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RpoE binds to target DNA in response to temperature changes <i>in vivo</i> and <i>in vitro</i>.

Abstract

<p>(<b>A</b>) ChIP assays were used to analyze RpoE binding to the <i>luxR</i>, <i>rpoE</i> and <i>rpoH</i> promoter <i>in vivo</i>. Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using PCR with the primers P<sub><i>luxR</i></sub>chip-F/R, P<sub><i>rpoE</i></sub>-chipF/R, P<sub><i>rpoH</i></sub>-chipF/R and control-F/R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005645#ppat.1005645.s010" target="_blank">S2 Table</a>). (<b>B</b>) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene <i>gyrB</i>. Results were calculated using the ΔΔ<i>C</i><sub>T</sub> method. * <i>P</i> <0.05, ** <i>P</i> <0.01, <i>t-</i>test. (<b>C</b>) Plot showing the affinity of RpoE binding to the promoters of <i>luxR</i>, <i>rpoE</i>, and <i>rpoH</i> at different temperatures as determined using EMSA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005645#ppat.1005645.s004" target="_blank">S4 Fig</a>). The intensities of the bound DNA fragments were determined using a densitometer and plotted against the RpoE concentrations. Triplicate assays were performed, and a representative plot is shown. (<b>D</b>) The promoter strength of <i>luxR</i>, <i>rpoE</i>, and <i>rpoH</i> in the presence of similar levels of RpoE at different temperatures in <i>E</i>. <i>coli</i> DH5α cells. The three indicated promoters were fused to different fluorescence reporters and cloned into the same cloning plasmid, pMD19T. The fluorescence reads for each of the promoters after incubation in the presence of arabinose inducing pBAD-driven <i>rpoE-flag</i> expression were subtracted from the reads obtained with no arabinose and normalized to both the corresponding reads at 22°C and the densitometry values of RpoE expression. The results are presented as the mean ± S.D. (<i>n</i> = 3).</p

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The Francis Crick Institute

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Last time updated on 12/02/2018

This paper was published in The Francis Crick Institute.

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