Results and Discussion

The PCR products corresponding to the joint points of predicted co-integrations among the three replicons (chromosome-megaplasmid, chromosome-pSym, megaplasmid-pSym) were detected in the culture of the wild-type strain NGR234. A derivative population, CFNX416, possessing a co-integration between the pSym and the megaplasmid was isolated by artificial selection and confirmed by different techniques: plasmid profile, pulse-field gel electrophoresis (PFGE), Southern hybridization with specific replicón probes (nifH and exoBDFLK genes). The size of this co-integrated plasmid is estimated to be more than 2500 kb. Such rearrangement has no apparent influence on the growth in both rich and minimal media. After 30 days' culture (almost 200 generations) in liquid medium, transferred every day, most of the cells (more than 90%) still contained the co-integrated structure. Reversion to the wild type genome architecture and the partition of the co-integrate into new sized replicons were also observed. Considering that no exogenous DNA was introduced and that the experiments were performed without any selective pressure, the results suggest that: (i) the natural genomic design strategy is efficient for the artificial selection of alternative genome structures; and (ii) the bacterial genome is capable of rapid evolution that can be observed in action.

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