An Excretory Function for theEscherichia coliOuter Membrane Pore TolC: Upregulation ofmarAandsoxSTranscription and Rob Activity Due to Metabolites Accumulated intolCMutants

Rosner, Judah L.; Martin, Robert G.

doi:10.1128/jb.00507-09

Cited by 55 publications

(71 citation statements)

References 62 publications

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“…Although this is the initial report of direct regulation at the posttranscriptional level, tolC is known to have complex transcriptional regulation involving four promoters and regulation of some promoters by MarA, SoxS, and Rob, increasing tolC transcription in response to some substrates (12,46). Our results suggest that transcripts from all of these promoters should be subject to regulation by SdsR.…”

Section: Discussionmentioning

confidence: 74%

“…The function of ygiBC-encoded proteins is unknown, but at least one phenotype of a tolC mutant, impaired growth on glucose minimal medium, was exacerbated by the deletion of ygiBC and their homologs, yjfM and yjfC (9,10). Both the acrAB and tolC promoters are members of the marA, soxS, and rob transcriptional regulon and are upregulated during superoxide stress and exposure to heavy metals, bile salts, and antibiotics (11,12), consistent with the role of the efflux pump in exporting these molecules from the cell.…”

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confidence: 99%

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Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

Parker

Gottesman

2016

J Bacteriol

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Bacteria use multidrug efflux pumps to export drugs and toxic compounds out of the cell. One of the most important efflux pumps in Escherichia coli is the AcrAB-TolC system. Small regulatory RNAs (sRNAs) are known to be major posttranscriptional regulators that can enhance or repress translation by binding to the 5= untranslated region (UTR) of mRNA targets with the help of a chaperone protein, Hfq. In this study, we investigated the expression of acrA, acrB, and tolC translational fusions using 27 Hfq-dependent sRNAs overexpressed from plasmids. No significant sRNA regulation of acrA or acrB was detected. SdsR (also known as RyeB), an abundant and well-conserved stationary-phase sRNA, was found to repress the expression of tolC, the gene encoding the outer membrane protein of many multidrug resistance efflux pumps. This repression was shown to be by direct base pairing occurring upstream from the ribosomal binding site. SdsR overexpression and its regulation of tolC were found to reduce resistance to novobiocin and crystal violet. Our results suggest that additional targets for SdsR exist that contribute to increased antibiotic sensitivity and reduced biofilm formation. In an effort to identify phenotypes associated with single-copy SdsR and its regulation of tolC, the effect of a deletion of sdsR or mutations in tolC that should block SdsR pairing were investigated using a Biolog phenotypic microarray. However, no significant phenotypes were identified. Therefore, SdsR appears to modulate rather than act as a major regulator of its targets. IMPORTANCEAcrAB-TolC is a major efflux pump present in E. coli and Gram-negative bacteria used to export toxic compounds; the pump confers resistance to many antibiotics of unrelated classes. In this study, we found that SdsR, a small RNA expressed in stationary phase, repressed the expression of tolC, resulting in increased sensitivity to some antibiotics. This extends the findings of previous studies showing that sRNAs contribute to the regulation of many outer membrane proteins; manipulating or enhancing their action might help in sensitizing bacteria to antibiotics.

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Section: Discussionmentioning

confidence: 74%

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confidence: 99%

Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

Parker

Gottesman

2016

J Bacteriol

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“…In the outer membrane, TolC represents a pore that is tightly coupled to several export systems localized in the cytoplasmic membrane. TolC is known to be implicated in the export of xenobiotics and to serve as an emergency valve for toxic metabolites (14). We demonstrate here that TolC is also coupled to the export of intracellular cAMP.…”

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confidence: 58%

Escherichia coli Exports Cyclic AMP via TolC

2011

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In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-␤-Dthiogalactopyranoside) was sufficient to induce ␤-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce ␤-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.In Escherichia coli the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex affects the expression of more than 180 genes (16,18). Hence, the intracellular levels of cAMP need to be tightly controlled (5). Recently, a critical review of published data concluded that the rate of cAMP biosynthesis in E. coli is determined mainly by the cellular energy charge (13). At low ATP levels cAMP formation is enhanced. cAMP-CRP then stimulates transcription of catabolic enzymes and inhibits transcription of anabolic enzymes to ensure that the energy demands required for continual proliferation are met (13). The high affinity between cAMP and CRP requires E. coli to keep intracellular cAMP levels very low, which makes determination of intracellular cAMP levels in E. coli difficult and variable. Actually, whenever investigated thoroughly, most of the cAMP produced by E. coli was detected in the medium (2). Using bacterial membrane vesicles it was demonstrated that cAMP export is an active, energy-dependent process, whereas cAMP uptake appeared to be controlled by diffusion. However, the data further indicated that efflux and uptake may use identical protein components for cAMP trafficking (8). Another possibility to reset the cAMP system in E. coli may be degradation by a known cAMP phosphodiesterase activity (CpdA). However, the K m of this enzyme was reported to be 47 M or even 500 M cAMP (6, 9). Since CRP has an apparent dissociation constant of about 400 nM for cAMP (15), it is unlikely that CpdA with its low substrate affinity has a major impact in setting intracellular cAMP concentrations in E. coli.Thus, E. coli appears to rapidly quench the intracellular cAMP levels mainly by export and to a much smaller extent by degradation to reset the cellular regulatory systems. Thus far, the mechanism of cAMP export from E. coli that results in considerable cAMP concen...

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“…In E. coli, the mar/sox/rob regulon includes the major multidrug efflux pump AcrAB and the outer membrane channel TolC. It has been found that transcription of marA, soxS, and rob is increased in tolC mutants (57), and this prompted us to investigate whether the expression of the regulators pliA, rob, soxS, and opiA is also influenced in an acrB or tolC mutant of E. amylovora Ea1189. However, our analysis revealed that the expression of pliA, rob, soxS, and opiA was not significantly changed in mutants defective in AcrB or TolC (data not shown).…”

Section: Resultsmentioning

confidence: 99%

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

2014

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bTranscriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/ XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob-overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS-deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora. PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA, including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses.

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An Excretory Function for theEscherichia coliOuter Membrane Pore TolC: Upregulation ofmarAandsoxSTranscription and Rob Activity Due to Metabolites Accumulated intolCMutants

Cited by 55 publications

References 62 publications

Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

Escherichia coli Exports Cyclic AMP via TolC

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

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