Burkholderia cenocepacia infections in the lungs of cystic fibrosis (CF) patients are difficult to treat due to resistance to multiple antibiotics, implying the need to identify new therapeutic targets to eradicate lung infections. The sputum of CF patients is rich in amino acids and was previously shown to increase flagellar gene expression in B. cenocepacia. However, the molecular mechanisms by which B. cenocepacia regulates swimming motility and flagellar biosynthesis are not understood. In the first part of this thesis, I elucidate how CF sputum conditions regulate swimming motility and flagellar biosynthesis in B. cenocepacia K56-2. Electron microscopy and flagella staining of B. cenocepacia demonstrated that the increased motility and flagellin corresponds to multiple flagella expressed under CF sputum conditions. I created a deletion ∆flhF mutant that exhibited downregulation in flagellin expression and displayed 80% aflagellated bacterial subpopulations. These results demonstrate that CF sputum nutritional conditions regulate flagellin and flagellation pattern through flhF in B. cenocepacia K56-2.

In the second part of the thesis, I investigated the role of c-di-GMP metabolizing genes in the swimming motility of B. cenocepacia K56-2 in CF sputum conditions. Overexpression of the wspR gene that causes an increase in c-di-GMP levels resulted in a significant reduction in the motility of B. cenocepacia K56-2. The increased c-di-GMP levels did not affect flagellin expression and flagellar biosynthesis. Screening of the putative c-di-GMP genes insertional mutants for defective motility led us to identify a BCAL1069 gene, which encodes a protein with a putative sensory signaling PAS domain, and c-di-GMP metabolizing GGDEF and EAL domains. Further, the reduced swimming motility of the BCAL1069 mutant was evident in CF nutritional conditions and in the presence of arginine and glutamate. The increase in intracellular c-di-GMP levels in the BCAL1069 mutant indicated that BCAL069 possesses an EAL domain (phosphodiesterase) activity. Thus it appears that BCAL1069 mediates swimming motility of B. cenocepacia K56-2 in response to CF sputum nutritional cues by changing intracellular c-di-GMP levels. Taken together, the findings of this thesis suggest that CF sputum conditions regulate number and functioning of flagella in B. cenocepacia K56-2 through the flhF and BCAL1069 genes, respectively.