title: Stability by multimer resolution of pJHCMW1 is due to the Tn1331 resolvase and not to the Escherichia coli Xer system.
abstract: The plasmid pJHCMW1 encodes resistance to several aminoglycosides and beta-lactams and consists of a copy of the transposon Tn1331, a region including the replication functions, and a sequence with homology to ColE1 cer, designated mwr. In this work, the role of this cer-like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied. The Escherichia coli Xer site-specific recombination system acts at sites such as ColE1 cer to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance. Despite its high similarity to ColE1 cer, the pJHCMW1 mwr was a poor substrate for Xer recombination in E. coli and did not contribute significantly to plasmid stability. Instead, the Tn1331 co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1. Although Xer recombination at pJHCMW1 mwr was inefficient in E. coli, the recombination that did occur was dependent on ArgR, PepA, XerC and XerD. A supercoiled circular DNA molecule containing two pJHCMW1 mwr sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD in vitro, confirming that pJHCMW1 mwr is a functional recombination site. However, unlike cer, some Holliday-junction-containing product could be detected for mwr in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions. Binding experiments demonstrated that XerD bound to pJHCMW1 mwr core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD.
authors.affiliation: Institute of Molecular Biology and Nutrition, Department of Biological Science, School of Natural Science and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA2.|Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
authors.forename: Marcelo E
authors.initials: ME
authors.lastname: Tolmasky
authors.affiliation: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
authors.forename: Sean
authors.initials: S
authors.lastname: Colloms
authors.affiliation: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
authors.forename: Garry
authors.initials: G
authors.lastname: Blakely
authors.affiliation: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
mesh_terms: D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D019426:Integrases; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D045522:Recombinases; D011995:Recombination, Genetic; D019895:Transposases / Q000378:metabolism*; D045564:Transposon Resolvases
chemical_list: D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D045522:Recombinases; C473360:XerC protein, E coli; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D019895:Transposases; D045564:Transposon Resolvases; C473361:XerD protein, E coli; C115006:integron integrase IntI1