Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Belcheva, A.; Verma, Vidhu; Korenevsky, Artyom; Fridman, Michael D.; Kumar, Krishan; Golemi-Kotra, Dasantila

doi:10.1128/jb.06143-11

Cited by 20 publications

(27 citation statements)

References 35 publications

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“…Briefly explained, given our knowledge of the HK/RR paradigm (41), our data, and data from other published studies, cell wall damage first imparts a modification of VraT which in turn influences autophosphorylation of VraS, ultimately leading to the phosphorylation of VraR. VraR then activates or represses genes through binding to target promoters (23,24,42). Recent data demonstrating that portions of VraS and VraT interact in vivo support this idea (28).…”

Section: Discussionmentioning

confidence: 78%

“…VraS responds to antimicrobial-elicited cell wall stress by autophosphorylation and subsequent transphosphorylation of VraR, its cognate RR (23). Phosphorylated VraR binds to its own promoter and presumably to genes in the vra stimulon to activate or repress their transcription (24). It is known that cell wall stress is required, but the specific molecular signal responsible for induction of activation of VraS and VraR is still unknown.…”

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confidence: 99%

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VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

Boyle-Vavra

Yin

et al. 2013

Antimicrob Agents Chemother

100

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Staphylococcus aureus infections caused by strains that are resistant to all forms of penicillin, so-called methicillin-resistant S. aureus (MRSA) strains, have become common. One strategy to counter MRSA infections is to use compounds that resensitize MRSA to methicillin. S. aureus responds to diverse classes of cell wall-inhibitory antibiotics, like methicillin, using the two-component regulatory system VraSR (vra) to up-or downregulate a set of genes (the cell wall stimulon) that presumably facilitates resistance to these antibiotics. Accordingly, VraS and VraR mutations decrease resistance to methicillin, vancomycin, and daptomycin cell wall antimicrobials. vraS and vraR are encoded together on a transcript downstream of two other genes, which we call vraU and vraT (previously called yvqF). By producing nonpolar deletions in vraU and vraT in a USA300 MRSA clinical isolate, we demonstrate that vraT is essential for optimal expression of methicillin resistance in vitro, whereas vraU is not required for this phenotype. The deletion of vraT also improved the outcomes of oxacillin therapy in mouse models of lung and skin infection. Since vraT expressed in trans did not complement a vra operon deletion, we conclude that VraT does not inactivate the antimicrobial. Genome-wide transcriptional microarray experiments reveal that VraT facilitates resistance by playing a necessary regulatory role in the VraSR-mediated cell wall stimulon. Our data prove that VraTSR comprise a novel three-component regulatory system required to facilitate resistance to cell wall agents in S. aureus. We also provide the first in vivo proof of principle for using VraT as a sole target to resensitize MRSA to ␤-lactams.

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Section: Discussionmentioning

confidence: 78%

mentioning

confidence: 99%

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

Boyle-Vavra

Yin

et al. 2013

Antimicrob Agents Chemother

100

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“…Both S. aureus σ A and E . coli σ 70 RNA polymerase subunits recognize canonical promoters and are interchangeable (49). It has already been reported that srn_9340 transcription depends on σ A (46).…”

Section: Resultsmentioning

confidence: 99%

Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs inStaphylococcus aureus

Mauro¹,

Rouillon²,

Felden³

2016

Nucleic Acids Res

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The opportunistic pathogen Staphylococcus aureus expresses transcription factors (TFs) and regulatory small RNAs (sRNAs) which are essential for bacterial adaptation and infectivity. Until recently, the study of S. aureus sRNA gene expression regulation was under investigated, but it is now an expanding field. Here we address the regulation of Srn_3610_SprC sRNA, an attenuator of S. aureus virulence. We demonstrate that SarA TF represses srn_3610_sprC transcription. DNase I footprinting and deletion analyses show that the SarA binding site on srn_3610_sprC belongs to an essential 22 bp DNA region. Comparative analysis also revealed another possible site, this time in the srn_9340 promoter. SarA specifically binds these two sRNA promoters with high affinity in vitro and also represses their transcription in vivo. Chromatin immunoprecipitation (ChIP) assays confirmed SarA attachment to both promoters. ChIP and electrophoretic mobility shift assays targeting σA RNA polymerase subunit or using bacterial RNA polymerase holoenzyme suggested that SarA and the σA bind srn_3610_sprC and srn_9340 promoters in a mutually exclusive way. Beyond the mechanistic study of SarA repression of these two sRNAs, this work also suggests that some S. aureus sRNAs belong to the same regulon and act jointly in responding to environmental changes.

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“…It is puzzling however that Efm LiaR W73C does not actually confer the same extent of dimerization as BeF 3 − stimulated wild type Efm LiaR (wild type Efm LiaR dimerization K d was 15 μM compared to 236 μM for Efm LiaR W73C ). Since in vivo concentrations of LiaR are estimated to be in the low micromolar range [37], the weak in vitro dimerization of Efm LiaR W73C is less than expected for constitutive hyperactivation. Alternatively, the Efm LiaR W73C mutant may interact more efficiently with its cognate kinase LiaS to produce a higher extent of phosphorylation than the comparable wild type LiaR interaction with LiaS.…”

Section: Discussionmentioning

confidence: 99%

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State

Davlieva

Tovar-Yanez

DeBruler

et al. 2016

Journal of Molecular Biology

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The cyclic antimicrobial lipopeptide daptomycin triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, nonsusceptibility to daptomycin by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated Enterococcus faecium LiaR protein at 3.2 Å resolution and, in combination with solution studies, show that activation of LiaR induces formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to daptomycin by upregulation of the LiaFSR regulon. A comparison of the Enterococcus faecalis LiaR, Enterococcus faecium LiaR and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with daptomycin resistance suggest that physicochemical properties such as oligomerization state and DNA specificity, though tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.

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Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Cited by 20 publications

References 35 publications

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs inStaphylococcus aureus

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State

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