ISOLATION AND CHARACTERIZATION OF A PSEUDOMONAS-AERUGINOSA GENE, PTXR, WHICH POSITIVELY REGULATES EXOTOXIN-A PRODUCTION

Exotoxin A production in Pseudomonas aeruginosa is a complicated and h ighly regulated process that involves several genes. In this report, w e describe the isolation of a new toxA regulatory gene (ptwR) which af fects exotoxin A production in P. aeruginosa, In an iron-deficient med ium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 res ulted in a four- to fivefold increase in exotoxin A synthesis, No effe ct was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease, Using subcloning and complementation experim ents, ptxR was localized to a 2.1 kb KpnI-Bg/II fragment, Nucleotide s equence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR com pares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system, The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino-terminus region of PtxR contain s a putative helix-turn-helix DNA-binding motif, Specific ptxR-deletio n mutants in P. aeruginosa strains PAO1 and PA103 were constructed, In comparison with their parent strains, both mutants showed a significa nt reduction in the level of exotoxin A activity, However, upon extens ive subculturing, the level of exotoxin A produced by the PAO1::ptxR m utant was similar to that of PAO1, Transcriptional studies, using both toxA-lacZ fusion and RNA analysis, confirmed that ptwR increases toxA and regA transcription, These results suggest that ptxR regulates (th rough regA) exotoxin A production at the transcriptional level.