In order to understand the regulatory mechanisms involved in the coordinate expression of the PBS subunits, a search for a means of introducing exogenous DNA into Microcystic was initiated. The primary goal of this project is to develop a genetic system for studying this ecologically important cyanobacterium. We recently isolated an 8kb plasmid, named pMa025, from M.aeruginosa UV025. A hybrid plasmid was constructed by ligating pMa025 into pUC19. The resultant recombinant plasmid, pMaUC, is being maintained in E. coli XLI Blue. Our objective is to use this potential shuttle vector to introduce altered genes in Microcystic. The work to be carried out during this project includes the following. 1) Restriction mapping of pMa025 to enable us to a) delete segments to determine the approximate location of replicon sequences to ensure a viable plasmid and b) determine insertion sites for constructing recombinants. 2) Complete sequencing of the plasmid to ascertain if any information, such as open reading frames and possible procaryotic consensus promoter sequence, are present on the plasmid. This should provide insight regarding plasmid function(s). 3) Construction of a shuttle vector using the hybrid plasmid pMaUC. The appearance of ampicillin resistance provides evidence that pMaUC can transform M. aeruginosa. The vector will be streamlined to induce only pUC19, the genes involved in the replication and maintenance of pMa025 and a kanamycin resistance cassette. The resulting shuttle vector will be tested for its ability to introduce normal or altered genes into Microcystis and into E. coli. 4) Introduction of modified genes (after deletion or inactivation of endogenous genes) into Microcystic using the shuttle vector. Isolation of PBS subunit genes will be undertaken and methods will be developed for successful transformations using these or modified genes. 5) Characterization of pMa025 in association with its host. Very little is presently known regarding how cyano bacterial plasmids are maintained in vivo. In addition to identifying the replicon region, studies of the in vivo properties of pMa025 will be undertaken. These will include examining the mechanisms of plasmid partitioning and stability, quantitating copy number and assaying for variations in copy number with growth phase. 6) The possible role of plasmids in Microcystic toxin production will be evaluated.
