Identification and characterization of a second, inducible promoter of relA in Escherichia coli

Nakagawa, Akira; Oshima, Taku; Mori, Hirotada

doi:10.1266/ggs.81.299

Cited by 22 publications

(20 citation statements)

References 45 publications

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“…2A). Two promoters driving relA transcription (P1 from −178 nt; P2 from −626 nt) have been mapped (Metzger et al ., 1988; Nakagawa et al ., 2006). In contrast to the translational fusion, expression from a transcriptional fusion containing the upstream non‐coding region through the upstream transcriptional start of relA (−880 nt to −626 nt) was unaltered by the csrA mutation (Fig.…”

Section: Resultsmentioning

confidence: 99%

Circuitry linking the Csr and stringent response global regulatory systems

Edwards

Patterson-Fortin

Vakulskas

et al. 2011

Molecular Microbiology

166

306

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Summary CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR, and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

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Section: Resultsmentioning

confidence: 99%

Circuitry linking the Csr and stringent response global regulatory systems

Edwards

Patterson-Fortin

Vakulskas

et al. 2011

Molecular Microbiology

166

306

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“…The relA P2 promoter contains a cAMP receptor protein (CRP) binding site that is activated by CRP (Nakagawa et al , 2006) and the relA P2 (−100+50) reporter included this CRP site. The microarray data also indicated that crp mRNA levels were increased (1.9-fold) in cells lacking 6S RNA compared with wild-type cells (A. T. Cavanagh & K. M. Wassarman, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

“…In agreement with CRP activation at this promoter, the overall level of expression of the minimal relA P2 (−41+2) was decreased relative to relA P2 (−100+50) . hns has also been reported to activate transcription from relA P2 (Nakagawa et al , 2006). However, we observed a decrease in hns mRNA levels in cells lacking 6S RNA compared with wild-type cells (A. T. Cavanagh & K. M. Wassarman, unpublished data), suggesting that a 6S RNA-dependent decrease in hns cannot account for the observed 6S RNA-dependent increase in relA expression.…”

Section: Resultsmentioning

confidence: 99%

6S RNA regulation of relA alters ppGpp levels in early stationary phase

2010

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6S RNA is a small, non-coding RNA that interacts directly with σ70-RNA polymerase and regulates transcription at many σ70-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from σ70-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of σS activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions.

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“…cAMP, whose physiological level is associated with nutrient availability, increases cspD transcription in complex with its receptor protein Crp (Uppal et al, 2014 ). Moreover, the cAMP-Crp complex also activates the expression of relA , resulting in a further increase of the intracellular ppGpp level (Nakagawa et al, 2006 ). This example illustrates how the metabolic state of a cell can be coupled to persister formation via different pathways to achieve a subtle and precise regulation.…”

Section: Genes Linking Metabolism and The Persister Statementioning

confidence: 99%

Metabolic aspects of bacterial persisters

Prax

Bertram

2014

Front. Cell. Infect. Microbiol.

105

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Persister cells form a multi-drug tolerant subpopulation within an isogenic culture of bacteria that are genetically susceptible to antibiotics. Studies with different Gram negative and Gram positive bacteria have identified a large number of genes associated with the persister state. In contrast, the revelation of persister metabolism has only been addressed recently. We here summarize metabolic aspects of persisters, which includes an overview about the bifunctional role of selected carbohydrates as both triggers for the exit from the drug tolerant state and metabolites which persisters feed on. Also alarmones as indicators for starvation have been shown to influence persister levels via different signaling cascades involving the activation of toxin-antitoxin systems and other regulatory factors. Finally, recent data obtained by 13C-isotopolog profiling demonstrated an active amino acid anabolism in Staphylococcus aureus cultures challenged with high drug concentrations. Understanding the metabolism of persister cells poses challenges but also paves the way for the development of anti-persister compounds.

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Identification and characterization of a second, inducible promoter of relA in Escherichia coli

Cited by 22 publications

References 45 publications

Circuitry linking the Csr and stringent response global regulatory systems

Circuitry linking the Csr and stringent response global regulatory systems

6S RNA regulation of relA alters ppGpp levels in early stationary phase

Metabolic aspects of bacterial persisters

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