PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa

Abstract

PfeR (Regulator) and PfeS (Sensor), members of the superfamily of so-called two-component regulatory protein pairs, are required for the enterobactin-inducible production of the ferric enterobactin receptor (PfeA) in Pseudomonas aeruginosa. A pfeR knockout mutant failed to demonstrate enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that PfeR acts at the level of pfeA gene expression. Consistent with this, PfeR overexpressed in P. aeruginosa bound, in bandshift assays, the promoter region of pfeA. Such binding was enhanced when PfeR-containing extracts were prepared from cells cultured in the presence of enterobactin, consistent with a model of PfeR as an enterobactin-responsive activator of pfeA expression. A region showing homology to the consensus binding sequence for the global iron repressor Fur was identified upstream of pfeR, suggesting that the pfeRS operon is iron regulated. As expected, expression of a pfeR-lacZ fusion in P. aeruginosa was increased under conditions of iron limitation. Enterobactin failed, however, to provide any enhancement of pfeR-lacZ expression under iron-limiting conditions, indicating that PfeR does not positively regulate pfeRS expression. A pfeA knockout mutant demonstrated enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that the receptor is not required for the enterobactin inducibility of pfeA gene expression. Such mutants show growth, albeit reduced, in enterobactin-supplemented iron-limiting minimal medium, indicating that a second route of uptake across the outer membrane exists for ferric enterobactin in P. aeruginosa and may be important for the initial induction of pfeA in response to enterobactin.