Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa

Abstract

Exoenzyme S is an ADP-ribosyltransferase enzyme distinct from exotoxin A that is synthesized and secreted by Pseudomonas aeruginosa. Yields of exoenzyme S are variable and depend on strain and growth conditions. Since certain medium additives are required for exoenzyme S production, its regulation may be influenced by environmental stimuli. In this study, we have cloned a region that complements the exoenzyme S-deficient phenotype of strain 388 exs1::Tn1, a chromosomal Tn1 insertional mutation. A large clone (28 kb) was shown to restore both synthesis and secretory functions to the mutant strain. Subcloning and Tn501 mutagenesis experiments localized the region required for exoenzyme S synthesis to a 3.2-kb fragment. Nucleotide sequence analysis demonstrated several open reading frames. Comparison of the N-terminal amino acid sequence of purified exoenzyme S with predicted amino acid sequences of all open reading frames indicated that the structural gene was not encoded within the sequenced region. Homology studies suggested that the region encoded three regulatory genes, exsC, exsB, and exsA. ExsA was homologous to the AraC family of transcriptional activator proteins, with extensive homology being found with one member of this family, VirF of Yersinia enterocolitica. VirF and ExsA both contain carboxy-terminal domains with the helix-turn-helix motif of DNA-binding proteins. The ExsA gene product appeared to be required for induction of exoenzyme S synthesis above a low basal level. Expression of ExsA was demonstrated by cloning the region under the control of the T7 promoter. Gene replacement experiments suggested that the expression of ExsC affects the final yield of exoenzyme S.

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury

“…Individual colonies were picked onto assay medium for exoenzyme S production and screened for ExoU expression using a rabbit polyclonal antisera to gel-purified ExoU. Bound IgG was detected using affinity-purified, horseradish peroxidase-conjugated goat-anti-rabbit immunoglobulin G (Boehringer Mannheim) and 4-chloro-1-naphthol (Sigma) as the colorimetric substrate (Frank and Iglewski, 1991). ExoU-deficient strains were rescreened by Western blot analysis using rabbit-anti-ExoS, which recognizes ExoS and ExoT, to select for strains that retained expression of ExoT.…”

Early Immune Response to the Components of the Type III System of Pseudomonas aeruginosa in Children with Cystic Fibrosis

“…P. aeruginosa cell lysate was prepared from a 6-h or overnight culture supernatant of P. aeruginosa PA01 that was provided by Steven Lory (Harvard Medical School, Boston, MA). The bacterium was grown in a deferrated dialysate of Trypticase soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma Chemical Company, St. Louis, MO), 1% glycerol, and 100 mM monosodium glutamate at 32°C (15). The cells were pelleted (14,000 ϫ g for 40 min), and the spent culture supernatant was subjected to ammonium sulfate precipitation (65% final concentration).…”

The exoenzyme S regulon of Pseudomonas aeruginosa

“…The genetic basis for the induction of exoenzyme S synthesis was identified with the cloning and functional analysis of the exoenzyme S trans-regulatory locus (Frank and Iglewski, 1991). The trans-regulatory locus encodes two proteins, ExsC and ExsA, and a cis-acting RNA region termed exsB .…”

“…The trans-regulatory locus encodes two proteins, ExsC and ExsA, and a cis-acting RNA region termed exsB . ExsA, a member of the AraC family of transcriptional regulators, exhibits 56% overall amino acid identity to the Yersinia VirF / LcrF proteins (Cornelis et al, 1989;Frank and Iglewski, 1991). Alignment of the C-terminal domains, containing putative helix-turn-helix motifs, results in a 92.5% identity score, suggesting that ExsA and LcrF / VirF may bind similar DNA sequences .…”