Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon

Abstract

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 analyses 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.

Anti-activator ExsD Forms a 1:1 Complex with ExsA to Inhibit Transcription of Type III Secretion Operons

“…Like most AraC/XylS proteins, ExsA is largely insoluble when overproduced and tends to aggregate when concentrated, both properties which are challenging for biochemical studies. Although a maltose-binding protein-ExsA fusion was used to study the DNA binding activity (41), only recently was the biochemical characterization of His 6 -ExsA at a low concentration and in presence of Tween 20 reported (29). However, in these conditions separately produced and purified ExsA and ExsD proteins were not able to form any complex in vitro, and our attempts to see an effect of ExsD on the in vitro binding activity of purified ExsA repeatedly failed (data not shown).…”

Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS

“…ExsA is a global regulator of the type III secretion machinery (Hovey & Frank, 1995). Examination of monolayer infections with PAK or an isogenic exsA mutant revealed a profound defect in the ability of the type III mutant to provoke apoptosis.…”

“…To date, four such factors have been characterized, including ADP-ribosylating enzymes ExoS and ExoT (Frank, 1997 ;Frithz-Lindsten et al, 1997 ;Yahr et al, 1996a), an acute cytolytic factor ExoU (Finck-Barbancon et al, 1997 ;Hauser et al, 1998) an adenylate cyclase ExoY (Yahr et al, 1998). Expression of these secreted effector molecules as well as components of the type III secretory apparatus are under the control of the transcriptional activator, ExsA (Hovey & Frank, 1995). From a survey of various clinical isolates, Fleiszig et al (1997) have divided isolates of P. aeruginosa into two categories, invasive and noninvasive strains, based on their abilities to invade mammalian cells.…”

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

“…AraCfamily transcription regulators generally function as dimers to either activate or repress transcription at a given locus (57,58). The AraC family members canonically regulate genes involved in metabolism (e.g., AraC and XylS for regulation of arabinose and xylene/benzoate metabolism, respectively [59,60]) and/or virulence (61) (e.g., ExsA regulation of type III secretion in P. aeruginosa [62] and ToxT regulation of cholera toxin and the toxincoregulated pilus in Vibrio cholerae [63]). Evidence suggests that GbdR senses GB and dimethylglycine levels in the cell and induces transcription of genes involved in virulence, GB transport, GB catabolism, and detoxification of the catabolic byproducts, hydrogen peroxide and formaldehyde (48,56,(64)(65)(66)(67)(68) (Fig.…”