CHARACTERIZATION OF PTXS, A PSEUDOMONAS-AERUGINOSA GENE WHICH INTERFERES WITH THE EFFECT OF THE EXOTOXIN-A POSITIVE REGULATORY GENE, PTXR

The complicated process of exotoxin A production by Pseudomonas aerugi nosa is controlled by several genes. We have recently described a toxA positive regulatory gene, ptxR. We also proposed the presence of anot her gene which is adjacent to ptxR and interferes with ptxR function o n exotoxin A production. In the presence of a fragment that carries th e putative gene, the enhancement in exotoxin A production by ptxR was reduced threefold. In this study, we describe the characterization of this gene. Nucleotide sequence analysis of the 2.1-kbp fragment at the 5' end of ptxR revealed the presence of an open reading frame designa ted ptxS (the gene next to ptxR) which encodes a 37.4-kDa protein. The gene ptxS is transcribed in the opposite orientation to ptxR from the other DNA strand. The deduced amino acid sequence of ptxS exhibited a strong homology to several proteins of the GalR-LacI family of repres sors. A putative helix-turn-helix DNA binding motif was identified at the amino-terminus region of PtxS. When PtxS was overexpressed in Esch erichia coli using the T7 expression system, a single protein of 38-kD a molecular weight was detected. An isogenic mutant defective in ptxS was constructed in PAO1 using the gene replacement technique. The loss of ptxS resulted in a twofold increase in exotoxin A production compa red to PAO1. The effect of ptxS on ptxR was examined using a ptxR-lacZ fusion. In the presence of ptxS, the level of P-galactosidase activit y produced by the ptxR-lacZ fusion was significantly reduced. These re sults suggest that ptxS encodes a protein which negatively regulates p txR expression in P. aeruginosa.