Transcription of the pap pilin (papA) gene in Escherichia coli is subject to control by a heritable phase variation mechanism in which alternation between transcriptionally active (phase on) and inactive (phase off) states occurs. Our results suggest that phase switching occurs without DNA rearrangement of pap DNA sequences, distinguishing this system from those described for E. coli type 1 pili and Salmonella flagellar phase variation. Analysis of the regulatory region upstream of papA in DNAs isolated from phase off and phase on cell populations showed that two deoxyadenosine methylase (Dam) sites, GATC1028 and GATC1130, were present. Southern blot analysis of MboI and DpnI restriction digests of DNAs showed that the GATC1028 site was unmethylated only in DNA isolated from phase on populations. Conversely, GATC1130 sites were unmethylated in DNA isolated from phase off populations. The presence of unmethylated GATC sites in E. coli is unusual and to our knowledge has not been previously reported. These results suggest that the methylation states of GATC1028 and GATC1130 may regulate pap transcription. Consistent with this hypothesis, Dam methylase levels affected the regulation of pap transcription; papA transcription was absent in dam‐ E. coli. Moreover, transition from the phase off to phase on state was not observed in E. coli expressing aberrantly high levels of Dam. A basic model is presented which outlines a possible mechanism by which alternation between phase off and phase on methylation states could occur.

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The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using paplac and ilvIH-ac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucineresponsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator ofothers in E. coli. DNA sequence analysis of an mbf::mTnlO insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing thepap andfan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K9 pili expression, similar to results obtained with the ilvlH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other frp-regulated genes whose expression is modulated by leucine.Escherichia coli is an opportunistic pathogen that can cause meningitis, urinary tract infection, bacteremia, and diarrheal disease (1). Most E. coli strains that cause pyelonephritis express P adhesin (2), which binds the bacteria to the P blood group antigen of host epithelial cells lining the intestinal and urinary tracts (3). At least 11 genes, forming the pyelonephritis-associated pilus operon (pap), are involved in expression of the P pilus-adhesin complex and these include the papI and papB genes, which regulate pap transcription (4, 5). P-pilus expression is under phase variation control in which individual cells within a bacterial population either express (ON) or do not express (OFF) P pili (6, 7).Pap pili phase variation is controlled at the transcriptional level by a mechanism involving specific methylation inhibition at two deoxyadenosine methylase (Dam) sites (GATC1028 and GATC1130) near the pap pilin transcription start site (8,9).The GATC1028 site is unmethylated in ON cells but is methylated in OFF cells. Conversely, the GATC1130 site is unmethylated in OFF cells and methylated in ON cells (9). Recent evidence indicates that a gene denoted mbf (methylation blocking factor) is required for inhibiting methylation of both the GATC1028 and GATC1130 sites since transposon mTnlO insertions within this gene result in full methylation of both pap GATC sites (10). mbf: :mTnlO insertion mutants display a 40-fold reduction in pap pilin transcript levels based on analysis of single-copy pap-lac operon fusion strains, indicating that mbf is required for both pap GATC methylation inhibition and pap pilin transcription (10).In this paper, we show that mbf is identical to lrp, the leucine-responsive regulatory protein gene, which i...

The expression of the pap pilus operon of Escherichia coli is under a phase-variation control mechanism in which cells undergo a reversible transition between transcriptionally active (phase ON) and inactive (phase OFF) states. In this study, we explore the roles of leucine-responsive regulatory protein (Lrp) and the histone-like protein H-NS in the regulation of pap phase variation. Our data indicate that the phase OFF state results from repression of the intrinsically active papBA promoter by Lrp and H-NS, each of which can act independently as transcriptional repressors. Lrp requires pap DNA sequences upstream of the papBA promoter for its repressor activity whereas H-NS does not. In contrast, in the ON state, Lrp, in conjunction with PapI, activates pap transcription. This activation is not merely a result of alleviating the H-NS mediated repression, but induces a level of transcription that is eightfold higher than the basal level of transcription from the papBA promoter measured in the absence of both H-NS and Lrp. Analysis of Lrp activation mutants indicates that binding of Lrp to pap DNA sequences is not sufficient for transcription activation, consistent with a model in which an additional domain of Lrp interacts with the transcriptional apparatus. Together, our results show that Lrp functions as a transcriptional activator in phase-ON cells and as a repressor of basal transcription in phase-OFF cells. Because pap phase variation occurs in the absence of H-NS, it is not clear what role this regulatory protein plays in pap gene regulation.

The initial pathogenic step in nonobstructive Escherichia coli pyelonephritis usually involves the binding of a bacterial adhesin with host uroepithelial glycoprotein receptors containing the D-Gal pal --4 D-Gal pfi1 (Gal-Gal) moiety. In this study, groups of mice were immunized with Gal-Gal pili and challenged 2 wk later intravesicularly with E. coli strains expressing homologous or heterologous pili. 63 of 129 pili-immunized mice (49%) were protected from subsequent E. coli renal colonization compared with 5 of 85 control mice (6%). Among mice that had E. coli cultured from their right kidney, control mice had greater bacterial colony counts than pili-immunized animals (P < 0.05). Light microscopic examination of kidneys demonstrated less histopathology among pili immunized mice than among control mice (P < 0.05). Protection correlated with the presence of specific IgG antibodies in the urine and serum that bind to the major pilin structural polypeptide and not to the Gal-Gal pili tip adhesin per se. These results support the concept that immunization with a bacterial surface-coat constituent can prevent mucosal infection by interfering with colonization. Also Gal-Gal pili of E. coli represent a suitable candidate for immunoprophylaxis against pyelonephritis. Introduction The Gal-Gal binding pili phenotype of piliated Escherichia coli correlates with uropathogenicity because it is a critical ligand for upper urinary tract epithelial colonization in the anatomically normal urinary tract (1-5). The gene for Gal-Gal binding encodes for a major pilin protein (pap A), assembly and transporter proteins, and three other pilus tip proteins, one of which (pap G) constitutes the putative adhesin (6)(7)(8)