The leucine-responsive regulatory protein, Lrp, modulates microcin J25 intrinsic resistance in Escherichia coli by regulating expression of the YojI microcin exporter.
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ABSTRACT:
SUBMITTER: Socias SB
PROVIDER: S-EPMC2632010 | biostudies-other | 2009 Feb
REPOSITORIES: biostudies-other
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Project description:CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell.
| S-EPMC4135631 | biostudies-literature
Project description:Procedures were developed to facilitate the identification of genes that belong to a given regulon and characterization of their responses to the regulator. The regulon controlled by the Escherichia coli leucine-responsive regulatory protein (Lrp) was studied by isolating random transcriptional fusions to lacZ, using lambda placMu53 and a strain in which lrp is under isopropylthio-beta-D-galactopyranoside (IPTG)-inducible control. Fusions exhibiting IPTG-responsive beta-galactosidase activity were cloned by integrating the suicide vector pIVET1 via homologous recombination at lacZ, followed by self-ligating digested chromosomal DNA. We verified the patterns of lacZ expression after using the plasmid clones to generate merodiploid strains with interrupted and uninterrupted copies of the same sequence. If the merodiploid expression pattern was unchanged from that shown by the original fusion strain, then the cloned fusion was responsible for the regulatory pattern of interest; a difference in the expression pattern could indicate that the original strain carried multiple fusions or that there were autogenous effects of having interrupted the fused gene. Using these procedures, we generated a fusion library of approximately 5 x 10(6) strains; approximately 3,000 of these strains were screened, yielding 84 Lrp-responsive fusions, and 10 of the 84 were phenotypically stable and were characterized. The responses of different fusions in a given operon to in vivo Lrp titrations revealed variations in expression with the position of insertion. Among the newly identified members of the regulon is an open reading frame (orf3) between rpiA and serA. Also, expression of a fusion just downstream of dinF was found to be Lrp dependent only in stationary phase.
| S-EPMC179537 | biostudies-other
Project description:CS31A produced by septicemic and diarrheic Escherichia coli belongs to the Pap-regulatory family of adhesive factors, which are under methylation-dependent transcriptional regulation. Common features of operons encoding members of this family include two conserved GATC sites in the upstream regulatory region, and transcriptional regulators homologue to the PapB and PapI proteins. Methylation protection of GATC sites was previously shown to be dependent on the leucine-responsive regulatory protein (Lrp). Lrp and ClpB, the PapB equivalent, repressed clp basal transcription. A PapI homologue (AfaF) was required together with Lrp to establish the phase variation control, which gave rise to phase-ON cells that expressed CS31A and phase-OFF cells that did not express CS31A. In phase-OFF cells, the GATC(dist) site was methylated and the GATC(prox) site was protected from methylation, whereas in phase-ON cells, the inverse situation was found. Unlike Pap fimbriae, CS31A synthesis was dramatically reduced in media containing L-alanine or L-leucine. L-Alanine prevented the OFF-to-ON switch, locking clp expression in the OFF phase, whereas L-leucine repressed transcription without obvious effect on the switch frequency of phase variation. In phase-variable cells, leucine and alanine promoted methylation of GATC(dist) and methylation protection of GATC(prox), increasing the methylation pattern characteristic of repressed cells. Furthermore, alanine prevented the AfaF-dependent methylation protection of GATC(dist) and thus the appearance of phase-ON cells. In addition, analysis of clp expression in a Lrp-negative background indicated that alanine and leucine also repressed clp transcription by a methylation-independent mechanism.
| S-EPMC150119 | biostudies-literature
Project description:Broad-acting transcription factors (TFs) in bacteria form regulons. Here, we present a 4-step method to fully reconstruct the leucine-responsive protein (Lrp) regulon in Escherichia coli K-12 MG 1655 that regulates nitrogen metabolism. Step 1 is composed of obtaining high-resolution ChIP-chip data for Lrp, the RNA polymerase and expression profiles under multiple environmental conditions. We identified 138 unique and reproducible Lrp-binding regions and classified their binding state under different conditions. In the second step, the analysis of these data revealed 6 distinct regulatory modes for individual ORFs. In the third step, we used the functional assignment of the regulated ORFs to reconstruct 4 types of regulatory network motifs around the metabolites that are affected by the corresponding gene products. In the fourth step, we determined how leucine, as a signaling molecule, shifts the regulatory motifs for particular metabolites. The physiological structure that emerges shows the regulatory motifs for different amino acid fall into the traditional classification of amino acid families, thus elucidating the structure and physiological functions of the Lrp-regulon. The same procedure can be applied to other broad-acting TFs, opening the way to full bottom-up reconstruction of the transcriptional regulatory network in bacterial cells.
| S-EPMC2614783 | biostudies-literature
Project description:The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp. The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein. We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions. Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth. Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium. Lrp binds specifically to DNA fragments containing the gltBDF promoter region. Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM. Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine. On the basis of our present results, we present a model for positive regulation of target genes by Lrp. Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene. At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine. This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.
| S-EPMC206857 | biostudies-other
Project description:BackgroundBitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4″-O-isovaleryltransferase gene (ist). Exogenous L-Leucine (L-Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and L-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus.ResultsSSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23, bsm42 and acyB2, in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the ΔSSP_Lrp-SP strain containing an SSP_Lrp deficient of putative L-Leu binding domain was higher than that of S. spiramyceticus 1941 (476.2 ± 3.1 μg/L versus 313.3 ± 25.2 μg/L, respectively), especially SP III increased remarkably. The yield of BT in ΔSSP_Lrp-BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the ΔSSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the ΔSSP_Lrp-SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine.ConclusionSSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative L-Leu binding domain was advantageous for production of BT and SP, especially their III components.
| S-EPMC6379999 | biostudies-literature
Project description: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 pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 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 the pap and fan 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 K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.
| S-EPMC49059 | biostudies-other
Project description:The tdh promoter of Escherichia coli is induced seven- to eightfold when cells are grown in the presence of exogenous leucine. A scheme was devised to select mutants that exhibited high constitutive expression of the tdh promoter. The mutations in these strains were shown to lie within a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein). By deletion analysis, the site of action of Lrp was localized to a 25-bp region between coordinates -69 and -44 of the tdh promoter. Disruption of a 12-bp presumptive target sequence found in this region of tdh resulted in constitutively derepressed expression from the tdh promoter. Similar DNA segments (consensus, TTTATTCtNaAT) were also identified in a number of other promoters, including each of the Lrp-regulated promoters whose nucleotide sequence is known. The sequence of the promoter region of serA, an Lrp-regulated gene, was determined. No Lrp consensus target sequence was present upstream of serA, suggesting that Lrp acts indirectly on the serA promoter. A previously described mutation in a leucine-responsive trans-acting factor, LivR (J. J. Anderson, S. C. Quay, and D. L. Oxender, J. Bacteriol. 126:80-90, 1976), resulted in constitutively repressed expression from the tdh promoter and constitutively induced expression from the serA promoter. The possibility that LivR and Lrp are allelic is discussed.
| S-EPMC208338 | biostudies-other
Project description: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.
| S-EPMC2894021 | biostudies-literature
Project description:BackgroundLeucine supplementation might have therapeutic potential in preventing diet-induced obesity and improving insulin sensitivity. However, the underlying mechanisms are at present unclear. Additionally, it is unclear whether leucine supplementation might be equally efficacious once obesity has developed.Methodology/principal findingsMale C57BL/6J mice were fed chow or a high-fat diet (HFD), supplemented or not with leucine for 17 weeks. Another group of HFD-fed mice (HFD-pairfat group) was food restricted in order to reach an adiposity level comparable to that of HFD-Leu mice. Finally, a third group of mice was exposed to HFD for 12 weeks before being chronically supplemented with leucine. Leucine supplementation in HFD-fed mice decreased body weight and fat mass by increasing energy expenditure, fatty acid oxidation and locomotor activity in vivo. The decreased adiposity in HFD-Leu mice was associated with increased expression of uncoupling protein 3 (UCP-3) in the brown adipose tissue, better insulin sensitivity, increased intestinal gluconeogenesis and preservation of islets of Langerhans histomorphology and function. HFD-pairfat mice had a comparable improvement in insulin sensitivity, without changes in islets physiology or intestinal gluconeogenesis. Remarkably, both HFD-Leu and HFD-pairfat mice had decreased hepatic lipid content, which likely helped improve insulin sensitivity. In contrast, when leucine was supplemented to already obese animals, no changes in body weight, body composition or glucose metabolism were observed.Conclusions/significanceThese findings suggest that leucine improves insulin sensitivity in HFD-fed mice by primarily decreasing adiposity, rather than directly acting on peripheral target organs. However, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhans's physiology might help prevent type 2 diabetes development. Differently, metabolic benefit of leucine supplementation is lacking in already obese animals, a phenomenon possibly related to the extent of the obesity before starting the supplementation.
| S-EPMC3783457 | biostudies-literature