Evaluation of the biological containment system based on the Escherichia coli gef gene in Pseudomonas aeruginosa W51D.

Abstract

The cell-killing gef gene was introduced, under the control of the lac promoter and as part of a transposon, into Pseudomonas aeruginosa W51D, a strain able to degrade branched-chain alkylbenzene sulfonates. Only 1% of the cells that inherited the transposon (Tngef) showed a conditional lethal phenotype, and this phenotype was lost at a high frequency without apparent loss of the tetracycline resistance encoded by the transposon. Southern blot analysis of two W51D::Tngef derivatives that expressed the cell-killing function showed multiple insertions of the transposon. These data suggest that Gef protein is able to kill P. aeruginosa W51D, but it seems that the level of resistance to Gef toxin in this stain is higher than that of previously reported bacteria, and that the expression of multiple copies of the gef gene is necessary to attain cell death. The higher level of resistance does not seem to be particular to strain W51D since two other P. aeruginosa strains analyzed (PAO2003 and ATCC 9027) also presented a small proportion of cells expressing the conditional lethal phenotype when they inherited the Tngef transposon.