The role of pseudopilins during transformation in Bacillus subtilis

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DESCRIPTION (provided by applicant): The study of natural transformation, along with conjugation and transduction, has gained importance since genomic sequencing revealed horizontal gene transfer to be a significant factor in the evolution of bacteria and antibiotic resistance. The machinery that mediates transformation in Bacillus subtilis is highly conserved among competent microbes, both gram-positive and negative. Also, B. subtilis pseudopilins and assembly proteins, which are essential components of the transformation machinery, share homology to proteins of type 2 secretion systems (T2SS) and type IV pill (T4P), found in many gram-negative human pathogens. Ultimately, studying the competence proteins in B. subtilis will shed light on general characteristics of transformation proteins and the functions of T2SS and T4P proteins. The focus of this proposal is to decipher the functions of ComGD, ComGE, and ComGG, collectively called the minor pseudopilins, and ComGC, the major pseudopilin which comprises the competence pseudopilus, of Bacillus subtilis. These proteins are essential for both DNA-binding and transport to the cytosol during transformation, but their specific roles are unknown. We hypothesize that the minor pseudopilins interact to form a heterotrimeric complex similar to the one formed by the minor pseudopilins of Escherichia coli T2SS. The E. coli minor pseudopilin complex is believed to bind to the tip of the T2SS pseudopilus and mediate protein-protein interactions. Accordingly, we believe the B. subtilis complex localizes at the tip of the competence pseudopilus, comprised of ComGC, and binds to ComEA, the integral membrane DNA-binding protein. As a result, ComEA will be connected to the competence pseudopilus such that retraction of the pseudopilus will cause movement in ComEA, which will allow for delivery of the DNA to the membrane channel composed of ComEC. Analysis of our model will require in vitro and in vivo biochemical protein-protein interaction studies like pull-downs and immunoprecipitiation. Fluorescence microscopy will serve to evaluate the localization and possible co-localization of ComEA and the pseudopilins with other competence proteins. Overall, we seek to decipher the function and interactions of the pseudopilins during transformation. Bacillus subtilis shares DNA using the same mechanism and machinery that some human pathogens use to become antibiotic resistant. The focus of this research is on studying this conserved machinery in B. subtilis because it can be easily manipulated.