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Title: Genetic analysis of ecgonine degradation by Pseudomonas fluorescens (MBER)
Author: Griffiths, M. B.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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This study details a genetic investigation of ecgonine degradation by P. fluorescens MBER. Transposon (Tn5) insertional inactivation mutagenesis was performed to create mutants blocked in the ecgonine degradative pathway. By this method, 87 such mutants were isolated and analysed to identify where in the genome the interruption had occurred. At the whole cell and cell-free extract level, no intermediary metabolites were detected in these mutants by either TLC or GC/MS analysis. The genetic location of Tn5 in the DNA was therefore investigated to obtain sequence data around the insertion site which might identify the gene into which insertion occurred. This genetic analysis revealed that the transposon had inserted in an identical fragment in all 87 mutants, indicating a hyper-insertion region in the DNA. Sequence data flanking the insertion site did not exhibit homology to any known sequences in world-wide databases; the identity of the interrupted gene currently remains unknown. Co-isolated with the unidentified sequence, however, was DNA exhibiting high homology to a phenol hydroxylase, and a benzene monooxygenase. Initial enzyme induction studies revealed that the meta-cleavage pathway associated with catechol metabolism in P. fluorescens MBER was induced by growth on ecgonine as the sole source of carbon. Putative evidence obtained during the course of this study infers that aromatic degradative enzymes may be involved in the later stages of ecgonine degradation. The results obtained are also discussed in the light of subsequent work, which discovered the unidentified DNA interrupted by Tn5 insertion resulting in loss of ecgonine degrading ability was not co-located with the putative benzene monooxygenase and phenol hydroxylase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available