Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4.
The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.
Eberl
L
L
Institut für Biotechnologie, Arbeitsgruppe Genetik, Technische Universität Graz, Austria.
Kristensen
C S
CS
Givskov
M
M
Grohmann
E
E
Gerlitz
M
M
Schwab
H
H
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D027101:DNA Topoisomerase IV; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D016366:Open Reading Frames; D009876:Operon; D011485:Protein Binding; D014176:Protein Biosynthesis; D011815:R Factors / Q000235:genetics*; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D019895:Transposases
D001426:Bacterial Proteins; D003062:Codon; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C052883:chromosome partition proteins, bacterial; D009713:Nucleotidyltransferases; D019895:Transposases; D027101:DNA Topoisomerase IV; D004264:DNA Topoisomerases, Type I