The Pseudomonas aeruginosa exotoxin a regulatory gene, ptxS: Evidence for negative autoregulation

We have previously described a Pseudomonas aeruginosa gene, ptxR, which enh ances exotoxin A production at the transcriptional level. We have also desc ribed another gene, pfxS, which is transcribed divergently from ptxR and in terferes with the enhancement of exotoxin A synthesis by ph;R, However, the mechanisms through which ptxR and/or ptxS are regulated is not known. In t his study, we attempted (by using the DNA gel shift assay) to determine if P. aeruginosa contains a potential regulatory protein that binds specifical ly to the ptxR or ptxS upstream region. In the initial analysis, different- sized gel shift bands Here detected when a probe containing the ptxR-ptxS i ntergenic region was incubated with the lysate of P, aeruginosa PAO1, The s trongest binding activity was detected with a smaller fragment that represe nts the ptxS upstream region. Additional deletion analysis localized the bi nding to a 52-bp fragment immediately upstream of ptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of t he ptxS isogenic mutant PAO1::ptxS. However, the binding band was regenerat ed when a plasmid carrying ptxS intact was introduced into PAO1::ptxS. In a ddition, the gel shift hand was detected when the 52-bp fragment,vas incuba ted with a lysate of Escherichia coli in which ptxS was overexpressed from the T7 promoter. The effect of PtxS on pfxS expression was examined by usin g a ptxS-lacZ fusion plasmid, The level of P-galactosidase activity produce d by PAO1::pfxS carrying the fusion plasmid was four- to fivefold higher th an that produced by PAO1 carrying the same plasmid, Using DNase I footprint ing analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a Pt xS binding site. These results suggest that PtxS autoregulates its synthesi s by binding to a specific sequence within the ptxS upstream region.