A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator.
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ABSTRACT:
SUBMITTER: Hay NA
PROVIDER: S-EPMC179319 | biostudies-other | 1997 Aug
REPOSITORIES: biostudies-other
Publications
A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator.
Hay N A NA Tipper D J DJ Gygi D D Hughes C C
Journal of bacteriology 19970801 15
Proteus swarming is the rapid cyclical population migration across surfaces by elongated cells that hyperexpress flagellar and virulence genes. The mini-Tn5 transposon mutant mns2 was isolated as a tight nonswarming mutant that did not elongate or upregulate flagellar and hemolysin genes. Individual cell motility was retained but was reduced. The transposon had inserted in the gene encoding the global transcriptional regulator Lrp (leucine-responsive regulatory protein), expression of which was ...[more]
Similar Datasets
Project description:The Lrp/AsnC family of transcription factors links gene regulation to metabolism in bacteria and archaea. Members of this family, collectively, respond to a wide range of amino acids as coregulators. In Escherichia coli, Lrp regulates over 200 genes directly and is well known to respond to leucine and, to a somewhat lesser extent, alanine. We focused on Lrp from Proteus mirabilis and E. coli, orthologs with 98% identity overall and identical helix-turn-helix motifs, for which a previous study nevertheless found functional differences. Sequence differences between these orthologs, within and adjacent to the amino acid-responsive RAM domain, led us to test for differential sensitivity to coregulatory amino acids. In the course of this investigation, we found, via in vivo reporter fusion assays and in vitro electrophoretic mobility shift experiments, that E. coli Lrp itself responded to a broader range of amino acids than was previously appreciated. In particular, for both the E. coli and P. mirabilis orthologs, Lrp responsiveness to methionine was similar in magnitude to that to leucine. Both Lrp orthologs are also fairly sensitive to Ile, His, and Thr. These observations suggest that Lrp ties gene expression in the Enterobacteriaceae rather extensively to physiological status, as reflected in amino acid pools. These findings also have substantial implications for attempts to model regulatory architecture from transcriptome measurements or to infer such architecture from genome sequences, and they suggest that even well-studied regulators deserve ongoing exploration.
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Project description:BackgroundBacterial genome sequences are being determined rapidly, but few species are physiologically well characterized. Predicting regulation from genome sequences usually involves extrapolation from better-studied bacteria, using the hypothesis that a conserved regulator, conserved target gene, and predicted regulator-binding site in the target promoter imply conserved regulation between the two species. However many compared organisms are ecologically and physiologically diverse, and the limits of extrapolation have not been well tested. In E. coli K-12 the leucine-responsive regulatory protein (Lrp) affects expression of approximately 400 genes. Proteus mirabilis and Vibrio cholerae have highly-conserved lrp orthologs (98% and 92% identity to E. coli lrp). The functional equivalence of Lrp from these related species was assessed.ResultsHeterologous Lrp regulated gltB, livK and lrp transcriptional fusions in an E. coli background in the same general way as the native Lrp, though with significant differences in extent. Microarray analysis of these strains revealed that the heterologous Lrp proteins significantly influence only about half of the genes affected by native Lrp. In P. mirabilis, heterologous Lrp restored swarming, though with some pattern differences. P. mirabilis produced substantially more Lrp than E. coli or V. cholerae under some conditions. Lrp regulation of target gene orthologs differed among the three native hosts. Strikingly, while Lrp negatively regulates its own gene in E. coli, and was shown to do so even more strongly in P. mirabilis, Lrp appears to activate its own gene in V. cholerae.ConclusionThe overall similarity of regulatory effects of the Lrp orthologs supports the use of extrapolation between related strains for general purposes. However this study also revealed intrinsic differences even between orthologous regulators sharing >90% overall identity, and 100% identity for the DNA-binding helix-turn-helix motif, as well as differences in the amounts of those regulators. These results suggest that predicting regulation of specific target genes based on genome sequence comparisons alone should be done on a conservative basis.
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Project description:Proteus mirabilis urease catalyzes the hydrolysis of urea, initiating the formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Purified whole-cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences was probed with a 4.2-kb BglI fragment from within the urease operon. Autoradiographs of slot blots demonstrated 4.2- and 5.8-fold increases, respectively, in urease-specific RNA upon induction with urea. Structural and accessory genes necessary for urease activity, ureD, A, B, C, E, and F, were previously cloned and sequenced (B. D. Jones and H. L. T. Mobley, J. Bacteriol. 171:6414-6422, 1989). A 1.2-kb EcoRV-BamHI restriction fragment upstream of these sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame of 882 nucleotides, designated ureR, which is transcribed in the direction opposite that of the urease structural and accessory genes and encodes a 293-amino-acid polypeptide predicted to be 33,415 Da in size. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels of [35S]methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band with an apparent molecular size of 32 kDa. Fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence contains a potential helix-turn-helix motif and an associated AraC family signature and is similar to that predicted for a number of DNA-binding proteins, including E. coli proteins that regulate acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). These data suggest that UreR governs the inducibility of P. mirabilis urease.
| S-EPMC196161 | biostudies-other
Project description:Proteus mirabilis contributes to a significant number of catheter-associated urinary tract infections, where coordinated regulation of adherence and motility is critical for ascending disease progression. Previously, the mannose-resistant Proteus-like (MR/P) fimbria-associated transcriptional regulator MrpJ has been shown to both repress motility and directly induce the transcription of its own operon; in addition, it affects the expression of a wide range of cellular processes. Interestingly, 14 additional mrpJ paralogs are included in the P. mirabilis genome. Looking at a selection of MrpJ paralogs, we discovered that these proteins, which consistently repress motility, also have nonidentical functions that include cross-regulation of fimbrial operons. A subset of paralogs, including AtfJ (encoded by the ambient temperature fimbrial operon), Fim8J, and MrpJ, are capable of autoinduction. We identified an element of the atf promoter extending from 487 to 655 nucleotides upstream of the transcriptional start site that is responsive to AtfJ, and we found that AtfJ directly binds this fragment. Mutational analysis of AtfJ revealed that its two identified functions, autoregulation and motility repression, are not invariably linked. Residues within the DNA-binding helix-turn-helix domain are required for motility repression but not necessarily autoregulation. Likewise, the C-terminal domain is dispensable for motility repression but is essential for autoregulation. Supported by a three-dimensional (3D) structural model, we hypothesize that the C-terminal domain confers unique regulatory capacities on the AtfJ family of regulators.Balancing adherence with motility is essential for uropathogens to successfully establish a foothold in their host. Proteus mirabilis uses a fimbria-associated transcriptional regulator to switch between these antagonistic processes by increasing fimbrial adherence while simultaneously downregulating flagella. The discovery of multiple related proteins, many of which also function as motility repressors, encoded in the P. mirabilis genome has raised considerable interest as to their functionality and potential redundancy in this organism. This study provides an important advance in this field by elucidating the nonidentical effects of these paralogs on a molecular level. Our mechanistic studies of one member of this group, AtfJ, shed light on how these differing functions may be conferred despite the limited sequence variety exhibited by the paralogous proteins.
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Project description:This series of microarrays compares gene expression by the bacterial pathogen Proteus mirabilis when the transcriptional regulator mrpJ is deleted or induced to levels found during experimental urinary tract infection. The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections. Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for successful disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects a wide array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking expression levels that occur during UTI leads to differential expression of 217 genes related to, among others, bacterial virulence, type VI secretion and metabolism. We probed the molecular mechanism of transcriptional regulation through MrpJ using reporter assays and chromatin immunoprecipitation (ChIP). Two virulence-associated target genes, the flagellar master regulator flhDC and mrp itself, appear to be regulated through a binding site proximal to the transcriptional start, complemented by a more distantly situated enhancer site. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observe that mrpJ is widely conserved in a collection of recent clinical isolates, leading us to conclude that our results elucidate an unanticipated role of MrpJ as a global regulator of P. mirabilis virulence.
2015-04-13 | GSE63321 | GEO
Project description:This series of microarrays compares gene expression by the bacterial pathogen Proteus mirabilis when the transcriptional regulator mrpJ is deleted or induced to levels found during experimental urinary tract infection. The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections. Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for successful disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects a wide array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking expression levels that occur during UTI leads to differential expression of 217 genes related to, among others, bacterial virulence, type VI secretion and metabolism. We probed the molecular mechanism of transcriptional regulation through MrpJ using reporter assays and chromatin immunoprecipitation (ChIP). Two virulence-associated target genes, the flagellar master regulator flhDC and mrp itself, appear to be regulated through a binding site proximal to the transcriptional start, complemented by a more distantly situated enhancer site. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observe that mrpJ is widely conserved in a collection of recent clinical isolates, leading us to conclude that our results elucidate an unanticipated role of MrpJ as a global regulator of P. mirabilis virulence. Four biological replicates were analyzed for each set of arrays (P. mirabilis HI4320 wild type vs. ΔmrpJ, and vector pLX3607 vs. mrpJ plasmid pLX3805).
2015-04-13 | E-GEOD-63321 | biostudies-arrayexpress
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2018-08-10 | GSE96042 | GEO
Project description:We report a comparison of the transcriptional profiles between a wild-type P. mirabilis strain and an isogenic rcsB mutant
2015-12-25 | GSE76341 | GEO
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| S-EPMC8229911 | biostudies-literature
Project description:Swarming motility by the urinary tract pathogen Proteus mirabilis has been a long-studied but little understood phenomenon. On agar, a P. mirabilis colony grows outward in a bull's-eye pattern formed by consecutive waves of rapid swarming followed by consolidation into shorter cells. To examine differential gene expression in these growth phases, a microarray was constructed based on the completed genome sequence and annotation. RNA was extracted from broth-cultured, swarming, and consolidation-phase cells to assess transcription during each of these growth states. A total of 587 genes were differentially expressed in broth-cultured cells versus swarming cells, and 527 genes were differentially expressed in broth-cultured cells versus consolidation-phase cells (consolidate). Flagellar genes were highly upregulated in both swarming cells and consolidation-phase cells. Fimbriae were downregulated in swarming cells, while genes involved in cell division and anaerobic growth were upregulated in broth-cultured cells. Direct comparison of swarming cells to consolidation-phase cells found that 541 genes were upregulated in consolidate, but only nine genes were upregulated in swarm cells. Genes involved in flagellar biosynthesis, oligopeptide transport, amino acid import and metabolism, cell division, and phage were upregulated in consolidate. Mutation of dppA, oppB, and cysJ, upregulated during consolidation compared to during swarming, revealed that although these genes play a minor role in swarming, dppA and cysJ are required during ascending urinary tract infection. Swarming on agar to which chloramphenicol had been added suggested that protein synthesis is not required for swarming. These data suggest that the consolidation phase is a state in which P. mirabilis prepares for the next wave of swarming.
| S-EPMC2876570 | biostudies-literature