5' end (+1) located 72 bp upstream of the translationa l 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 p romoter that could be transcriptionally activated by CRP and/or FNR. Accord ing to DNA bending analyses, the clyA promoter region has high intrinsic cu rvature. We suggest that it represents a regulatory region which is particu larly susceptible to H-NS silencing, and its features are discussed in rela tion to regulation of other silenced operons.