Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu

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Abstract
Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants. By incubating the strains 302 and 328 at 42°C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.