Studies on the molecular biology of the cyanobacteria Spirulina maxima

Study on the cyanobacterium Spirulina sp., originated from a growing interest in the commercial production of this algae in the Hawaiian islands. Although the molecular biology and techniques of genetic engineering in cyanobacteria has advanced rapidly in the past few years, the application to Spirulina has been minimal. The objective of this work was therefore to generate a shuttle vector a method of mobilization and clone and characterize the phycobiliprotein genes.;Available strains were screened for plasmids. The uniform response of the test strains to heavy metal and antibiotic tolerance, encouraged all avenues of detection, with only negative results. An attempt to clone a labile megaplasmid or origin of replication was investigated. "Shotgun" library experiments were established to transform the algae and select for the chloramphenicol marker.;Transformation conditions for cyanobacteria are numerous and we attempted to repeat most of them using different vectors and libraries. The only conditions tested showing any increased survivability was when the algae was grown in iron depleted media. However, no cultures survived reselection.;The presence of a restriction system required investigation. Whitehead's procedure for the rapid screening for type II restriction enzymes show an extremely powerful DNAse is present in Spirulina. Wether this is a restriction system or a nonspecific DNAse is not clear. This would limit transformation as a method for gene transfer. The alternative method of conjugation has been attempted, however the plating of the mating mixture on mixed media (Spirulina media + 0.5% LB) resulted in mixotrophic lysis of the algae.;Finally, for the cloning of the phycobiliprotein genes, using the clone pTP1, from Agmenellum quad. (Pilot) as probe DNA, only the hybridization conditions of Lemeaux and Grossman were able to produce signals. An explanation for this is not evident from the base composition, degree of methylation, and C-PC N-terminal amino acid sequence data. Further, colony hybridization was unsuccessful. Only through Southern blots of minipreps were we able to isolate four unique clones. The restriction patterns and preliminary sequence data are unique to each clone. Digesting with BstYI(XhoII) gave identical fragments, common to the alpha probe, for every clone.