Dataset Information

CsrA (BB0184) is not involved in activation of the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

ABSTRACT: Borrelia burgdorferi encodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes to B. burgdorferi infectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA in B. burgdorferi gene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulate rpoS expression and, thus, how CsrA may influence the RpoN-RpoS pathway in B. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an inducible csrA expression system in B. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels of bosR, rpoS, and RpoS-dependent genes (such as ospC and dbpA). In addition, we constructed isogenic csrA mutants in two widely used infectious B. burgdorferi strains. When expression of bosR, rpoS, ospC, and dbpA was compared between the csrA mutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that the csrA mutants remained infectious for and virulent in mice. Analyses of B. burgdorferi gene expression in mouse tissues showed comparable levels of rpoS transcripts by the csrA mutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out by B. burgdorferi.

SUBMITTER: Ouyang Z

PROVIDER: S-EPMC3993398 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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CsrA (BB0184) is not involved in activation of the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Ouyang Zhiming Z Zhou Jianli J Norgard Michael V MV

Infection and immunity 20140122 4

Borrelia burgdorferi encodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes to B. burgdorferi infectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA in B. burgdorferi gene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulate rpoS expression and, thus, how CsrA m ...[more]

PMID: 24452681

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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

CsrA (BB0184) is not involved in activation of the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Publications

CsrA (BB0184) is not involved in activation of the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Similar Datasets

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