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  • 1
    Language: English
    In: Journal of bacteriology, February 2017, Vol.199(3), pp.e00691-16
    Description: The L-arabinose-inducible araBAD promoter (P) allows tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been successfully used to study bacterial sRNA regulation, where P drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA can regulate P promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of P at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, that is through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by cAMP-CRP complex, reinforces the catabolite repression network. The bacterial arabinose inducible system is widely used for titratable control of gene expression. We demonstrate here that a post-transcriptional mechanism mediated by the Spot 42 sRNA contributes to the functionality of the P system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the P system, and has implications for improving its usage for tunable gene expression.
    Keywords: Gene Expression ; Translation ; Promoters ; Growth Conditions ; Cyclic Amp ; Catabolite Repression ; Arabinose ; Transcription ; Post-Transcription ; Bacteriology ; Escherichia Coli ; Cell Biology ; RNA;
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 2
    Language: English
    In: Journal of Bacteriology, Feb, 2014, Vol.196(3-4), p.754(8)
    Description: The stationary phase/general stress response sigma factor RpoS (sigma S) is necessary for adaptation and restoration of homeostasis in stationary phase. As a physiological consequence, its levels are tightly regulated at least at two levels. Multiple small regulatory RNA molecules modulate its translation, in a manner that is dependent on the RNA chaperone Hfq and the rpoS 5' untranslated region. ClpXP and the RssB adaptor protein degrade RpoS, unless it is protected by an anti-adaptor. We here find that, in addition to these posttranscriptional levels of regulation, tRNA modification also affects the steady-state levels of RpoS. We screened mutants of several RNA modification enzymes for an effect on RpoS expression and identified the miaA gene, encoding a tRNA isopentenyltransferase, as necessary for full expression of both an rpoS750-lacZ translational fusion and the RpoS protein. This effect is independent of rpoS, the regulatory RNAs, and RpoS degradation. RpoD steady-state levels were not significantly different in the absence of MiaA, suggesting that this is an RpoS-specific effect. The rpoS coding sequence is significantly enriched for leu codons that use MiaA-modified tRNAs, compared to rpoD and many other genes. Dependence on MiaA may therefore provide yet another way for RpoS levels to respond to growth conditions.
    Keywords: Escherichia Coli -- Research ; Escherichia Coli -- Physiological Aspects ; Post-translational Modifications -- Analysis ; Transfer Rna -- Research ; Translational Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 3
    Article
    Article
    Language: English
    In: Journal of bacteriology, 15 October 2017, Vol.199(20)
    Description: Bacteria have robust responses to a variety of stresses. In particular, bacteria like have multiple cell envelope stress responses, and generally we evaluate what these responses are doing by the repair systems they induce. However, probably at least as important in interpreting what is being sensed as stress are the genes that these stress systems downregulate, directly or indirectly. This is discussed here for the Cpx and sigma E systems of .
    Keywords: Cpx ; Hfq ; Escherichia Coli -- Genetics ; Escherichia Coli Proteins -- Genetics
    E-ISSN: 1098-5530
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  • 4
    Language: English
    In: Journal of Bacteriology, 2016, Vol.198(7-8), p.1101(13)
    Description: 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. IMPORTANCE AcrAB-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.
    Keywords: RNA – Research ; Gram-Negative Bacteria – Research ; Gram-Negative Bacteria – Genetic Aspects ; Escherichia Coli – Research ; Escherichia Coli – Genetic Aspects
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 5
    Language: English
    In: Journal of bacteriology, 25 January 2016, Vol.198(7), pp.1101-13
    Description: 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. AcrAB-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.
    Keywords: Bacterial Outer Membrane Proteins -- Metabolism ; Carrier Proteins -- Metabolism ; Escherichia Coli -- Metabolism ; Escherichia Coli Proteins -- Metabolism ; Gene Expression Regulation, Bacterial -- Physiology ; Membrane Transport Proteins -- Metabolism ; RNA, Bacterial -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 6
    Language: English
    In: The Journal of Bacteriology, 2010, Vol. 192(21), p.5559
    Description: Small noncoding RNAs (sRNAs) regulate gene expression in Escherichia coli by base pairing with mRNAs and modulating translation and mRNA stability. The sRNAs DsrA and RprA stimulate the translation of the stress response transcription factor RpoS by base pairing with the 5' untranslated region of the rpoS mRNA. In the present study, we found that the rpoS mRNA was unstable in the absence of DsrA and RprA and that expression of these sRNAs increased both the accumulation and the half-life of the rpoS mRNA. Mutations in dsrA, rprA, or rpoS that disrupt the predicted pairing sequences and reduce translation of RpoS also destabilize the rpoS mRNA. We found that the rpoS mRNA accumulates in an RNase E mutant strain in the absence of sRNA expression and, therefore, is degraded by an RNase E-mediated mechanism. DsrA expression is required, however, for maximal translation even when rpoS mRNA is abundant. This suggests that DsrA protects rpoS mRNA from degradation by RNase E and that DsrA has a further activity in stimulating RpoS protein synthesis, rpoS mRNA is subject to degradation by an additional pathway, mediated by RNase III, which, in contrast to the RNase E-mediated pathway, occurs in the presence and absence of DsrA or RprA. rpoS mRNA and RpoS protein levels are increased in an RNase III mutant strain with or without the sRNAs, suggesting that the role of RNase III in this context is to reduce the translation of RpoS even when the sRNAs are acting to stimulate translation. doi: 10.1128/JB.00464-10
    Keywords: Messenger Rna -- Properties ; Protein Synthesis -- Research ; Bacterial Genetics -- Research ; Translation (Genetics) -- Research ; Escherichia Coli -- Genetic Aspects;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 7
    In: The Journal of Bacteriology, 2007, Vol. 189(13), p.4872
    Description: Escherichia coli cell viability during starvation is strongly dependent on the expression of the rpoS gene, encoding the RpoS sigma subunit of RNA polymerase. RpoS abundance has been reported to be regulated at many levels, including transcription initiation, translation, and protein stability. The regulatory RNA SsrA (or tmRNA) has both tRNA and mRNA activities, relieving ribosome stalling and cotranslationally tagging proteins. We report here that SsrA is needed for the correct high-level translation of RpoS. The ATP-dependent protease Lon was also found to negatively affect RpoS translation, but only at low temperature. We suggest that SsrA may indirectly improve RpoS translation by limiting ribosome stalling and depletion of some component of the translation machinery.
    Keywords: Biology;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 8
    Language: English
    In: Journal of Bacteriology, Jan, 2009, Vol.191(1-2), p.461(16)
    Description: Small noncoding regulatory RNAs (sRNAs) play a key role in regulating the expression of many genes in Escherichia coli and other bacteria. Many of the sRNAs identified in E. coli bind to mRNAs in an Hfq-dependent manner and stimulate or inhibit translation of the mRNAs. Several sRNAs are regulated by well-studied global regulators. Here, we report characterization of the CyaR (RyeE) sRNA, which was previously identified in a global search for sRNAs in E. coli. We demonstrated that CyaR is positively regulated by the global regulator Crp under conditions in which cyclic AMP levels are high. We showed by using microarray analysis and Northern blotting that several genes are negatively regulated by CyaR, including ompX, encoding a major outer membrane protein; luxS, encoding the autoinducer-2 synthase; nadE, encoding an essential NAD synthetase; and yqaE, encoding a predicted membrane protein with an unknown function. Using translational lacZ fusions to yqaE, ompX, nadE, and luxS, we demonstrated that the negative regulation of these genes by CyaR occurs at the posttranscriptional level and is direct. Different portions of a highly conserved 3' region of CyaR are predicted to pair with sequences near the ribosome binding site of each of these targets; mutations in this sequence affected regulation, and compensatory mutations in the target mRNA restored regulation, confirming that there is direct regulation by the sRNA. These results provide insight into the mechanisms by which Crp negatively regulates genes such as luxS and ompX and provide a link between catabolite repression, quorum sensing, and nitrogen assimilation in E. coli.
    Keywords: Escherichia Coli -- Genetic Aspects ; Genetic Regulation -- Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Journal of bacteriology, February 2014, Vol.196(4), pp.754-61
    Description: The stationary phase/general stress response sigma factor RpoS (σ(S)) is necessary for adaptation and restoration of homeostasis in stationary phase. As a physiological consequence, its levels are tightly regulated at least at two levels. Multiple small regulatory RNA molecules modulate its translation, in a manner that is dependent on the RNA chaperone Hfq and the rpoS 5' untranslated region. ClpXP and the RssB adaptor protein degrade RpoS, unless it is protected by an anti-adaptor. We here find that, in addition to these posttranscriptional levels of regulation, tRNA modification also affects the steady-state levels of RpoS. We screened mutants of several RNA modification enzymes for an effect on RpoS expression and identified the miaA gene, encoding a tRNA isopentenyltransferase, as necessary for full expression of both an rpoS750-lacZ translational fusion and the RpoS protein. This effect is independent of rpoS, the regulatory RNAs, and RpoS degradation. RpoD steady-state levels were not significantly different in the absence of MiaA, suggesting that this is an RpoS-specific effect. The rpoS coding sequence is significantly enriched for leu codons that use MiaA-modified tRNAs, compared to rpoD and many other genes. Dependence on MiaA may therefore provide yet another way for RpoS levels to respond to growth conditions.
    Keywords: Gene Expression Regulation, Bacterial ; Alkyl and Aryl Transferases -- Metabolism ; Bacterial Proteins -- Biosynthesis ; Escherichia Coli -- Enzymology ; RNA, Transfer -- Metabolism ; Sigma Factor -- Biosynthesis
    E-ISSN: 1098-5530
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  • 10
    Language: English
    In: Journal of Bacteriology, March, 2007, Vol.189(5-6), p.2238(11)
    Description: SgrR is the first characterized member of a family of bacterial transcription factors containing an N-terminal DNA binding domain and a C-terminal solute binding domain. Previously, we reported genetic evidence that SgrR activates the divergently transcribed gene sgrS, which encodes a small RNA required for recovery from glucose-phosphate stress. In this study, we examined the regulation of sgrR expression and found that SgrR negatively autoregulates its own transcription in the presence and absence of stress. An SgrR binding site in the sgrR-sgrS intergenic region is required in vivo for both SgrR-dependent activation of sgrS and autorepression of sgrR. Purified SgrR binds specifically to sgrS promoter DNA in vitro; a mutation in the site required for in vivo activation and autorepression abrogates in vitro SgrR binding. A plasmid library screen identified clones that alter expression of a [P.sup.sgrS]-lacZ fusion; some act by titrating endogenous SgrR. The yfdZ gene, encoding a putative aminotransferase, was identified in this screen; the yfdZ promoter contains an SgrR binding site, and transcriptional fusions indicate that yfdZ is activated by SgrR. Clones containing role, which encodes a glucose-specific repressor protein, also downregulate [P.sup.sgrS]-lacZ. The talc clones do not appear to titrate the SgrR protein, indicating that MIc affects sgrS expression by an alternative mechanism.
    Keywords: Dna Binding Proteins -- Research ; Gene Expression -- Research ; Transcription (Genetics) -- Research ; Dna Binding -- Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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