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Doctoral Dissertation Announcement
Candidate: Miles Rogers
Doctor of Philosophy
Department: Biological Sciences
Title: A Tale of Two Proteins: Regulation of the Enterohemolysin Operon by the Histone-Like Nucleoid Structuring Protein and Characterization of the Novel Protein YghJ in Shiga-Toxin Producing Escherichia Coli
Dr. John Spitsbergen, Chair
Dr. Maria Scott
Dr. Susan Stapleton
Dr. David Huffman
Date: Tuesday, July 3, 2012 10:30 a.m. to 12:30 p.m.
1718 Wood Hall
Shiga toxin-producing Escherichia coli (STEC) are worldwide pathogens causing an estimated 200,000 infections per year in the United States. Infections with STEC sometimes progress to kidney failure ending in death. The two studies presented below describe a novel virulence factor and mechanisms required for pathogenesis of STEC seropathotype O91:H21.
First the novel protein, YghJ, was identified and found to be conserved amongst extraintestinal and diarrheal seropathotypes of E. coli with similar proteins carried by pathogens such as Vibrio cholerae. When the yghJ gene was disrupted creating a YghJ null mutant, the defect resulted in a significant growth deficiency in minimal media with L-malate as the sole carbon source. The null mutant growth defect was complemented by replacement of the mutated copy with an intact copy of yghJ expressed from a high copy number plasmid. Structure-function analysis of YghJ revealed lipidation restricts the YghJ protein to the inner membrane. Further studies suggest an amino-terminal domain involved in carbon binding and a carboxy-terminal region with mucinase activity. Taken together, the data indicates that the YghJ belongs to a new class of protein and plays an important role in carbon utilization since oxidation of L-malate to oxaloacetate is obligatory for survival of facultative anaerobes like E. coli.
The second study focused on regulation of expression of the enterohemolysin toxin gene (ehxA) carried by STEC. The role of the H-NS protein, a global inhibitor, was investigated. Insertional mutation of the hns gene was constructed in STEC O91:H21and revealed that the amount of EhxA increased in the hns knockout strain. The H-NS protein was found to control expression of the ehxA gene through direct binding of the promoter upstream of the enterohemolysin operon (ehxCABD). In total, the data indicates that in STEC the 88 bp DNA region upstream of ehxCABD contains a cis-acting element to which H-NS binds and negatively regulates expression of enterohemolysin.
In conclusion, YghJ participates in a key carbon metabolic pathway and provides a competitive advantage in carbon-limited environments where L-malate is present. Compelling evidence suggests that H-NS interferes with RNA polymerase binding blocking transcription of ehxCABD in STEC grown ex vivo.