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Xenorhabdus nematophila is a gamma-proteobacterial mutualist of an insect-pathogenic nematode, Steinernema carpocapsae. X. nematophila requires nilC, a gene predicted to encode an outer membrane lipoprotein of unknown function, for colonization of its nematode host. Characterization of NilC, described here, demonstrated it is a 28 kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Lipidation and processing of NilC occurs by a mechanism that is conserved in proteobacteria. This work also showed NilC is membrane associated and oriented towards the periplasm of X. nematophila and is produced as an outer membrane-associated protein when expressed in Escherichia coli. Expression analyses revealed that nilC transcription is directly or indirectly repressed by Lrp, and this regulatory link may explain the nematode mutualism defect of a previously identified lrp::Tn5 mutant. An lrp::Tn5 mutant produces an additional nilC transcript, not observed in wild-type cells growing in vitro, and produces approximately 75-fold more nilC than wild-type cells in late stationary phase. These fundamental characterizations of nilC expression and nilC localization and processing events have provided firm bases for understanding the role of this colonization factor in the X. nematophila/S. carpocapsae microbe-host interaction.

Genome analysis has revealed that members of the Lrp family of transcriptional regulators are widely distributed among prokaryotes, both bacteria and archaea. The archetype Leucine-responsive Regulatory Protein from Escherichia coli is a global regulator involved in modulating a variety of metabolic functions, including the catabolism and anabolism of amino acids as well as pili synthesis. Most Lrp homologues, however, appear to act as specific regulators of amino acid metabolism-related genes. Like most prokaryotic transcriptional regulators, Lrp-like regulators consist of a DNA-binding domain and a ligand-binding domain. The crystal structure of the Pyrococcus furiosus LrpA revealed an N-terminal domain with a common helix-turn-helix fold, and a C-terminal domain with a typical alphabeta-sandwich fold. The latter regulatory domain constitutes a novel ligand-binding site and has been designated RAM. Database analysis reveals that the RAM domain is present in many prokaryotic genomes, potentially encoding (1) Lrp-homologues, when fused to a DNA-binding domain (2) enzymes, when fused as a potential regulatory domain to a catalytic domain, and (3) stand-alone RAM modules with unknown function. The architecture of Lrp regulators with two distinct domains that harbour the regulatory (effector-binding) site and the active (DNA-binding) site, and their separation by a flexible hinge region, suggests a general allosteric switch of Lrp-like regulators.

Ss-Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine-responsive regulatory protein), which out of all genome-encoded proteins is most similar to Escherichia coli Lrp (E-value of 5.6 e-14). The recombinant protein has been purified as a 68 kDa homotetramer. The specific binding of Ss-Lrp to its own control region is suggestive of negative autoregulation. A high resolution contact map of Ss-Lrp binding was established by DNase I and hydroxyl radical footprinting, small non-intercalating groove-specific ligand-binding interference, and various base-specific premodification and base removal binding interference techniques. We show that Ss-Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA-box and BRE promoter elements. Therefore, Ss-Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB. Moreover, the results demonstrate profound Ss-Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.

Host-encoded functions that regulate the transfer operon (tra) in the virulence plasmid of Salmonella enterica (pSLT) were identified with a genetic screen. Mutations that decreased tra operon expression mapped in the lrp gene, which encodes the leucine-responsive regulatory protein (Lrp). Reduced tra operon expression in an Lrp- background is caused by lowered transcription of the traJ gene, which encodes a transcriptional activator of the tra operon. Gel retardation assays indicated that Lrp binds a DNA region upstream of the traJ promoter. Deletion of the Lrp binding site resulted in lowered and Lrp-independent traJ transcription. Conjugal transfer of pSLT decreased 50-fold in a Lrp- background. When a FinO- derivative of pSLT was used, conjugal transfer from an Lrp- donor decreased 1000-fold. Mutations that derepressed tra operon expression mapped in dam, the gene encoding Dam methyltransferase. Expression of the tra operon and conjugal transfer remain repressed in an Lrp- Dam- background. These observations support the model that Lrp acts as a conjugation activator by promoting traJ transcription, whereas Dam methylation acts as a conjugation repressor by activating FinP RNA synthesis. This dual control of conjugal transfer may also operate in other F-like plasmids such as F and R100.

Expression of the Escherichia coli serA gene is activated in vivo by the product of the lrp gene, leucine-responsive regulatory protein (Lrp), an effect partially reversed by L-leucine. We show here that serA is transcribed from two promoters, P1 45 bp upstream of the translation start site, and P2 92 bp further upstream. Lrp binds to a long AT-rich sequence from -158 to -82 from the start of the coding region, i.e. upstream of P1 and overlapping P2. It activates transcription from P1 and represses expression from P2. A second regulator, cAMP/CRP, activates P2, an effect that is largely inhibited by Lrp, such that catabolite repressor protein (Crp) and Lrp are rival activators of serA transcription.

Catabolite 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 leucine-responsive regulatory protein (Lrp) at binding sites that extend from -180 to -149 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 CAP-dependent promoters. Also, like class I promoters, the C-terminal domain of the alpha-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 C-terminal domain of the alpha-subunit of RNA polymerase.

The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth. Activation requires the LysR-like transcription factor SpvR and the alternative sigma factor RpoS. In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA-binding proteins contribute to the control of spv expression. These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine-responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon. Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold. It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter. Lrp represses spvA gene expression by up to fivefold and Lrp-mediated repression is antagonized by leucine. The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted. This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation. These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine-tuned in response to multiple environmental inputs.

The dadAX operon is expressed by multiple promoters that are repressed by leucine-responsive regulatory protein (Lrp) and activated by cyclic AMP-CRP. In previous work, we found that alanine or leucine acted as inducers to antagonize Lrp repression of the three major promoters directly. Here, we identify 11 Lrp binding sites located within 350 bp of dad DNA. A mutational analysis, coupled with in vivo and in vitro transcription experiments, indicated that Lrp sites that overlap the dad promoters were involved in repression. In contrast, sites upstream of the promoters did not appear to be necessary for repression, but were required for activation by Lrp plus alanine or leucine of one of the major dad promoters, P2. This activation by alanine or leucine was not simply relief of repression, as P2 transcription from a constitutive template was increased fivefold compared with the basal level of transcription found in the absence of Lrp and the co-activator cyclic AMP-CRP. Alanine or leucine decreased the affinity of Lrp to repressor sites, while having little or no effect on the binding of Lrp to activator sites. This differential effect of alanine and leucine on Lrp binding helps to explain how these modifiers influence both repression and activation of the dad operon.

The leucine-responsive regulatory protein (Lrp) is a global regulator that controls the expression of numerous operons in Escherichia coli. Lrp can act as a repressor or as an activator of transcription with its effects being potentiated, repressed or unaffected by the presence of exogenous leucine. The phase variation of type 1 fimbria in E. coli provides a unique system in which to investigate the effects of leucine on Lrp, as it is the only known example in which Lrp is a positive regulator and leucine potentiates this effect. Previous studies determined that Lrp binds with high affinity to two sites within the fim switch (fim sites 1 and 2), and binding to these sites stimulates recombination. Here, it is shown that, even though leucine stimulates the fim switch in vivo, it nevertheless causes a slight decrease in Lrp binding to the fim switch in vitro. These contradictory results are explicable by the finding that Lrp binding to a third region adjacent to fim sites 1 and 2 inhibits recombination. According to this model, leucine stimulates recombination by selectively disrupting Lrp binding to this newly characterized region, while having little or no effect on Lrp binding to fim sites 1 and 2.

Endospore formation in Bacillus subtilis is primarily dependent on the phosphorylation of the key transcription factor Spo0A by two major kinases, KinA and KinB, thought to be activated by distinct signals. Using a strategy designed to detect mutations that specifically affect the signalling pathway to KinB, we have isolated a Tn10 insertion mutant in one of two adjacent lrp-like genes coding for homologues of the Escherichia coli leucine-responsive regulatory protein (Lrp) and another mutant in the glyA gene encoding the serine hydroxymethyl transferase (SHMT). SHMT catalyses interconversion of serine and glycine while transferring the resulting one-carbon unit into the C1 pool through methylene tetrahydrofolate. Sporulation experiments performed in a series of supplemented media indicated that the role of SHMT in the KinB pathway is to feed the pool of C1 units recruited for the biosynthesis of key metabolites, which include the methyl donor S-adenosyl-methionine (SAM). The results of experiments using L-ethionine suggest that SAM is involved in post-synthetic methylation reactions or biosynthesis of metabolites that serve to activate KinB. Truncated LrpA and LrpB peptides that have retained the DNA-binding domain but have lost the C-terminal half of the protein appear to act as repressors of glyA transcription and KinB-dependent sporulation. However, deletions of lrpA, lrpB or lrpAB have little effect on glyA transcription or sporulation through KinB, suggesting that other effectors, such as additional Lrp homologues, may act in conjunction with LrpA and LrpB. Our results indicate that lrpA-lrpB together with the biosynthetic glyA gene lie on a common signalling pathway meant to activate the KinB sensor kinase.

Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II). The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site. The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion. The cloned DNA used in this study did not contain a related papl gene. In these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. In the presence of AfaF, a Papl equivalent, the phase-variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase. In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF.

The product of the Escherichia coli aidB gene is homologous to human isovaleryl-coenzyme A dehydrogenase (IVD), an enzyme involved in the breakdown of the amino acid leucine. The aidB gene is not expressed constitutively, but its transcription is induced via distinct mechanisms in response to: (i) exposure to alkylating agents; (ii) acetate at a slightly acidic pH; and (iii) anoxia. Induction by alkylating agents is mediated by the transcriptional activator Ada, in its methylated form (meAda); the other forms of induction are Ada independent and require sigma s, the alternative sigma factor mainly expressed during the stationary phase of bacterial growth. In this report we show that, in the absence of any transcriptional factor, aidB is efficiently transcribed in vitro by the sigma s, but not by the sigma 70, form of RNA polymerase holoenzyme. In the presence of meAda, levels of transcription by both forms of RNA polymerase are significantly increased. However, sigma s-dependent transcription of aidB is inhibited both in vitro and in vivo by binding of the transcriptional regulator Lrp (leucine responsive protein) to the aidB promoter region (PaidB). Lrp acts as a specific repressor for sigma s-dependent transcription of aidB. Leucine counteracts Lrp binding to P aidB, as does binding to P aidB of me Ada, which causes Lrp to dissociate from the promoter. The physiological significance of aidB transcription regulation is discussed.

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.

Expression of the K88 (fae) operon is negatively controlled by the co-operative binding of Lrp and FaeA to the fae regulatory region and is dependent on the methylation status of three GATC sites present in this region. In this paper, we describe the binding of Lrp to a T-rich DNA helix between GATC site I and site II. FaeA stabilized and modified the Lrp binding, thereby extending the Lrp footprint over GATC site I and site III. Methylation of GATC site I prevented the binding of Lrp/FaeA at this site and appeared to be essential for the cells, since mutation of this site into GTTC resulted in a lethal overproduction of K88 fimbriae. Methylation of GATC site II and site III reduced the stability of Lrp/FaeA binding. Moreover, methylation of GATC site III stimulated faeB promoter activity. The plasmid population in cells harbouring multiple copies of a K88 plasmid consisted of two differentially methylated forms. Form A plasmids with a methylated GATC site I and site III and a nonmethylated site II (+,-,+) represented 20% of the population and were responsible for high-level expression. Form B plasmids with a methylated GATC site I and a non-methylated site II and site III (+,-,-) represented 80% of the population and were responsible for low-level expression. Apparently, K88 fimbriae expression in vivo is balanced at its maximal possible level by modulation of the methylation status of GATC site III. The ratio (1:4) between these populations is stabilized by a constitutive synthesis of FaeA resulting from the presence of an IS1 insertion upstream of faeA. This IS1 insertion separates the faeA promoter from the FaeB-binding sites, thereby neutralizing the control by FaeB activity on expression of FaeA. Instead, faeA transcription is stimulated by binding of FaeA to the faeA promoter region.

The Escherichia coli operons daa and sfa encode F1845 and S pili, respectively. In this paper we show that the expression of these operons is under phase variation control at a transcriptional level. The transcription of both operons is dependent on the global regulator leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons. These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state. We also show that Lrp binds to the daa and sfa regulatory regions and that this binding is modulated by the methylation of the GATC sites. These results indicate that the phase variation of the daa and sfa operons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap operon.

Nucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted IS1 insertions. The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae. Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co-operative activity of FaeA and the leucine-responsive regulatory protein (Lrp). Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression. Mutations in faeB had no effect on the biosynthesis of K88 fimbriae. The presence of the two IS1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp. Like pap, the fae operon does not respond to exogenous leucine.

Lysyl-tRNA synthetases are synthesized from two distinct genes in Escherichia coli, lysS (constitutively) and lysU (inducibly); however, the physiological significance and the differential control mechanism of these two genes have been a long-standing puzzle. Recent studies have successfully uncovered a significant control mechanism of lysU expression, which involves the leucine-responsive regulatory protein (Lrp) and a translational enhancer element called 'downstream box'. Moreover, it is likely that there is a mechanism underlying co-ordinate expression of lysU with other genes outside the leucine-Lrp regulon under harsh conditions such as low pH and anaerobiosis. A possible mechanism of lysyl-tRNA synthetase expression and function is reviewed.

The transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea-associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papI genes of the P fimbrial adhesin, respectively. The 5' termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine-responsive regulatory protein (Lrp)-binding sites which contained differentially methylated GATC sequences, a cAMP-CRP-binding site, and an integration host factor (IHF)-binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvIH. Lrp binds to six sites upstream of ilvIH, and Lrp binding is required for ilvIH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvIH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvIH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.

Pyelonephritis-associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp and Papl bind to a specific non-methylated pap regulatory DNA region containing the sequence 'GATC' and facilitate the formation of an active transcriptional complex. Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam. However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited. These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states. The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved 'GATC-box' domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.