Association of Mu-containing plasmids with the Escherichia coli chromosome upon prophage induction

Abstract

To determine the structure of a prophage-containing plasmid during Mu transposition, the physical state of pSC101::Mucts after thermoinduction was monitored. The fate of a mini Mu plasmid constructed in vitro by deleting 27 kbases from the center of the Mu prophage in pSC101::Mucts was also examined. At various times after prophage induction, DNA was extracted from Mu or mini Mu plasmid-containing strains and subjected to electrophoresis in low concentration agarose gels followed by transfer of the DNA to nitrocellulose paper. Separate hybridization with 32P-labeled pSC101 and Mu DNA revealed the position of the plasmids and the replication of Mu DNA. At times after induction when Mu replication was clearly visible, Mu and mini Mu plasmids were found to migrate with E. coli DNA. This Mu-specific association requires the phage coded A and B proteins. EM showed that some of the associated DNA is comprised of circular plasmid molecules which appear to be in contact with the chromosomal DNA. These structures may represent intermediates or end products of the replication-integration process. The finding that Mu and mini Mu plasmids do not give rise to any detectable excision products and apparently remain intact during Mu transposition supports the proposal that the predominant event after Mu induction is the replication of Mu DNA in situ to generate integrative intermediates.