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Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1
1Department of Biochemistry, School of Biological Sciences, University College of North Wales, Gwynedd, UK.
Abstract:
Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.
Insights
Researchers created cointegrate plasmids by combining the R-plasmid RP4 with TOL catabolic plasmids from Pseudomonas putida. This study details the genetic makeup and evolution of these TOL (toluene/xylene) plasmids.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Broad-host-range R-plasmid RP4 was used to create cointegrate plasmids.
- Two catabolic plasmids from Pseudomonas putida HS1, pDK1 (wild-type TOL) and pDKT1 (deletion derivative), were utilized.
Purpose of the Study:
- To characterize cointegrate plasmids formed in vivo between RP4 and TOL catabolic plasmids.
- To investigate the genetic organization and evolutionary implications of TOL plasmids.
Main Methods:
- Formation of cointegrate plasmids in vivo.
- Restriction mapping and subcloning of plasmid DNA.
- Enzyme assays in Escherichia coli and Pseudomonas hosts.
- Hybridization studies.
Main Results:
- Two cointegrate plasmids, pDK2 (40 kbp insert) and pDKT2 (20 kbp insert), were constructed.
- pDK2 contained both upper and meta pathway operons and the xylS regulator gene.
- pDKT2 contained only the upper pathway operon and the regulatory region.
- High similarity was found with TOL plasmids pWW0 and pWW53-4 in gene order and restriction sites, but differing orientations.
Conclusions:
- The study provides detailed genetic maps of novel TOL plasmid cointegrates.
- Findings offer insights into the structural organization and potential evolution of TOL plasmids.
- Comparative analysis highlights similarities and differences with other known TOL plasmids.