List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.

<p>List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.</p

Transcriptome changes elicited by glucose in GBS.

<p>Comparison of gene expression changes (log2) between mid-log cultures of 2603 V/R wild-type strain and <i>CovRS</i> mutant following challenge with or without glucose. Data for the wild type and mutant strains are shown on the <i>x</i> axis and <i>y</i> axis, respectively.</p

CovR binds to <i>bibA</i> promoter <i>in vivo</i>. (A)

<p>Quantification by qRT-PCR of <i>bibA</i> promoter immunoprecipitated with CovR antiserum in 2603 V/R wild type strain grown in medium devoid of glucose or in the presence of 55mM glucose. <i>cfb</i> promoter and <i>cylX</i> promoter were used as a positive control while <i>sag0017</i> promoter was used as a negative control. The level of PCR products of eluate from the isogenic Δc<i>ovRS</i> deletion mutant grown with or without glucose was negligible. The data are representative of 3 independent experiments, each in triplicate. Error bars, SD. (<b>B)</b> Competitive EMSA experiment. Labelled <i>PbibA</i> fragment (3.3 nM) was incubated without <i>(lane1)</i> or with CovR (2 µM) <i>(lane2</i>–<i>6)</i>, in the presence of different amounts of unlabelled <i>PbibA (lane 3</i>–<i>4)</i>, as a specific competitor, and <i>Psag0017 (lane5</i>–<i>6)</i>, as a non-specific competitor. The labelled DNA was detected by chemioluminescence. <b>(C)</b> CovR phosphorylation increases its affinity for <i>bibA</i> promoter. Electrophoretic mobility shift assay using recombinant CovR (left) and chemically phosphorylated recombinant CovR (right). Labelled <i>PbibA</i> DNA fragment (3.3 nM) was incubated without or with the indicated amounts of CovR. The labelled DNA was detected by chemioluminescence.</p

Real-time RT-PCR evaluation of <i>bibA</i> expression in 2603V/R and Δc<i>ovRS</i> strains grown in medium containing 55 mM glucose or in sugar-free medium.

<p>Transcript levels were normalized to the expression level of <i>gyrA</i>. Syber green runs were performed with cDNAs from the same reverse transcription reaction from 1 µg of total RNA. The ΔΔCT method was applied as a comparative method of quantification, using strains grown in sugar free medium as reference. The data are representative of 2 independent experiments, each in triplicate. Error bars, SD.</p

Graphical representation summarizing adaptive regulation of GBS in high glucose conditions.

<p>Genes of interest are color-grouped according to main functional categories. Arrows indicate up- or down-regulation relative to time of 30′ in high glucose <i>vs.</i> no glucose.</p

Differential regulation of gene expression in GBS strain 2603 V/R versus the isogenic Δ<i>CovRS</i> mutant strain after incubation in medium with 55 mM glucose versus a sugars-free complex medium.

<p>White bars indicate the number of glucose-regulated genes in the wild-type strain; black bars indicate the number of genes that are glucose- dependent and CovRS-dependent; grey bars indicate the number of genes that are glucose-dependent and CovRS-independent.</p