Analysis of the Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS
Swanson, Britta L.
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Exotoxin A production in Pseudomonas aeruginosa is a complex, highly regulated process which includes both environmental and genetic factors. Several genes have been identified which increase the expression of exotoxin A, the most prominent being regAB. Another regulatory gene, ptxR, has been shown to positively influence exotoxin A synthesis by acting through regAB. A negative exotoxin A regulatory gene was located upstream of ptxR in the opposite orientation. This gene, ptxS, interferes with the effect of ptxR on exotoxin A expression through an unknown mechanism. PtxS belongs to the GalR-LacI family of transcriptional repressors. Due to the nature of this family of proteins, it is possible that ptxS acts as a negative regulator of ptxR expression by binding to its upstream region. Alternatively, ptxS may regulate another gene which then exerts its effects on ptxR transcription. The aim of this study is to determine how ptxR and/or ptxS are regulated. The ptxS gene was found to autoregulate its synthesis by binding to a 14 bp palindromic sequence within the ptxS upstream region. This palindrome represents the PtxS operator site. Any change within this sequence affects either the binding of PtxS to the operator, the expression of the ptxS gene, or both. However, the autoregulation is most likely not the only means of regulation within the ptxR-ptxS intergenic region. Deletion analysis of the ptxS upstream region localized the promoter region to approximately 700 bp upstream of the ptxS translational start site. Within this sequence, two additional regions of DNA binding activity were localized which were not due to PtxS binding. These results suggest that the ptxR-ptxS intergenic region is under the control of several potential factors. A second PtxS operator sequence was found downstream of ptxS which specifically bound PtxS. This target site was located upstream of four putative carbohydrate utilization genes. A P. aeruginosa ptxS isogenic mutant was unable to grow in the presence of 2- ketogluconate as a sole carbon source. Therefore, it was tested and proved that PtxS negatively regulates a putative 2-ketogluconate utilization operon by binding to its upstream region. This binding activity is modulated in response to the specific inducer metabolite, 2-ketogluconate. It appears that/^/Jc^ is the first gene in the 2-ketogluconate utilization operon.