Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains

Abstract

Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of E. coli before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of P. putida KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of P. putida-TRANS ( transference of RP4- activated nucleotide segments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a P. putida double mutant Δ pyrF (uracil auxotroph) Δ edd (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the pyrF ⁺/ edd ⁺ phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10 ^–6 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for P. putida, but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.