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 aeruginosa is controlled by several genes. We have recently described a toxA positive regulatory gene, ptxR. We also proposed the presence of another gene which is adjacent to ptxR and interferes with ptxR function on exotoxin A production. In the presence of a fragment that carries the 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 designated 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 repressors. A putative helix-turn-helix DNA binding motif was identified at the amino-terminus region of PtxS. When PtxS was overexpressed in Escherichia coli using the T7 expression system, a single protein of 38-kDa 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 compared to PAO1. The effect of ptxS on ptxR was examined using a ptxR-lacZ fusion. In the presence of ptxS, the level of beta-galactosidase activity produced by the ptxR-lacZ fusion was significantly reduced. These results suggest that ptxS encodes a protein which negatively regulates ptxR expression in P. aeruginosa.
Colmer
J A
JA
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Hamood
A N
AN
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005098:Exotoxins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D056968:Lac Repressors; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D056968:Lac Repressors; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C050993:Galactose repressor proteins; C113064:PtxS protein, Pseudomonas aeruginosa