Analysis of catabolic plasmids in pseudomonas

Abstract

The experimental work carried out for this thesis concentrated on two separate aspects of the molecular biology of toluenedegradative (Tot) strains of Pseudomonas.
Pseudomonas strain Mr74 harbours the single TOt plasmid pWW74, and this plasmid carries two related genes for catechol 2,3-oxygenase (C230) enzymes. One of these genes had been previously cloned on a 2.05 Kb XhoI DNA fragment to yield the recombinant plasmid pWW74-3016. Internal regions of this cloned XhoI fragment carrying the C230 gene were sub-cloned into 1413 based vectors and analysed by DNA sequencing. The nucleotide sequence of the 5' end portion of the C230 gene comprising 231 nucleotides was determined. Taken together with previous nucleotide sequence data, the first 445 nucleotides of the gene, and its upstream region, are presented here. Comparison of this region with sequence data for other C230 genes has lead to the classification of the pW\474 genes as belonging to the C230 lb gene family.
Pseudomonas cepacia strain MT84 also harbours a large Tot plasmid, pWW84, which also carries two related C230 genes. However, my interest in this strain and its plasmid concentrated on the conjugative transmission of the plasmid pWW84. It was demonstrated that pWW84 could transfer between MT84 donor cells and plasmid-free recipient strains derived from MT84. Analogous matings were carried out using the archetypal Tot plasmid pWWO, which specifies a plate-preferred conjugation system. pWW84 transfer occurred most efficiently in liquid broth matings. rather than plate matings. This observation contrasts with all previous studies on conjugative plasmid transfer, and pWW84 is unique in this respect. Additionally, pWW84 appears to participate in a predominantly strain-specific conjugation process, and is unable to transfer to other Pseudomonas recipients.
Electron microscope analysis of 14T84 and plasmid-free derivatives lead to the discovery of a novel filamentous extracellular
organelle. here designated as a cilus. The probable role of the cilus in conjugal transfer of pWW84 was examined, and models are presented for its possible role in this process.