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Towards a genetic system for the genus Sulfobacillus

Joubert, Tertia Magdalena (2008-03)

Thesis (MSc (Microbiology))--Stellenbosch University, 2008.


Members of the genus Sulfobacillus form an important part of the microbial consortia that are active in the biooxidation of sulphide ores in biomining processes, yet very little is known about these industrially important organisms. The study of sulfobacilli, and other biomining organisms, is hampered by the absence of effective gene cloning and inactivation systems. During this study, the groundwork was laid for the development of a genetic system for the genus Sulfobacillus. The plasmid diversity present in industrial and environmental isolates of sulfobacilli was assayed. Plasmids were plentiful in the assayed strains, providing the basis for development of cloning vectors for sulfobacilli. Plasmid DNA isolated from Sulfobacillus thermosulfidooxidans strain DSM 9293T was methylated at dam and dcm sites. Whether the methylase enzymes responsible for this methylation pattern form part of restriction-methylation systems or only play a regulatory role is unknown, but it does indicate the appropriate methylation state of DNA for the transformation of this strain. The DNA sequences of three plasmids originating from sulfobacilli were analysed and compared. There was no significant similarity between the three plasmid sequences, indicating diversity in plasmid genetic load and replication mechanisms. Plasmid pSulfBC1 was predicted to replicate via the rolling circle mechanism, while the replication mechanisms of pKara and pTHWX could not be predicted from sequence data. Two antibiotics, chloramphenicol and tetracyline, were found to be suitable for selection of Sulfobacillus transformants. E. coli – Sulfobacillus shuttle vectors were constructed using the Sulfobacillus plasmid, pKara, as the backbone with a Gram-positive chloramphenicol resistance marker and appropriate elements allowing replication in, and mobilization from, E. coli. These shuttle vectors were used in the evaluation of electroporation and conjugation as methods for the delivery of DNA to Sulfobacillus. Transformants of sulfobacilli could not be obtained by either transformation method, although some progress was made towards determining the optimal conditions for both methods. The most promising finding was that cells of E. coli and Sulfobacillus could be maintained on the same medium for a theoretically sufficient time to allow mating. It is likely that Sulfobacillus transconjugants can be obtained with the right combination of donor, mobilizable vector, selectable marker and treatment to neutralize restriction systems.

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