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Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi.

ABSTRACT: The RpoN-RpoS regulatory pathway plays a central role in governing adaptive changes by B. burgdorferi when the pathogen shuttles between its tick vector and mammalian hosts. In general, transcriptional activation of bacterial RpoN (?54)-dependent genes requires an enhancer binding protein. B. burgdorferi encodes the putative enhancer binding protein Rrp2. Previous studies have revealed that the expression of ?54-dependent rpoS was abolished in an rrp2 point mutant. However, direct evidence linking the production of Rrp2 in B. burgdorferi and the expression of rpoS has been lacking, primarily due to the inability to inactivate rrp2 via deletion or insertion mutagenesis. Herein we introduced a regulatable (IPTG-inducible) rrp2 expression shuttle plasmid into B. burgdorferi, and found that the controlled up-regulation of Rrp2 resulted in the induction of ?54-dependent rpoS expression. Moreover, we created an rrp2 conditional lethal mutant in virulent B. burgdorferi. By exploiting this conditional mutant, we were able to experimentally manipulate the temporal level of Rrp2 expression in B. burgdorferi, and examine its direct impact on activation of the RpoN-RpoS regulatory pathway. Our data revealed that the synthesis of RpoS was coincident with the IPTG-induced Rrp2 levels in B. burgdorferi. In addition, the synthesis of OspC, a lipoprotein required by B. burgdorferi to establish mammalian infection, was rescued in the rrp2 point mutant when RpoS production was restored, suggesting that Rrp2 influences ospC expression indirectly via its control over RpoS. These data demonstrate that Rrp2 is required for the synthesis of RpoS, presumably via its action as an enhancer binding protein for the activation of RpoN and subsequent transcription of rpoS in B. burgdorferi.

SUBMITTER: Ouyang Z

PROVIDER: S-EPMC4014564 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi.

Ouyang Zhiming Z Zhou Jianli J Norgard Michael V MV

PloS one 20140508 5

The RpoN-RpoS regulatory pathway plays a central role in governing adaptive changes by B. burgdorferi when the pathogen shuttles between its tick vector and mammalian hosts. In general, transcriptional activation of bacterial RpoN (σ54)-dependent genes requires an enhancer binding protein. B. burgdorferi encodes the putative enhancer binding protein Rrp2. Previous studies have revealed that the expression of σ54-dependent rpoS was abolished in an rrp2 point mutant. However, direct evidence linki ...[more]

PMID: 24810170

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Project description:The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, ostensibly is required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN, and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN, and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN, or rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS, therefore providing additional avenues for future research into the genetic determinants contributing to virulence expression by B. burgdorferi. Keywords: gene profiling 297 (wt) vs rpoS mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoN mutant: 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rrp2 mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 5A18 (wt) vs XY424.3 (rrp2 mutant): 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoS mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoN mutant: 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rrp2 mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 5A18 (wt) vs XY424.3 (rrp2 mutant): 5 slides, 10 hybridizations (including dyeswaps), three biological replicates

Project description:Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoS(Bb)) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoS(Bb) recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoS(Bb) dependent, while the ospE and flaB reporters were RpoS(Bb) independent. To compare promoter recognition by RpoS(Bb) with that of the prototype RpoS (RpoS(Ec)), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoS(Ec) the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoS(Bb) under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoS(Ec) and RpoS(Bb) each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(-14) in the presumptive -10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoS(Bb) as it is for RpoS(Ec); (ii) the nucleotide at the -15 position determines RpoS or sigma(70) selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoS(Ec)- or RpoS(Bb)-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.

Dataset Information

Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi.

Publications

Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi.

Similar Datasets

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