Thesis--Ph. D., University of Hong Kong. / Typewritten.

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When Pseudomonas aeruginosa ATCC 9027 var. RCII, a chloramphenicol-tolerant substrain, is grown in a phosphate limited, complex medium, along with the drug (150 (mu)g/ml), it accumulates high intracellular levels of inorganic phosphate and fails to synthesize normal levels of alkaline phosphatase and pyocyanine. Glucose transport is additionally hindered, and, accordingly, extracellular glucose is mainly oxidized to 2-ketogluconate. The preference of NAD(H)-linked enzymatic activities suggests the absence of transhydrogenase activity. The cytochrome content and intracellular ATP pool of this substrain are also greater. The ATP pool is further augmented when chloramphenicol is omitted from the medium. H('+)/O analysis confirmed that the substrain gained one additional ATP conservation site. Drug tolerance in P. aeruginosa ATCC 9027 is clearly accompanied by greater energy production. Slower growth arises since more energy is delegated towards maintenance and survival in the presence of the drug.

Intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa has mainly been attributed to the presence of several chromosomally-encoded multidrug efflux systems. The MexAB-OprM system exports the largest range of structurally unrelated antimicrobial agents and its expression is modulated by multiple regulatory controls. To develop a better understanding of mexAB-oprM overexpression in nalC mutants, which characteristically produce the effector protein PA3719 that binds and disrupts MexR transcriptional repression of mexAB-oprM, the PA3719-MexR interaction domains were investigated. Using a bacterial two-hybrid system, the C-terminus of PA3719 was found to be sufficient to mediate MexR-binding, and the binding region was found to be distinct from the MexR DNA-binding motif. The two-hybrid system was also used in an attempt to understand the role of PA3720, a protein of unknown function that is also overexpressed in nalC mutants. Results from this study confirm that PA3720 does not function to bind and alleviate NalC transcriptional repression of the PA3720-PA3719 operon. This study also attempted to identify the signals involved in overexpressing PA3720-PA3719, in the hopes to elucidate the natural function of MexAB-OprM. Random transposon mutagenesis using a PA3720-PA3719 promoter-lacZ fusion containing P. aeruginosa strain was conducted, but failed to clearly identify any disrupted genes associated with PA3720-PA3719 overexpression. Using the same PA3720-PA3719 promoter-lacZ fusion, expression of these genes was assessed as a function of growth in both wildtype and nalC mutant P. aeruginosa strains. Interestingly, PA3720-PA3719 expression was found to be growth-regulated, with an increased amount of expression occurring in late log/early stationary phase, even in the absence of nalC. This suggests that another regulator(s) is/are involved in modulating PA3720-PA3719 levels in late log/early stationary phase. Since PA3719 ultimately influences mexAB-oprM expression, its involvement in mediating growth-phase mexAB-oprM expression was assessed by examining mexA expression in both wildtype and PA3719 deletion P. aeruginosa strains. PA3719 was found to be involved in some, but not all, of the growth phase control of mexAB-oprM. These results suggest that mexAB-oprM growth-phase regulation is complex, as both MexR-dependent and MexR-independent regulatory pathways seem to exist. Overall, this study has produced a better understanding of mexAB-oprM regulation in nalC mutant P. aeruginosa strains. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-09-25 19:00:42.929