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"Multiple roles of MecA in regulation of stationary phase development in Bacillus subtilis"

by
Melissa DeFrancesco
Department of Microbiology and Molecular Genetics
B.A. 2003, Drew University


Thesis Advisor: David Dubnau, Ph.D.

Professor

Department of Microbiology and Molecular Genetics - PHRI

Friday, December 18, 2009
10:30 a.m., ICPH Seminar Room


Abstract

The MecA protein of Bacillus subtilis is an important regulatory factor involved in modulating the activities of the cell through protein-protein interactions. MecA acts as a negative regulator of competence, defined as the ability to take up DNA. MecA acts on competence by targeting the transcriptional regulator ComK for degradation by the ClpCP protease, thus preventing transcription of all the genes required to bind and internalize DNA. MecA releases ComK when ComS, the product of a quorum sensing mechanism, is produced and competes with ComK for binding to the N terminal domain of MecA. In this study we have performed a mutational analysis of MecA using alanine scanning mutagenesis to identify residues that are important for binding to ComK, ComS and ClpC. We were also able to identify residues that were important for the function of MecA in the regulation of exopolysaccharide (EPS) production, an essential early step in biofilm formation. It had been observed that deleting mecA resulted in overproduction of EPS from the eps promoter. We have characterized the regulation of eps by MecA demonstrating that the negative effect of MecA can be bypassed by knocking out two of the transcriptional regulators of eps, SinI and AbrB. Spo0A is required for eps expression because, when activated via phosphorylation, Spo0A inhibits transcription of abrB and activates transcription of sinI, the product of which is required to alleviate direct repression of eps by SinR. We have shown that MecA, in conjunction with ClpC, acts to inhibit the DNA binding activity of Spo0A, and hence by implication the ability of Spo0A to act as a transcription factor. This mechanism is also sufficient to explain MecA’s negative regulation of sporulation. Our results suggest a new activity for MecA and ClpC, different from their mode of action in regulating competence. We propose that MecA acts a buffer to regulate the transition of individual cells to a number of stationary phase adaptations, preventing cells from committing valuable resources to energy intensive pathways while conditions are favorable for growth. We also shed light on the roles of the positively acting competence, sporulation and biofilm regulatory proteins YlbF and YmcA. We show that both proteins act to destabilize MecA, explaining their effects on competence and that YlbF acts to stabilize SinI, explaining its necessity for EPS production.


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