Screening for trbB- and traG-like sequences by PCR for the detection of conjugative plasmids in bacterial soil isolates

Abstract

The transfer regions of different conjugative plasmids show significant similarities in the genetic organization and in the amino acid sequence of some gene products, especially of proteins from the traG or trbB family. These similarities are also evident on the level of the nucleotide sequences. On the basis of conserved DNA regions we designed degenerate PCR primer pairs to detect specifically tra regions within a collection of bacterial clones isolated from an agricultural soil. Most of the potential transfer-proficient indigenous bacterial isolates were able to mobilize a derivative of the nonconjugative IncQ plasmid RSF1010 into recipient strains. With the help of the primers it should be possible to evaluate the genetic potential for horizontal gene transfer carried out by conjugative plasmids.

Diversity and linkage of replication and mobilisation genes in Bacillus rolling circle-replicating plasmids from diverse geographical origins

“…Many DNA sequences are now available for Bacillus replicons and transfer region, facilitating the development of a series of oligonucleotide primers for use in PCR to permit the assessment of the distribution and diversity of replication and transfer functions in Bacillus isolates from natural environments and enable investigation of the linkage of such elements. Such approaches have been utilised successfully in the study of plasmids from Gram-negative bacteria, for example for a series of broad host range plasmids [6], and more recently to study IncN-related plasmid replicons [7] and conjugative plasmid transfer regions [8].…”

Distribution and characteristics of plasmid mobilizers in aquatic and soil environments and activated sludge

“…On the other hand, in Chryseomonas luteola IU-7, the visible plasmids were smaller than the 30 kb needed for encoding the conjugative function (Rochelle et al, 1989). In this strain, plasmids with the conjugative function may be present on non-visible plasmids or megaplasmids (Disqué-Kochem et al, 2001), which are difficult to detect by the method employed in this study.…”

“…Recently, Disqué-Kochem et al (2001) designed universal PCR primers to detect the traG-and trbB-like genes in the tra region, and demonstrated that they could detect bacteria with plasmids capable of mobilizing a Tra Ϫ plasmid from an agricultural soil sample.…”

“…Then, colonies used for screening mobilizers were randomly chosen considering their morphology. Screening of mobilizers was carried out by colony PCR (Joshi et al, 1991) targeting trbB-like DNA sequences by using a universal primer set [B-1, B-2] (Disqué- Kochem et al, 2001). PCR amplifications were performed by the thermal profile of Disqué-Kochem et al (2001) in a Mastercycler Standard (Eppendorf).…”

“…Screening of mobilizers was carried out by colony PCR (Joshi et al, 1991) targeting trbB-like DNA sequences by using a universal primer set [B-1, B-2] (Disqué- Kochem et al, 2001). PCR amplifications were performed by the thermal profile of Disqué-Kochem et al (2001) in a Mastercycler Standard (Eppendorf). Aliquots (5 ml) of PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with 0.5 mg/ml of ethidium bromide solution.…”