Lrp acts as both a positive and negative regulator for type 1 fimbriae production in Salmonella enterica serovar Typhimurium.

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrp(C)) mutant strains. In this work, we used chromosomal P(fimA)-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to P(fimA) was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in P(fimA) play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to P(fimA) without competitive exclusion. In addition, both Lrp and FimZ binding to P(fimA) caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1.

BACKGROUND: Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. RESULTS: It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. CONCLUSION: The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.

Competitive activation of the Escherichia coli argO gene coding for an arginine exporter by the transcriptional regulators Lrp and ArgP.

In vivo and in vitro analyses indicate that transcription of the argO gene coding for an arginine exporter is regulated by the global transcriptional regulator Lrp, an effect that went by unnoticed in previous genome-scale screenings of the Lrp regulatory network in Escherichia coli. Lrp activates the argO promoter fourfold; exogenous leucine antagonizes, but does not completely eliminate this effect. Activation by Lrp interferes with the previously demonstrated activation of the argO promoter by ArgP. This interference results from the mutual inhibitory binding of the two activators to overlapping targets. As a consequence, each regulator acts more potently in the absence of the other. Dimeric Lrp binds cooperatively to at least three regularly spaced semi-palindromic binding sites. Leucine reduces complex formation approximately twofold but concomitantly enhances the cooperativity of the binding. Footprinting data suggest a severe Lrp-induced deformation of the argO control region. Combined, the effector modulated activation of argO transcription by ArgP and Lrp must ensure an adapted and fine-tuned synthesis of the transporter in response to environmental conditions. The repertoire of bacterial transcription regulation mechanisms is vast, but the competitive activation of a single promoter by two activator proteins as described here appears to be rare.

Studies on the expression of 6S RNA from E. coli: involvement of regulators important for stress and growth adaptation.

The small bacterial 6S RNA has been recognized as a transcriptional regulator, facilitating the transition from exponential to stationary growth phase by preferentially inhibiting E sigma 70 RNA polymerase holoenzyme transcription. Consistent with this function, the cellular concentration of 6S RNA increases with stationary phase. We have studied the underlying mechanisms responsible for the growth phase-dependent differences in 6S RNA concentration. To this aim, we have analyzed the effects of the typical bacterial growth phase and stress regulators FIS, H-NS, LRP and StpA on 6S RNA expression. Measurements of 6S RNA accumulation in strains deficient in each one of these proteins support their contribution as potential regulators. Specific binding of the four proteins to DNA fragments containing 6S RNA promoters was demonstrated by gel retardation and DNase I footprinting. Moreover, in vitro transcription analysis with both RNA polymerase holoenzymes, E sigma 70 and E sigma 38, demonstrated a direct inhibition of 6S RNA transcription by H-NS, StpA and LRP, while FIS seems to act as a dual regulator. In vitro transcription in the presence of ppGpp indicates that 6S RNA promoters are not stringently regulated. Our results underline that regulation of 6S RNA transcription depends on a complex network, involving a set of bacterial regulators with general importance in the adaptation to changing growth conditions.

The leucine-responsive regulatory protein, Lrp, activates transcription of the fim operon in Salmonella enterica serovar typhimurium via the fimZ regulatory gene.

The fim operon of Salmonella enterica serovar Typhimurium encodes type 1 fimbriae. The expression of fim is controlled in response to environmental signals through a complex regulatory cascade involving the proteins FimW, FimY, and FimZ and a genetic locus, fimU, that encodes a rare arginine tRNA. We discovered that a knockout mutation in lrp, the gene that codes for the leucine-responsive regulatory protein (Lrp), inhibited fim transcription. The loss of fim gene expression was accompanied by a corresponding loss of the mannose-sensitive hemagglutination that is a characteristic of type 1 fimbriae. Normal type 1 fimbrial expression was restored following the introduction into the knockout mutant of a plasmid carrying a functional copy of the lrp gene. Electrophoretic mobility shift analysis revealed no interactions between purified Lrp protein and the regulatory region of the fimA, fimU, or fimW gene. Instead, Lrp produced protein-DNA complexes with the regulatory region of the fimZ gene, and the nature of these complexes was leucine sensitive. DNase I footprinting showed that Lrp binds within a region between -65 and -170 with respect to the fimZ transcription start site, consistent with the binding and wrapping of the DNA in this upstream region. Ectopic expression of the fimZ gene from an inducible promoter caused Lrp-independent type 1 fimbriation in serovar Typhimurium. These data show that Lrp makes a positive contribution to fim gene expression through direct interaction with the fimZ promoter region, possibly by antagonizing the binding of the H-NS global repressor protein.

The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila.

Xenorhabdus nematophila is a Gram-negative bacterium that leads both pathogenic and mutualistic lifestyles. In this study, we examine the role of Lrp, the leucine-responsive regulatory protein, in regulating both of these lifestyles. lrp mutants have attenuated virulence towards Manduca sexta insects and are defective in suppression of both cellular and humoral insect immunity. In addition, an lrp mutant is deficient in initiating colonization of and growth within mutualistic host nematodes. Furthermore, nematodes reared on lrp mutant lawns exhibit decreased overall numbers of nematode progeny. To our knowledge, this is the first demonstration of virulence attenuation associated with an lrp mutation in any bacterium, as well as the first report of a factor involved in both X. nematophila symbioses. Protein profiles of wild-type and mutant cells indicate that Lrp is a global regulator of expression in X. nematophila, affecting approximately 65% of 290 proteins. We show that Lrp binds to the promoter regions of genes known to be involved in basic metabolism, mutualism and pathogenesis, demonstrating that the regulation of at least some host interaction factors is likely direct. Finally, we demonstrate that Lrp influences aspects of X. nematophila phenotypic variation, a spontaneous process that occurs during prolonged growth in stationary phase.

[Export of metabolites by the proteins of the DMT and RhtB families and its possible role in intercellular communication].

The earlier published and new experimental data are summarized on the properties of the genes encoding the membrane proteins of the DMT family (RhtA (YbiF), EamA (YdeD), YijE, YddG, YedA, PecM, eukaryotic nucleoside phosphate sugar and hexose phosphate transporters), the RhtB/LysE family (RhtB, RhtC, LeuE, YahN, EamB (YfiK), ArgO (YggA), CmaU), as well as some other families (YicM, YdhC, YdeAB, YdhE (NorE)). These proteins are involved in the export of amino acids, purines, and other metabolites from the cell. The expression of most of the genes encoding these proteins is not induced by the substrates they transport but is controlled by the global regulation systems, such as the Lrp protein, and activated by the signal compounds involved in the intracellular communication. The level of expression, assessed in experiments on translational fusion of the corresponding bacterial genes with the beta-galactosidase gene, depends on the growth phase of the bacterial culture, composition of the medium, and some stress factors, such as pH osmolarity or decreased aeration. The efflux of normal cell metabolites is assumed to be the natural function of these proteins. This function may play a role in density-dependent behavior of cell populations (quorum sensing). It may have been enhanced in the course of evolution via specialization of these proteins in the efflux of compounds derived from metabolic intermediates and adjusted to the role of transmitters.