The transfer and stability of the dehalogenase I gene of Pseudomonas putida PP3

• Main Author: Beeching, John Rutland
• Published: University of Warwick 1984
• Subjects: 577; QR Microbiology
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354605

An R-prime, pUU2, derived from the broad host-range plasmid R68.44+ was generated carrying the dehalogenase I gene from Pseudomonas putida PP3. This R-prime enabled its host to use 2-monoch1oropropionic acid (2MCPA) as sole carbon and energy source. The R-prime was studied by restriction endonuclease analysis. The process of R-prime formation was examined in detail and was shown to have suffered from interference from an insertion sequence originating from the PP3 chromosome. The dehalogenase I PP3 chromosomal insert was shown to interfere with the plasmid transfer functions and to be capable of translocation within the plasmid. A Pseudomonad containing the R-prime pUU2 was grown under phosphate limitation on 2MCPA in a chemostat. During 2600h. a range of mutants were isolated in which the plasmid was shown to have undergone several modifications including the acquisition of lOkb of novel DNA, the transfer of the dehalogenase I gene to the chromosome, and in some cases the concomitant loss of the plasmid. A new range of 2Mcpa+ R-prime plasmids were generated using a chemostat-derived plasmid-minus strain and R68.A5. These demonstrated that though the insertion sequence IS21 of R68.A5 may be involved in R-prime formation it need not be intimately associated with the chromosomal insert into the plasmid, at least in the case of the dehalogenase I gene. This, together with the frequency of R-prime formation, lends support to the view that the dehalogenase I gene may be on a transposable element. Restriction fragments from the R-prime pUU2 were cloned into pAT153 and pKT231. Clones were isolated capable of growth of 2MCPA and their plasmids were analysed by restriction digestion.