Date of Award
2007
Degree Type
Thesis
Degree Name
Doctor of Philosophy
Program
Microbiology and Immunology
Abstract
Characterization of Burkholderia cenocepacia K56-2 stress response genes using novel mutagenesis plasmids
Burkholderia cenocepacia is an opportunistic pathogen that causes serious infections in cystic fibrosis patients. The widespread distribution of this bacterium in the environment suggests that B. cenocepacia must adapt to stress to survive. To investigate this we sought to characterize the genes encoding HtrA proteases and the alternative sigma factor RpoE, which are predicted to play a role in the B. cenocepacia stress response.
In B. cenocepacia K56-2 we identified a gene encoding an HtrA serine protease (BCAL2829). Genetic analyses employing mutagenesis and complementation studies revealed that HtrABCAL2829 was required for growth of B. cenocepacia upon exposure to in vitro stresses. Characterization of HtrABCAL2829 also demonstrated that the protein required a predicted catalytic serine residue and the PDZ domains for function. The HtrABCAL2829 protein was shown to localize to the periplasmic compartment and it was demonstrated that inactivation of htrA is associated with a survival defect in vivo.
A gene predicted to encode RpoE, the regulator of the extracytoplasmic stress response was also identified. Mutagenesis and complementation studies revealed that RpoE is required for growth under stress. Inactivation of rpoE also altered the expression of a cell surface associated carbohydrate and outer membrane proteins. Using a macrophage model of infection it was demonstrated that rpoE-deficient B. cenocepacia, unlike wild type bacteria, are trafficked to phago/lysosomes.
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B. cenocepacia carries three htrA genes in addition to htrABCAL2829; however, the inability to easily create multiple mutations in a single B. cenocepacia strain limited our ability to demonstrate a role for the htrA genes BCAL0326, mucD and BCAM1695 for growth under stress. To rectify this we created the plasmids pGPI-Scel and pDAI-SceI for the mutagenesis of B. cenocepacia in an I-Scel-dependent manner. To demonstrate the utility of these plasmids we created several unmarked gene deletions and made multiple mutations in a single B. cenocepacia strain. This tool will permit further investigation of the B. cenocepacia htrA genes and significantly enhances our ability to genetically manipulate B. cenocepacia.
Characterization of the B. cenocepacia stress response has only just begun but our data demonstrate it is important for infection. Further investigation of how this bacterium adapts to stress will help shed light on the molecular mechanisms that B. cenocepacia employs to survive in diverse ecological niches including the respiratory tract of CF patients.
Recommended Citation
Flannagan, Ronald S., "Characterization of Burkholderia cenocepacia K56-2 stress response genes using novel mutagenesis plasmids" (2007). Digitized Theses. 4995.
https://ir.lib.uwo.ca/digitizedtheses/4995