As Pseudomonas aeruginosa adapts to the Cystic Fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lung environments, mucoid strains often appear as a result of alginate overproduction. Such mucoid conversion is associated with the establishment of a chronic pulmonary infection. Alginate confers resistance to phagocytosis and has other pathogenic properties. The regulation of alginate production is complex and involves an alternate sigma factor, anti-sigmas and several DNA-binding transcriptional regulators. Here we examined the possibility that the catabolic repression control (Crc) protein repressor may affect alginate gene expression. A putative Crc binding site was observed adjacent to the ribosome binding site of algD, the first gene in the 12-gene alginate biosynthetic operon. We hypothesized that Crc binding here would act as a repressor of algD expression. Taking a genetic approach, Gateway technology was used to construct crc::GmR (nonpolar) mutants of P. aeruginosa strain PAO1 and its mucoid (mucA) mutant derivative, PDO300. The crc mutation had the expected phenotypes with respect to pyocyanin production, biofilm formation and diauxic growth. When a PalgD-lacZ (translational) fusion was tested, the crc mutant showed increased algD expression as predicted for a mRNA-binding repressor. Another Ptrc-algD-lacZ (translational) construct, but missing the upstream promoter (PalgD) elements, also showed increased activity in crc mutants as predicted if Crc was acting directly as a repressor of algD transcriptional / translational expression. However, this was not consistent with the production of alginate. The crc mutant of mucoid PDO300 showed lower levels of alginate production than its parent strain. Under conditions were the algD operon was induced by ammonium metavanadate in the growth medium to produce alginate, the crc mutation again resulted in a lower level of alginate production than wild-type, which was again inconsistent with the algD-lacZ expression data. This suggests that crc mutation, which has global effects on carbon flow in the cell, could be affecting metabolic pathways that feed precursors into the alginate biosynthetic pathway. Future directions for this research are discussed.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1537 |
| Date | 26 July 2013 |
| Creators | Alam, Arfeen |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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