title: The Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS: evidence for negative autoregulation.
abstract: We have previously described a Pseudomonas aeruginosa gene, ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene, ptxS, which is transcribed divergently from ptxR and interferes with the enhancement of exotoxin A synthesis by ptxR. However, the mechanisms through which ptxR and/or ptxS are regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine if P. aeruginosa contains a potential regulatory protein that binds specifically to the ptxR or ptxS upstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing the ptxR-ptxS intergenic region was incubated with the lysate of P. aeruginosa PAO1. The strongest binding activity was detected with a smaller fragment that represents the ptxS upstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream of ptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of the ptxS isogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carrying ptxS intact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate of Escherichia coli in which ptxS was overexpressed from the T7 promoter. The effect of PtxS on ptxS expression was examined by using a ptxS-lacZ fusion plasmid. The level of beta-galactosidase activity produced by PAO1::ptxS carrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within the ptxS upstream region.
authors.affiliation: Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
authors.forename: B L
authors.initials: BL
authors.lastname: Swanson
authors.forename: J A
authors.initials: JA
authors.lastname: Colmer
authors.forename: A N
authors.initials: AN
authors.lastname: Hamood
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005098:Exotoxins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006706:Homeostasis / Q000502:physiology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005098:Exotoxins; C113064:PtxS protein, Pseudomonas aeruginosa; D014157:Transcription Factors; C073470:LysR protein, Bacteria