Structural Studies of Bacterial Competence Proteins

North Carolina State University Raleigh, Raleigh NC

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Abstract

DESCRIPTION (provided by applicant): Bacteria have obtained a significant portion of their genetic diversity, metabolic fitness and lethality through the acquisition of nucleotide sequences from both closely and distantly related organisms. Horizontal gene transfer produces extremely dynamic genomes, in which substantial amounts of DNA are transferred between the chromosomes of different bacterial strains. Without question, such transfers have changed the ecological and pathogenic character of bacterial species. Transformation represents one of the main mechanisms by which DNA is incorporated into the recipients genome. It involves the uptake of naked DNA from the environment and has the potential to transmit DNA between distantly related organisms. For bacteria to be transformable, they must develop the physiological state referred to as competence. Genetic competence is defined as the ability of a cell to take up free DNA from the surrounding medium. If the cell is not competent, it cannot be transformed and its growth, survival and evolution are compromised. Research into the very nature of competence addresses both very fundamental and very practical questions and concerns. What mechanisms allow bacteria to incorporate new genes to develop novel, more sophisticated mechanisms for survival? By what means is genetic diversity provided, such that a microorganism can enlarge its own genomic complement? From a health related perspective, genetic transfer via competence is becoming more implicated in the development of the pathogenic character of bacteria. There is mounting evidence that virulent attributes and antibiotic resistance can be introduced and/or enhanced in this manner. The studies proposed here will focus on mechanistic characteristics of a set of interacting proteins that form the critical regulatory network initiating competence development in Bacillus subtilis. The goals of the research are to provide structural, dynamic and interaction information, in order to understand the regulatory effects of the proteins MecA, ComS, ClpP/C and ComK (and their complexes). This will be achieved by a combination of high-resolution NMR spectroscopy, electrospray mass spectrometry, surface plasmon resonance and other bioanalytical techniques, along with mutagenesis and protease foot-printing investigations. The results from these studies will provide a basis for the development of models to describe the mechanism of activation of competence, not only in B. subtilis, but also in other important gram-positive (e.g. Streptococcus pneumoniae) and gram-negative (e.g. Neisseria gonorrhoeae and Haemophihts influenza) bacteria.