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Title: Molecular genetic studies on the biosynthesis and regulation of the calcium-dependent antibiotic (CDA) production by Streptomyces coelicolor A3(2)
Author: Chong, P. P.
ISNI:       0000 0004 2679 0688
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 1998
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Among the four antibiotics known to be produced by Streptomyces coelicolor A3(2), the calcium-dependent antibiotic (CDA) is the least studied. CDA is a peptide antibiotic which is synthesized non-ribosomally by a putative multifunctional peptide synthetase enzyme. Based on this data, the putative cda biosynthetic gene cluster was localized via the PCR homology approach and several cosmids bearing fragments which hybridized to the PCR products were identified. The goals of this project were to characterise these cosmids; to delineate the cda biosynthetic genes and to clone the gene(s) which regulated the biosynthesis of CDA and confer resistance to CDA. To facilitate further analysis of the "cda cosmids", a rudimentary physical map was constructed through partial restriction mapping and conventional mapping. In order to assess the extent of the cda biosynthetic cluster, a gene replacement experiment was conducted; whereby an 8.5 kb region flanked by two Sal/I fragments known to bear the putative CDA peptide synthetase modules was deleted from the chromosome and replaced with a thiostrepton resistance marker. CDA plate bioassay results showed that CDA activity is completely abolished in the resultant Δcda 1 deletion mutant. In parallel, three other Sal/I fragments containing sequences homologous to peptide synthetase core motifs were used in single integration gene disruptions. Two of the fragments disrupted CDA production completely whereas the remaining fragment only disrupted CDA production partially. Two EcoRI-BamHI fragments from near the distal ends of the 85 kb "cda cosmid" cluster were also used in single-crossover gene disruptions and were found to be non-disruptive. DNA sequences from the ends of these fragments were obtained. The fragments which disrupted CDA production had sequences characteristic of non-ribosomal peptide synthetases. The distal fragments bore sequences which resemble those of ABC-transporter enzymes, and fatty acid condensation enzymes, respectively.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available