Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production

Production of exotoxin A, the most toxic of the virulence factors produced by the opportunistic pathogen Pseudomonas aeruginosa, is a complicated, highly regulated process involving several genes. In this study, we describe the isolation and characterization of two new regulatory genes for toxA expression, ptxR and ptxS. The presence of extra copies of ptxR in PA01 results in a fivefold increase in exotoxin A activity. Subcloning, complementation, nucleotide sequence analyses, and T7 expression experiments revealed that the ptxR open reading frame (ORE) encodes a 34 kDa protein. Computer analysis for amino acid homology revealed that the product of ptxR (RxR) belongs to the LysR family of transcriptional activators and contains a putative helix-turn-helix DNA binding motif. Transcriptional studies using RNA analysis and a toxAlacZ fusion disclosed that ptxR enhances both toxA and regA transcription. These results suggest ptxR acts through regA to enhance toxA expression. Further studies confirmed that the presence of a 2.1 kbp fragment 5' of ptxR reduced the enhancement in exotoxin A by threefold. Nucleotide sequence analysis and expression experiments revealed that this fragment carries an ORE, ptxS, encoding a 38 kDa protein, RxS. Computer analysis of the amino acid sequence indicated that RxS belongs to the GalR-LacI family of repressors. The presence of a helix-turn-helix motif suggests a DNA binding function for PtxS. Additional experiments, using ptxRlacZ fusions, provided evidence that ptxS affects ptxR expression directly. The presence of three potential GaIR binding sites in the ptxR upstream region suggests that RxS might interfere with pfxP function at the transcriptional level by binding to its upstream region. We have also examined ptxR expression and the effect of ptxR on toxA expression throughout the growth cycle of Pseudomonas aeruginosa strains PA01 and PA103 using toxAlacZ chromosomal integrates and the ptxRlacZ fusion plasmid. These experiments suggest that while the enhancement in toxA transcription by ptxR continues at later stages of the growth cycle, ptxS expression declines; and, that ptxR expression in Pseudomonas aeruginosa is iron-independent.