Rrp2, a sigma54-dependent transcriptional activator of Borrelia burgdorferi, activates rpoS in an enhancer-independent manner.
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ABSTRACT:
SUBMITTER: Blevins JS
PROVIDER: S-EPMC2668385 | biostudies-literature | 2009 Apr
REPOSITORIES: biostudies-literature
Publications
Rrp2, a sigma54-dependent transcriptional activator of Borrelia burgdorferi, activates rpoS in an enhancer-independent manner.
Blevins Jon S JS Xu Haijun H He Ming M Norgard Michael V MV Reitzer Larry L Yang X Frank XF
Journal of bacteriology 20090206 8
Rrp2 is the sole sigma(54)-dependent transcriptional activator present in the Borrelia burgdorferi genome. We showed that recombinant Rrp2 binds to DNA in a sequence-nonspecific manner. During infection, Rrp2 activates sigma(54)-dependent rpoS expression without an apparent upstream enhancer element commonly associated with other sigma(54)-dependent transcriptional activators. ...[more]
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Project description: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 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 σ54-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.
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Project description:Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the ?(54)-?(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl?P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl?P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl?P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl?P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl?P can serve as a global signal in bacterial pathogenesis.
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2008-09-09 | GSE11284 | GEO
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2008-09-09 | E-GEOD-11284 | biostudies-arrayexpress
Project description:Alteration of surface lipoprotein profiles is a key strategy that the Lyme disease pathogen, Borrelia burgdorferi, has evolved to be maintained within its enzootic cycle between arthropods and mammals. Accumulated evidence indicates that the central regulatory pathway controlling differential gene expression by B. burgdorferi is the RpoN-RpoS pathway (the sigma(54)-sigma(S) sigma factor cascade). It was previously shown that activation of the RpoN-RpoS pathway is controlled by Rrp2, a two-component response regulator and sigma(54)-dependent transcriptional activator. The role of Rrp2 in the infectious cycle of B. burgdorferi has not been determined heretofore. In this report, we demonstrate that an rrp2 mutant defective in activating sigma(54)-dependent transcription was unable to establish infection in mice, but the rrp2 mutant was capable of surviving within ticks during and after tick feeding. Because the rrp2 mutant was defective in the production of OspC, an outer surface lipoprotein essential for mammalian host infection, we further examined whether the loss of infectivity of the rrp2 mutant was solely due to the inability to produce OspC. While transformation with a shuttle vector carrying ospC under the control of a constitutive flaB promoter restored infection to an ospC mutant in immunodeficient SCID mice, it could not rescue the avirulent phenotype of the rrp2 mutant. These data indicate that, in addition to controlling OspC, Rrp2 controls another factor(s) essential for B. burgdorferi to establish infection in mammals. Furthermore, microarray analyses revealed that 125 and 19 genes were positively and negatively regulated, respectively, by Rrp2, which provides a foundation for future identification of additional Rrp2-dependent virulence determinants in B. burgdorferi.
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Project description:Rrp2 encodes a putative bacterial enhancer binding protein (bEBP) in Borrelia burgdorferi. Point mutation (G239C) of Rrp2 abolishes the transcriptional activation of σ54 -dependent rpoS. In contrast to canonical bEBPs that are dispensable for bacterial growth, Rrp2 is essential for borrelial growth in BSK medium. It has been believed that Rrp2's ATPase activity is not required for cell growth, but experimental evidence supporting this notion has been lacking. In particular, it has remained unclear whether the residue G239 is involved in Rrp2's presumptive ATPase activity. To address these information gaps, we examined the roles of Rrp2's potential strategic signatures including the G239 residue and the putative Walker A and Walker B motifs. Herein it was showed that Rrp2 has ATP binding and hydrolysis activities engendered by the Walker A and B motifs respectively. However, these activities were not significantly impaired by a G239C mutation. Further mutagenesis analyses indicated that Rrp2's Walker A and B motifs are required for borrelial growth; mutations of key residues in these two motifs were lethal to B. burgdorferi. The combined data suggest that the Walker A and Walker B motifs of Rrp2 are involved in the control of another unknown RpoS-independent gene product(s) associated with borrelial replication.
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Project description:Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment using a small repertoire of transcription factors that coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA, along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp), coordinate the stringent response to various environmental stresses, including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and its role in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp. Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but this relationship is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicate DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses in B. burgdorferi required for infectivity through DksA's interactions with RNA polymerase and post-transcriptional control of RpoS.
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