Trimethoprim-Produced F-Specific Insertion Mutations in Escherichia coli K-12 |
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| Authors: | Pradip Mukhopadhyay and Sunil Palchaudhuri |
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| Abstract: | Besides producing thymine-requiring mutants (thy), trimethoprim (TMP) cured the mini-ColE1 replicon pML21 at an appreciable frequency. The cured Escherichia coli K-12 cells behaved like polA mutants by failing to support the stable maintenance of the ColE1 plasmid. The mini-F replicon pSC138, which was lacking all three insertion sequences (IS3, gammadelta, and IS2) normally used for F-specific integration and excision, was not cured by TMP. Instead, it integrated into specific regions of the E. coli chromosome and thus caused auxotrophic mutations in operons which were always localized on either side of oriC (origin of chromosomal replication). The incompatibility and replication functions of the integrated plasmid in auxotrophs were retained, and the plasmid DNAs recovered from spontaneously occurring revertants did not show any alterations in their contour lengths as determined by electron microscopy. The F replicon (fragment 5) contained in plasmid pSC138 carried two origins of replication, the primary origin, oriV(1) at 42.6F and the secondary origin, oriV(2), at 44.1F. Another mini-F plasmid pMF21, deleted of the primary origin of replication (oriV(1)), was still capable of autonomous replication but failed to integrate onto the chromosome after TMP treatment. Furthermore, the composite plasmid pRS5, which normally uses only the replication origin and functions of the pSC101 component, was also insensitive to TMP. On the basis of these results, we propose a new scheme of F integration via the functional oriV(1) and suggest the involvement of a similar mechanism in the formation of Hfr strains by integrative suppression. |
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