Molecular analysis of quinoline metabolism in Pseudomonas aeruginosa QP
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Quinoline is a toxic nitrogenous heterocyclic compound that accumulates in the environment as a result of incomplete combustion of fossil fuels. Microbiological degradation of quinoline is an important mechanism in its removal. A Pseudomonas aeruginosa strain with this ability was isolated previously. This strain (QP) was transposon mutagenised to isolate quinoline degradation deficient mutants (Qln-). Putative mutants were isolated using a two-step screening process. Mutants failing to grow on minimal salts agar plates supplemented with quinoline as the sole source of carbon, nitrogen and energy were subsequently incubated in an analogous liquid medium. UV spectrophotometry of the cell free media was used to confirm their stability, as quinoline has a characteristic UV absorption spectrum in aqueous solution, any change in this spectrum revealed a modification to the molecule. Two of the five isolates judged Qln- following the plate assay, grew in the quinoline/minimal salts liquid medium with concomitant loss of quinoline, albeit slower than the wild type parent and were not studied further. The three remaining mutants could not grow in quinoline media without supplementary glucose. The UV spectra of cell-free media (containing both glucose and quinoline) taken from cultures of the remaining 3 Qln- mutants were identical. Spectral data suggested that the 3 mutants accumulated a single compound, this was identified by NMR analysis as 8-hydroxycoumarin. In all three Qln- mutants, the transposon had inserted into the same 8.6 kb Bg/11 fragment of the genome. This Bg/ll fragment containing the transposon insertion was cloned (pNHQ8) and a restriction map generated. Using subclones generated from pNHQ8 it was possible to screen a library of restriction fragments from the wild-type strain and clone the wild-type fragment. This fragment was extended using uninterrupted sequences from the mutant strain. The full size fragment could complement the Qln- mutants back to the quinoline degrading phenotype. The complementing plasmid was used to transform a non-quinoline degrading P. aeruginosa. This transformant could modify 8-hydroxycoumarin to a compound tentatively identified as 2,3-dihydroxyphenylpropionic acid, indicating that this plasmid encoded an enzyme. From these clones, approximately 3 kb of DNA sequence data was generated. A previous study of P. aeruginosa QP indicated that quinoline degradation was associated with four large plasmids. However, this study failed to isolate any plasmid DNA from this organism or connect the quinoline degrading phenotype with plasmids through a variety of techniques. During the course of the study it was observed that P. aeruginosa QP strains exhibited a high degree of DNA restriction, which could be partially overcome by growing the organism at 43°C. It was also observed that the organism rapidly degraded chromosomal DNA upon cell lysis. It was hypothesised that QP strains produced exonucleases. Both these characteristics provided challenges to the genetic manipulation of this organism.