REGULATION OF THE SALMONELLA-TYPHIMURIUM PEPT GENE BY CYCLIC-AMP RECEPTOR PROTEIN (CRP) AND FNR ACTING AT A HYBRID CRP-FNR SITE

The Salmonella typhimurium pepT gene is induced nearly 30-fold in resp onse to anaerobiosis. Anaerobic expression is dependent on the transcr iptional regulator encoded by fnr (previously oxrA). Primer extension analysis and site-directed mutagenesis experiments show that pepT is t ranscribed from two sigma(70) promoters. One promoter (P1) is FNR depe ndent and anaerobically induced, while the other (P2) appears to be co nstitutive. The potABCD operon is divergently transcribed from a promo ter near pepT P2. Sequence analysis of pepT promoter mutations which e ither elevate anaerobic expression or confer constitutive expression r evealed that these mutations affect the -10 region of the P1 or P2 pro moter, respectively. The pepT200 mutation, which changes the -10 regio n of the FNR-dependent P1 promoter to the consensus, has the surprisin g effect of allowing five- to sevenfold anaerobic induction in the abs ence of FNR We have shown that the anaerobic induction of pepT-lacZ in a pepT200 fnr strain is dependent on wild-type alleles of both crp an d cya, In a pepT200 pepT-lacZ strain, beta-galactosidase activity was elevated aerobically in the presence of exogenous cyclic AMP (cAMP) an d was elevated also in succinate minimal medium relative to its level in glucose minimal medium. Primer extension analysis confirmed that P1 is the cAMP receptor protein (CRP)-dependent promoter. Site-directed mutagenesis experiments indicated that a hybrid CRP-FNR binding site p ositioned at -41 of the P1 promoter is utilized by both FNR and CRP. C RP-cAMP also appeared to repress FNR-dependent transcription of pepT u nder anaerobic conditions in both the pepT(+) and pepT200 backgrounds. Although both CRP and FNR are capable of binding the hybrid site and activating transcription of pepT, CRP requires the consensus -10 seque nce for efficient activation.