An investigation of the replication of the broad-host-range plasmid pTF-FC2

Doctoral Thesis


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University of Cape Town

Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted.