| dc.contributor.author | Piraee, Mahmood. | en_US |
| dc.date.accessioned | 2014-10-21T12:33:47Z | |
| dc.date.available | 2002 | |
| dc.date.available | 2002 | |
| dc.date.issued | 2002 | en_US |
| dc.identifier.other | AAINQ67651 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/55825 | |
| dc.description | The highly conserved amino acids of FADH-dependent bacterial halogenases were selected as PCR primer target regions. Degenerate primers (MPF1 and MPR2) were designed taking account of codon usage in Streptomyces , and were used successfully with a touch-down (TD) PCR procedure to amplify a halogenase gene fragment from the chloramphenicol (Cm)-producer Streptomyces venezuelae ISP5230. This was the first PCR-assisted cloning of a halogenase gene fragment. The [alpha-32P]dCTP-labeled PCR product hybridized under high stringency conditions with a Southern blot of restriction-digested genomic DNA from S. venezuelae ISP5230, confirming that the amplicon originated from this organism. | en_US |
| dc.description | By screening a genomic library of S. venezuelae with the [alpha-32P]-labeled PCR product as a hybridization probe, a segment of DNA containing a cluster of three ORFs (11, 12 and 13) was isolated; the functions of three ORFs were investigated by gene disruption and HPLC analyses of the culture extracts. That ORF11 encoded an AMP-ligase (CmlK; 368 aa) involved in the chlorination reaction of Cm biosynthesis was determined from the results of gene disruption. CmlK lacks a thiolation site and is believed to be involved in activation of an unknown substrate for chlorination. ORF12 encoded a halogenase (CmlS; 535aa) responsible for chlorination of the antibiotic. Putative NAD(P)H-binding sites are conserved in most of known halogenases, and can be recognized in the N-terminal region of the deduced aa sequence of cmlS. A second motif, resembling the FAD-binding site of monooxygenases, is also present in CmlS. This is believed to be required for the chlorination reaction. Disruption of either cmlK in strain VS1101 or cmlS in strain VS1102 caused accumulation of non-chlorinated congeners of Cm. Culture extracts of the disrupted strains contained mainly corynecin II, which has a propionyl in place of the dichloroacetyl group of Cm. Corynecin II was purified from strain VS1102 and its structure was confirmed by 1H-NMR spectroscopy. The deduced amino acid sequence of ORF13 (CmlT; 292 aa) resembled the sequences of aldo/keto reductases that catalyze reduction of a carbonyl group to the corresponding alcohol. Since disruption of cmlT had no effect on antibiotic production, CmlT does not have a role in Cm biosynthesis. Extending the cloned chromosome sequence into the region downstream of ORF13 located two more ORFs (14 and 15), both of which encoded proteins with unknown functions, and thus could not be assigned a role in Cm biosynthesis. | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 2002. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Molecular. | en_US |
| dc.subject | Biology, Microbiology. | en_US |
| dc.title | Chlorination genes of chloramphenicol biosynthesis in Streptomyces venezuelae. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
