The object of this proposal is to develop a use a genetic system to study the synthesis and regulation of toxin and phycobilisome production in Microcystis aeruginosa UV027, a fresh water cyanobacterium implicated as a potential health hazard. A lambda ZAP II M. aeruginosa UV027 genomic DNA library, which we made, will be screened for relevant genes of interest. Heterologous probes will be used to isolate a peptide synthetase gene that is implicated in the biosynthesis of the toxin, microcystin, a protein phosphatase inhibitor. Microcystins are synthesized non-ribosomally. Once characterized, the peptide synthetase gene, under the control of an inducible promoter, will be cloned into the shuttle vector and used to transform Microcystis strains lacking the ability to make microcystins. Altered genes will be used to attempt to inhibit microcystin production in toxin-producing strains. Phycobilisome studies will be initiated by using a heterologous probe to isolate the genome for the alpha-subunit of allophycocyanin. Once characterized, this gene will be used to isolate and characterize the allophycocyanin operon. Mutated genes will be cloned into the shuttle vectors pMaL or pMaL-7, which we constructed, to transform Microcystis to study phycobilisome assembly. Regulatory gene and transcriptional DNA consensus sequences recently reported for several cyanobacteria will be used to search for similar sequences in Microcystis. Once found, downstream and upstream sequences will be obtained to determine which genes are being regulated and the regulatory mechanism(s) involved. The potential for controlling toxin production and growth of the cyanobacterium will also be examined.
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