A genome-scale phenotype screen to determine carbon source utilization and stress tolerance pathways in Francisella novicida

This dissertation presents investigations into the carbon source utilization pathways and stress condition hypersensitivities of the bacterium Francisella novicida. F. novicida is generally not considered pathogenic in humans but is closely related to, and a good surrogate for the study of Francisella tularensis. F. tularensis is a human pathogen and causative agent of the disease tularemia. This organism is able to persist and be infective as an aerosol, requires a very low exposure to cause disease, and the disease has a high mortality rate, making this organism potentially a very dangerous bioweapon. An annotation of F. novicida's genome has recently been constructed, and a transposon insertion gene disruption library has been created for all of the non-essential genes in the organism. In this investigation, genetic screens are performed on the F. novicida gene disruption library in order to create a genotype-phenotype map of many of the organism's metabolic and stress tolerance pathways. Chapter 1 is a general introduction. Chapter 2 presents methods used and tools developed for genome scale screening, phenotype quantification and data analysis. Chapter 3 presents a carbon source utilization screen of F. novicida. Mutants were assayed for their ability to grow on many different carbon sources including six carbon sugars, amino acids, and metabolic intermediates. Mutations in 251 ORFs (out of 1278 total) caused significant growth degradation. Analysis of mutant phenotypes revealed nineteen metabolic pathways containing sixty-six ORFs. The functions of seven previously unidentified ORFs were discovered and one mis-annotated gene was found. Two metabolic reactions not known to exist in F. novicida were discovered; the flow of carbon through the phosphofructokinase enzyme during glycolysis and the biosynthesis of proline from arginine. Chapter 4 presents a stress condition hypersensitivity screen of F. novicida. Mutants were assayed for their ability to grow in the presence of seven different antibiotics and six other stressful conditions. The hypersensitivity screen found 222 ORFs whose disruption caused a reduced ability of F. novicida to grow in at least one condition. Hierarchical clustering of mutant phenotypes revealed ten clusters of mutants closely related to each other. The ten clusters contain 109 ORFs, twenty-two of which are currently annotated as proteins of unknown function or hypothetical proteins.