The essential role of the promoter‐proximal subunit of CAP in pap phase variation: Lrp‐ and helical phase‐dependent activation of papBA transcription by CAP from −215

Abstract

SummaryCatabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP±CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP-dependent activation of papBA requires binding of the leucineresponsive regulatory protein (Lrp) at binding sites that extend from 2180 to 2149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAPdependent promoters. Also, like class I promoters, the C-terminal domain of the a-subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an`oriented heterodimer' approach with wild-type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter-distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H-NS. The results presented here show that AR1 of the promoter-proximal CAP subunit was required for papBA transcription even in the absence of the histone-like protein H-NS. These results show that the promoter-proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the Cterminal domain of the a-subunit of RNA polymerase.

ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

“…Interestingly, the closest homologue of OmpT, OmpH in the deep-sea bacterium Photobacterium profundum, was also found to be under catabolite repression and a putative CRP binding site was predicted at -330 of the ompH promoter, although functional data have not yet been reported (Bartlett and Welch, 1995). The greatest distance reported previously was for the papA promoter of uropathogenic E coli, in which binding of CRP centred at -215.5 of the promoter is essential for the activation of transcription (Weyand et al, 2001). The formation of a DNA loop involving two additional activators, Lrp and PapI, helps to eliminate the distance between CRP bound at -215.5 and the transcription apparatus, so that AR1 of CRP can contact the a-CTD of RNAP directly.…”

The Pix pilus adhesin of the uropathogenic Escherichia coli strain X2194 (O2 : K− : H6) is related to Pap pili but exhibits a truncated regulatory region

“…Expression is regulated by a region directly upstream of the gene of the major pilus subunit PapA encompassing the papI and papB genes. Transcription of the polycistronic mRNA is additionally influenced by global regulators such as the leucine-responsive protein (Lrp), the catabolite activator protein (CAP) and H-NS (Nou et al, 1995;Weyand et al, 2001;White-Ziegler et al, 1998). The different pilus systems show remarkable similarities in the coding sequences as well as in the protein structures of their different subunits (Smyth et al, 1996).…”

“…In the pap operon, differential methylation patterns of GATC sites located in the promoter region are responsible for inhibiting and inducing pilus transcription. Two pap operon genes (papB and papI) are involved in this process, acting in concert with global regulators such as Lrp (Nou et al, 1995;Weyand et al, 2001). …”

Self-perpetuating epigenetic pili switches in bacteria

“…CAP plays important roles in activation at the papBA and papI promoters (14)(15)(16)25). Binding of CAP to a single site located at Ϫ215.5 bp from the papBA start and Ϫ115.5 bp from the papI start is essential for activation of both papBA and papI transcription.…”

“…Binding of CAP to a single site located at Ϫ215.5 bp from the papBA start and Ϫ115.5 bp from the papI start is essential for activation of both papBA and papI transcription. CAP directly activates transcription at the papBA promoter (16). However, recent data suggest that CAP activates papI transcription indirectly by means of expression of PapB regulatory protein because activation of papI transcription occurs in the absence of CAP when papB is expressed from an independent promoter (manuscript in preparation).…”