Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis

Abstract

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xyleminhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.

Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis

“…To achieve the maximum field strength employing the Bio-Rad Gene Pulser (Bio-Rad, Richmond, Calif.), 0.2-cm cuvettes were used with settings of 100 fl, 25 ,uF, and 2.5 kV. Immediately after voltage application, the cells were diluted in 400 ,ul of cold S27 broth (23), spread onto a 0.2-p.m cellulose acetate membrane (Micro Filtration Systems, Dublin, Calif.), and placed on the agar surface of SC plates (23). After 16 h of incubation at 30°C, the membranes were transferred to SC plates containing 2 p.g of tetracycline per ml.…”

“…The occurrence of segregant colonies having lost the introduced genes has been studied with recombinant bacterial strains in both corn and bermuda grass. The methods for the culture, inoculation, sampling, and detection of recombinant bacterial strains in greenhouse-grown bermuda grass plants have been described previously (23). Eighteen bermuda grass plants were sampled at 6, 11, 18, and 25 weeks after inoculation.…”

“…In order to further characterize the segregants, two segregant cultures were arbitrarily chosen for in vitro growth rate comparisons with strains MDR1.586 and MDE1. Methods for the growth rate determination have been described previously (23).…”

“…kurstaki HD73 fused with a kanamycin resistance gene into the chromosome of C. xyli subsp. cynodontis by homologous recombination (23). This prototype strain (MDR1.3) exhibited activity against the ECB in diet assays but not in planta.…”

“…We have previously shown that growth rate differences between a recombinant strain of C. xyli subsp. cynodontis (strain MDR1.3) containing an integrated plasmid with multiple sites of homology to the chromosome and segregants of this strain were responsible for * the increase of segregant populations(23). rate studies must be done to determine whether this is iR1.586 DNA digeste actually the case for cells that have colonized bermuda grass uith Bglll.and corn.…”