The effect of ptxR on the production of exotoxin A, exoenzyme U, and the pyoverdine chromophore in the Pseudomonas aeruginosa strain PA103



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Texas Tech University


The production of exotoxin A and the pyoverdine siderophore m Pseudomonas aeruginosa is negatively regulated by iron. We have previously described a P aeruginosa gene ptxR that positively regulates exotoxin A and pyoverdine production. Previous studies from our laboratory and others reported that the presence of a pixR plasmid in P aeruginosa increased exotoxin A production by 4 to 5 fold and deregulated the production of the pyoverdine chromophore (pvc) with respect to iron. In this study, we examined toxA regulation by ptxR in the P. aeruginosa strain PA103, which has been extensively used in exotoxin A regulatory studies. To avoid problems associated with regulatory studies involving multiple copies of genes, we constructed a PA103 strain carrying two copies of ptxR (PA103-2R) by homologous recombination. Exotoxin A production in PA103-2R was dramatically increased and deregulated with respect to iron. The expression of the pvc operon remained deregulated with respect to iron. These effects, which were not due to ptxR overexpression, occurred at the transcnptional level. SDS-PAGE and amino acid sequencing analysis demonstrated that the type III secretion effector protein, ExoU, is over produced in PA103-2R. Further analysis confirmed that e.vof/expression in PA103-2R is deregulated with respect to calcium. We detected the same phenotype when the integrated plasmid together with adjacent PA103 chromosome (pNC105) was retrieved from PA103-2R and introduced into PA103 as a stable plasmid (pKHl). Nucleotide sequence analysis revealed that pNC105 carries genes that exist in PA103 but not PAOl. In addition, pNC105 carries an intact copy of ptxR, but is lacking 322 bp of the region 3' of it. The role of the deleted region was investigated by constructing a new plasmid with a 4 bp insertion at a unique site 3' of ptxR, pNC313. The presence of pNC313 in PA103 produced the same phenotype observed in PA103-2R. We have eliminated the possibility that changes within the region 3' of pi.xR abolished a binding site for a potential regulator. Examination of differences in the predicted mRNA secondary structure for ptxR due to the 3' changes eliminated this possibility as at least two mechanisms would be required to explain the phenotype. However, an open reading frame encoding an estimated 14 kDa protein, PtxQ, was identified 3' of ptxR. Changes in PA103-2R, pKHl, and pNC313 either eliminated p/.vg or produced a frame shift mutation within it. Based on the available results, we propose that PtxQ modulates PtxR function by forming a PtxR:PtxQ heterodimer. In the absence of PtxQ, excess molecules of PtxR increase toxA, pvc, and exoU expression beyond the repressive ability of either iron or calcium.



Pigments -- Biosynthesis, Bacterial, Genes, Virulence, Cytotoxins -- Biosynthesis, Pseudomonas aeruginosa -- Pathogenicity, Exotoxins -- Biosynthesis