Collections > Electronic Theses and Dissertations > A genetic and biochemical approach to identify function of novel proteins in Francisella tularensis

Francisella tularensis is a Gram-negative bacterial pathogen that can infect a broad range of hosts and is the etiologic agent of the human disease tularemia. Members of the family Francisellaceae have few close relatives, and about 30% of the F. tularensis genome encodes hypothetical proteins, or proteins with unknown function. We used biochemical techniques and developed genetic tools to identify and characterize two proteins of unknown function in F. tularensis. First we identified a conserved cytoplasmic membrane protein with unknown function, which we termed RipA. In F. tularensis, RipA is required for intracellular growth and virulence in the mouse model of tularemia. As a means to determine RipA function, we isolated and mapped independent extragenic suppressor mutants of a ∆ripA strain that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We found that the ratio of C:18 to C:16 fatty acids in lipid A was greater in the presence of RipA. Furthermore, LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. Together these data suggest RipA modulates the activity and abundance of LpxA in F. tularensis during adaptation to the host cell environment. Furthermore, we characterized the function, and named a second conserved protein in F. tularensis, PanG, which was required for pantothenate (vitamin B5) synthesis. PanG is a novel ketopantoate reductase (KPR) that can convert 2-dehydropantoate to pantoate. Both the homologous gene from Enterococcus faecalis (EF1861) and the analogous gene, panE, from Escherichia coli functionally complemented the Francisella novicida KPR mutant. Using genetic and chemical complementation we confirmed the in silico predicted genes involved in pantothenate synthesis in Francisella novicida and tularensis and identified a novel KPR, PanG. Together this work provided functional characterization of two F. tularensis proteins that were previously unknown. However, there remain over 500 genes in F. tularensis that are either annotated as hypothetical or unknown, which need to be characterized.