Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans

<jats:title>ABSTRACT</jats:title> <jats:p> In <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Streptococcus mutans</jats:named-content> , both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. mutans</jats:named-content> suggested that purified ComE binds to two 11-bp direct repeats in the <jats:italic>nlmC-comC</jats:italic> promoter region, where ComE activates <jats:italic>nlmC</jats:italic> and represses <jats:italic>comC</jats:italic> . In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of <jats:italic>cslAB</jats:italic> , <jats:italic>comED</jats:italic> , <jats:italic>comX</jats:italic> , <jats:italic>ftf</jats:italic> , <jats:italic>vicRKX</jats:italic> , <jats:italic>gtfD</jats:italic> , <jats:italic>gtfB</jats:italic> , <jats:italic>gtfC</jats:italic> , and <jats:italic>gbpB.</jats:italic> Quantitative binding studies were performed on each of these sequences and showed that only <jats:italic>cslAB</jats:italic> has a similar specificity and high affinity for ComE as that seen with the upstream region of <jats:italic>comC</jats:italic> . A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT. </jats:p>