22 research outputs found

    Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems

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    <div><p>The two-component signal transduction system BarA-UvrY of <i>Escherichia coli</i> and its orthologs globally regulate metabolism, motility, biofilm formation, stress resistance, virulence of pathogens and quorum sensing by activating the transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in <i>E</i>. <i>coli</i>. These sRNAs act by sequestering the RNA binding protein CsrA (RsmA) away from lower affinity mRNA targets. In this study, we used ChIP-exo to identify, at single nucleotide resolution, genomic sites for UvrY (SirA) binding in <i>E</i>. <i>coli</i> and <i>Salmonella enterica</i>. The <i>csrB</i> and <i>csrC</i> genes were the strongest targets of crosslinking, which required UvrY phosphorylation by the BarA sensor kinase. Crosslinking occurred at two sites, an inverted repeat sequence far upstream of the promoter and a site near the -35 sequence. DNAse I footprinting revealed specific binding of UvrY <i>in vitro</i> only to the upstream site, indicative of additional binding requirements and/or indirect binding to the downstream site. Additional genes, including <i>cspA</i>, encoding the cold-shock RNA-binding protein CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We conclude that the global effects of UvrY/SirA on gene expression are primarily mediated by activating <i>csrB</i> and <i>csrC</i> transcription. We also used <i>in vivo</i> crosslinking and other experimental approaches to reveal new features of <i>csrB/csrC</i> regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA. Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with <i>fliW</i>, which encodes a protein that binds to CsrA and antagonizes its activity in <i>Bacillus subtilis</i>. We propose that BarA-UvrY and orthologous TCS transcribe sRNA antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform this function in other species.</p></div

    Components of the nitrogen metabolism of <i>Ca</i>. N. evergladensis

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    <p>: ammonia oxidation (4, 5), ammonia assimilation (8, 9, 10), nitrite reduction (6), nitrous oxide production (7). Reactions are mediated by the following transporters and enzymes: urea transporters, urease (1, 2), ammonia transporters (3), archaeal ammonia monooxygenase (AMO) (4), candidate enzyme: multicopper oxidase (5), nitrite reductase (NirK) (6), nitric oxide reductase (NorD, NorQ), catalytic subunit (NorB) is missing (7), glutamate dehydrogenase (8), glutamine synthetase (9), glutamate synthase (10). NO may upregulate activity of AMO. * - experimental evidences are needed.</p

    Effect of DksA and RelA on UvrY-FLAG levels.

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    <p>Western blotting of UvrY-FLAG levels in strains MG1655 (no FLAG fusion), WT (MG1655 expressing UvrY-FLAG from the <i>uvrY</i> genomic locus), and isogenic ∆<i>dksA</i> and ∆<i>relA</i> strains. Proteins were collected from cultures grown in LB medium to mid-exponential growth phase (~OD<sub>600</sub> of 0.6). RpoB served as a loading control.</p

    Electrophoretic gel mobility shift assay showing UvrY binding to <i>csrB</i> and <i>csrC</i> DNA.

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    <p>Binding of phosphorylated (UvrY-P) and non-phosphorylated (UvrY) UvrY-His<sub>6</sub> to <i>csrB</i> DNA (A and B) and <i>csrC</i> DNA (C) was tested as shown. The <i>csrB</i> and <i>csrC</i> DNA probes (0.5 nM) used for this experiment (depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145035#pone.0145035.g001" target="_blank">Fig 1D and 1E</a>, respectively) were incubated with increasing concentrations of <i>in vitro</i> phosphorylated or non-phosphorylated UvrY-His6 protein for 30 min at room temperature. The DNA-protein complexes were resolved by electrophoresis on a non-denaturing 7% polyacrylamide gel. The phosphorylation state of the UvrY-His<sub>6</sub> protein used in these experiments was determined by Phos-tag SDS PAGE gel analysis (D).</p

    Comparison of protein coding sequences (CDS) of <i>Ca</i>. Nitrososphaera evergladensis with CDS of other ammonia-oxidizing archaea.

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    <p>(A) CDS of <i>Ca</i>. Nitrososphaera evergladensis were compared to CDS of <i>Ca</i>. N. gargensis. (B) CDS of the group I.1a (<i>N. maritimus</i>, <i>Ca</i>. N. sediminis, <i>C. symbiosum</i>, <i>Ca</i>. N. limnia, <i>Ca</i>. N. koreensis) were compared to CDS of the group I.1b (<i>Ca</i>. N. evergladensis and <i>Ca</i>. N. gargensis). Overlapping regions represent CDS with amino acid sequence identity 35% and higher.</p

    Effect of DksA and RelA on <i>in vivo</i> binding of UvrY to <i>csrB</i> promoter.

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    <p>The effect of DksA and RelA on <i>in vivo</i> binding of UvrY to <i>csrB</i> promoter was determined by ChIP-PCR assay in a WT (MG1655 expressing UvrY-FLAG) and isogenic ∆<i>dksA</i>, ∆<i>relA</i> and ∆<i>barA</i> strains. Agarose gel showing PCR amplification of <i>csrB</i> promoter region recovered from each strain. The <i>lacY</i> gene served as a negative control in this experiment.</p

    <i>In vitro</i> transcription-translation of a supercoiled plasmid-encoded <i>csrB-lacZ</i> transcriptional fusion.

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    <p>Reactions contained pLFXcsrB-lacZ plasmid (2 μg), UvrY-P (2.3 μM), ppGpp (250 μM) and/or DksA (2 μM) as indicated. Incorporation of <sup>35</sup>S-labeled methionine into protein products was detected by SDS PAGE followed by phosphorimaging. Signal intensity of the full length protein was determined using Quantity One software. The fold-effects of regulatory factors were determined with respect to the control reaction lacking the factors, after normalization against the internal control, β-lactamase, which was encoded on the same plasmid. Absolute deviation for each reaction was determined from two independent experiments.</p

    DNase I footprinting of <i>E</i>. <i>coli csrB</i> DNA using phosphorylated and non-phosphorylated UvrY.

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    <p>A <sup>32</sup>P-end labeled DNA probe that included both the upstream and downstream putative UvrY binding sites was used for these experiments (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145035#pone.0145035.g001" target="_blank">Fig 1D</a>). Reactions in all lanes except 1 contained DNase I (0.025U/12.5ul reaction). Reactions in lanes 3–6 and lanes 7–10 contained 0.25, 0.35, 0.5, 0.7 μM of phosphorylated or non-phosphorylated UvrY-His<sub>6</sub>, respectively. Lane 2 reaction contained no UvrY protein. A vertical black bar indicates a protected region, and the sequence corresponding to the protected region is shown in a vertical rectangular box. An alignment of sequences corresponding to the protected regions from DNase I and the ChIP-exo results is shown in the horizontal rectangular box. The 18nt-long palindromic sequence and the partially conserved palindromic sequences are marked with broken black lines.</p

    CsrA activates <i>uvrY</i> expression without affecting DeaD or SrmB RNA helicase levels.

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    <p>The <i>uvrY</i> gene fusions used in this study were previously depicted in (4): <i>lacUV5</i> promoter fused at the transcription start site to the <i>uvrY</i> mRNA leader and N (12 or 22) <i>uvrY</i> codons fused in frame to <i>lacZ</i> (A). The effect of <i>csrA</i> disruption on expression of P<i>lacUV5</i>-<i>uvrY’–’lacZ</i> reporter fusions is shown (B). Cells were grown in LB and harvested at various times throughout growth and assayed for β-galactosidase specific activity (A<sub>420</sub>/mg protein). The values represent the average of two independent experiments. Error bars depict standard error of the means. Western blots showing effects of <i>csrA</i> on the level of DeaD-FLAG (C) or SrmB-FLAG (D) in an MG1655 derivative that expresses the corresponding FLAG-tagged gene from its native genomic locus are shown. Effect of <i>csrA</i>::<i>kan</i> complemented by a <i>csrA</i> expression plasmid (pCRA16) or with a control plasmid (pBR322) is also shown for DEAD-FLAG. RpoB served as a loading control for these blots.</p
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