Characterization of Streptococcus mutans response regulator, ComE.

摘要

The development of a biofilm is part of the life cycle for many bacteria. The biofilm that forms on the surface of the tooth is commonly known as dental plaque and there are roughly 700 bacterial species detected amongst oral flora. The primary etiological agent for dental caries is the bacterium, Streptococcus mutans. In the presence of simple dietary sugars, this bacterium metabolizes sucrose into lactic acid, which results in the demineralization of the tooth surface. Extended exposure of lactic acid leads to dental caries.;In the oral cavity, there is a subpopulation of lysed bacteria where the DNA is released for uptake by the remaining cell populations. This flux of exogenous DNA is believed to be important for horizontal gene transfer in naturally competent bacteria such as S. mutans. Competence is defined as a physiological state where the bacterium is able to bind, transport and incorporate free DNA into its genome.;Based on the Streptococcus pneumoniae competence model, there are at least five genes involved in the early development of competence in S. mutans. comC encodes a competence-stimulating peptide (CSP) detected by its histidine kinase sensor protein, ComD and in turn activates its cognate response regulator, ComE. The other two genes, cslA and cslB encode a CSP-specific secretion apparatus consisting of an ATP-binding cassette (ABC) transporter (CslA) and its accessory protein (CslB), which are presumably involved in the processing and export of the CSP. This peptide-generating and responding pathway ensures the uptake of free DNA in the oral cavity for S. mutans.;I was interested in investigating ComE-DNA interactions. Here, I show that ComE is a DNA binding protein and specific to the promoter region of comC. Based on an in silico sequence analysis of other putative candidates' binding sites, the upstream regions of 9 genes were identified in the S. mutans genome. Further, biochemical analyses were used to determine if these nine target sequences are bona-fide ComE binding sites.;Since response regulators are often affected by their phosphorylation state, I have performed additional biochemical analyses. I have found that the phosphorylation state of ComE may not play a role in DNA binding affinity, but may induce the formation of an oligomer. The formation of this oligomer may be important in the regulation of gene expression because it increases the contact with DNA and possibly allows proteins to interact with RNA polymerase.