Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon.
ExsA has been implicated as a central regulator of exoenzyme S production by Pseudomonas aeruginosa. In this study, the DNA-binding and transcriptional activation properties of ExsA were investigated. ExsA was produced and purified as a fusion protein, MALA3A2, which was shown to bind specifically to promoter regions that regulated transcription of the exoenzyme S trans-regulatory locus (pC) and a locus located directly downstream of exsA (pD). Previously, MALA3A2 was shown to bind the exoS 5' PstI-NsiI region, which contained two independent but coordinately regulated (ExsA-mediated) promoters, pS' (now termed pORF1) and pS. DNase I footprint analysis of the promoter regions bound by ExsA revealed a common protected consensus sequence of TXAAAAXA. The consensus sequence was located -51 to -52 bp upstream of the transcriptional start sites for pD, pS, and pORF1. Promoter fusion, DNA-binding, and mutagenesis analysis indicated that the consensus sequence was important for transcriptional activation. Each ExsA-controlled promoter region contained at least two consensus sites in close proximity, similar to the arrangement of half-sites seen in AraC-controlled (Escherichia coli) or VirF-controlled (Yersinia enterocolitica) promoters. However, the results of this study suggested that only one consensus site was required in the exoenzyme S (pS) or ORF1 promoter (pORF1) to initiate transcription. These data suggest that members of the exoenzyme S regulon can be defined as possessing an ExsA consensus element which maps at bp -51 or -52 relative to the transcriptional start site.
Hovey
A K
AK
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Frank
D W
DW
D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018085:Regulon; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C095093:ExsA protein, Pseudomonas aeruginosa; C095092:ExsA protein, bacteria; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D015500:Chloramphenicol O-Acetyltransferase; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S