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Investigations into the microcystin gene cluster from Hapalosiphon hibernicus BZ-3-1 and Planktothrix agardhii CYA 126/8

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Title: Investigations into the microcystin gene cluster from Hapalosiphon hibernicus BZ-3-1 and Planktothrix agardhii CYA 126/8
Authors: Philmus, Benjamin
Keywords: Microcystins -- Hawaii
Cyanobacteria -- Genetics
Issue Date: 2006
Abstract: Hapalosiphon hibernicus BZ-3-1 is a terrestrial cyanobacterium isolated from a soil sample from Maui, Hawaii. It was shown to produce microcystin-LA during screening for protein phosphatase inhibitors. Using a PCR approach involving degenerate primer mixes, which were derived from comparison of the three known gene clusters previously sequenced from Microcystis aeruginosa, Planktothrix agardhii and Anabaeno circinalis. Using the primer mixes the genes mcyA. -B. -C. -G. -N, and -J were amplified in almost their entirety. The genes mcyD and -E did not give amplicons under similar conditions and therefore needed to be amplified in a slightly different manner. Fragments from the 5' and 3' end of each gene (I kb) were amplified with the degenerate primer mixes and then the known fragments were connected together using the gathered sequence. After sequencing the genes, the organization of the gene cluster was established by connecting PCR. The resulting gene cluster yielded two operons, mcyABC and mcyGDJEFIH, which are transcribed in opposite directions from a putative bi-directional promoter. The H. hibernicus cluster shows the greatest similarity to the Anabaena cluster (mcyA. -B. -D. -G. -J) and the nodularin gene cluster from Nodularia (mcy-E. F. -f). It was noticed that the Planktothrix cluster was the only one to contain mcyT and be deficient in mcyI and -F. Genetic studies were then undertaken to disrupt mcyT using homologous recombination. The DNA was introduced into the cyanobacterial cells via electroporation and resistance to chloramphenicol was used to select for the mutant. The mutant was analyzed for peptide production and it was discovered that microcystin biosynthesis had been reduced to 5-10% of that found in the wild type. During characterization of the mutant peptide production a new anabeanopeptin derivative was found Structure determination was accomplished using NMR and HR-MS techniques. Stereochemistry of the amino acid residues was determined by the advanced Marfey analysis using LC-MS.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.
Includes bibliographical references (leaves 72-75).
xiv, 75 leaves, bound ill. 29 cm
URI/DOI: http://hdl.handle.net/10125/20470
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:M.S. - Chemistry



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