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"A Tale of Two Functions: Characterization of a Novel Iron-Sulfur Regulatory Complex"

by
Andrew William Tanner
Microbiology and Molecular Genetics Program
B.S., Montclair State University, 2007, Montclair, NJ
M.S., Johns Hopkins University, 2010, Baltimore, MD


Thesis Advisor: David Dubnau, Ph.D.
Professor
Public Health Research Institute
Department of Microbiology, Biochemistry and Molecular Genetics

Wednesday, October 5, 2016
12:00 PM, ICPH Auditorium


Abstract

During times of environmental insult, Bacillus subtilis is capable of undergoing developmental changes such as biofilm formation, sporulation, the transition to motility and competence development. Each of these states is regulated in part by the phosphorylated form of the master response regulator Spo0A (Spo0A~P). The phosphorylation state of Spo0A is controlled by a multi-component phosphorelay. The phosphorelay is delicately controlled in order to achieve precise levels of Spo0A~P, as hundreds of regulated promoters have been shown to respond in a concentration-dependent manner. YlbF, YmcA and YaaT have previously been shown to be important regulatory proteins for multiple developmental fates. These proteins directly interact and form a stable complex. Through the use of suppressor mutations and promoter fusions it was proposed that the proteins accelerate the phosphorelay. Indeed, in vitro this complex is sufficient to stimulate the rate of phosphotransfer amongst the phosphorelay proteins.

Further investigation demonstrated that two [4Fe-4S]2+ iron-sulfur clusters can be assembled on the complex. As with other iron-sulfur cluster-binding proteins, the complex was also found to bind FAD, hinting that these cofactors may be involved in sensing the redox state within the cell. This work provides the first comprehensive characterization of an iron-sulfur protein complex that regulates Spo0A~P levels.

Although we have found that Bacillus subtilis sporulation is inhibited by both high and low levels of oxygen, this form of regulation may not occur through the complex. Phylogenetic evidence, correlation of gene expression levels, along with incomplete suppressor bypass of mutants strongly suggest that the complex may play a broader role in either iron-sulfur cluster biogenesis or RNA processing, in addition to its ability to stimulate the phosphorelay.


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