Department of Molecular Genetics and Microbiology
Bacteriophage lambda; Base Sequence; Chromosomes, Bacterial; DNA Primers; DNA, Bacterial; Escherichia coli K12; *Gene Duplication; Polymerase Chain Reaction; Recombination, Genetic
Microbiology | Molecular Biology | Molecular Genetics
BACKGROUND: An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage lambda Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating lambda vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants. RESULTS: A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case. CONCLUSIONS: The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.
DOI of Published Version
BMC Mol Biol. 2004 Dec 13;5(1):22. Link to article on publisher's site
BMC molecular biology
Poteete AR, Fenton AC, Nadkarni A. (2004). Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12. Open Access Articles. https://doi.org/10.1186/1471-2199-5-22. Retrieved from https://escholarship.umassmed.edu/oapubs/315