Dataset Information

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

ABSTRACT: The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene. The high frequencies of marker excision obtained with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event. Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida. This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.

SUBMITTER: Kristensen CS

PROVIDER: S-EPMC176555 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

Kristensen C S CS Eberl L L Sanchez-Romero J M JM Givskov M M Molin S S De Lorenzo V V

Journal of bacteriology 19950101 1

The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA fla ...[more]

PMID: 7798149

Similar Datasets

Project description:The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.

Dataset Information

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

Publications

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets