Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

Abstract

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...

Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12

“…1) (9). mbf, which maps at 19.6 min (9), is now known to be allelic to lrp (10). Transduction of insert 30 into the mbf::mTnJO-tet insertion mutant DL845 showed the close proximity of insert 30 to lrp; all 252 kanamycin-resistant transductants tested became tetracycline sensitive, implying loss of the resident mini transposon.…”

“…Using the approach outlined above, we have isolated insertions in lip, a gene that plays an important role in metabolism (for a review, see reference 41) and that affects both Pap and K99 fimbriation (9,10). Upon further analysis, we found that the frequency of switching of thefim invertible region, and consequently of phase variation, is sharply reduced in 1rp mutants.…”

Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues

“…Moreover, there is an absolute requirement of IHF for phase variation of type I fimbriae, such that him mutants are unable to switch phase (Dorman and Higgins, 1987;Eisenstein et al, 1987). The ETEC K99 and K88 fimbriae are also regulated by LRP, which represses the expression of K88 fimbriae and activates the expression of K99 fimbriae (Braaten et al, 1992;Huisman et al, 1994). Table 1 shows that, unlike these other systems, 987P fimbriae are unaffected by mutations in lrp, dam or either of the two subunits encoding IHF (himA and himD ).…”

The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12

“…Both fimB transcription and fimE transcription are affected only slightly by mutation of lrp (3). Therefore, it is likely that the marked stimulation of fim by lrp is either direct or occurs through factors other than FimB and FimE. Lrp, a site-specific DNA-binding protein, controls a regulon of at least 40 genes in Escherichia coli, including fimbrial expression and amino acid transport, degradation, and biosyn-thesis (3,6,9,10,18,19,(22)(23)(24). The most extensive studies of Lrp have examined its role in the regulation of ilvIH transcription (19,22,23).…”

“…Lrp binds to the promoter region of ilvIH with a high degree of cooperativity, bending the DNA to form a nucleoprotein complex (22,23). Lrp, also known as Mbf (methylation-blocking factor), participates directly in phase variation ofpapBA transcription (6,18). Pap phase variation is associated with alternate dam methylation-protection patterns and requires Lrp (18).…”