Construction of an Efficient Biologically Contained Pseudomonas putida Strain and Its Survival in Outdoor Assays

Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function. We constructed a mini-Tn5 transposon bearing a fusion of the P_lac promoter to the gef killing gene and a fusion of the Pm promoter to the lacI gene plus the positive regulator of the Pm promoter, the xylS gene. This mini-Tn5 transposon was transferred to the chromosome of Pseudomonas putida CMC4, and in culture this strain survived in the presence of 3-methylbenzoate (an XylS effector) and committed suicide in the absence of this aromatic compound. The rate of killing escape was on the order of 10⁻⁸ per cell and per generation. This contained strain and an uncontained control strain were used in outdoor tests performed in the spring-summer and autumn-winter periods to determine their survival in planted and unplanted soils with and without 3-methylbenzoate. In unplanted soils the numbers of both the contained strain and the uncontained strain per gram of soil tended to decrease, but the numbers of the contained strain decreased faster in soils without 3-methylbenzoate. The decrease in the number of CFU per gram of soil was faster in the spring-summer period than in the autumn-winter period. In planted soils survival in the rhizosphere and survival in bulk soil were studied. In the rhizosphere the uncontained control strain tended to become established at levels on the order of 10⁵ to 10⁶ CFU/g of soil regardless of the presence of 3-methylbenzoate. In the bulk soil the numbers of bacterial cells were 2 to 3 orders of magnitude lower. In planted soils the contained strain tended to disappear, but this tendency was more pronounced in the absence of 3-methylbenzoate and occurred faster in the summer assay than in the winter assay. We found no evidence of dispersal of the test strains outside the experimental plots.