Environmental Performance of Pseudomonas putida with a Uracylated Genome

A variant of the soil bacterium Pseudomonas putida with a genome containing a similar to 20 % replacement of the whole of thymine (T) by uracil (U) was made by deleting genesung(uracil DNA glycosylase) and dut(deoxyuridine 5 '-triphosphate nucleotide hydrolase). Proteomic comparisons revealed that, of 281 up-regulated and 96 down-regulated proteins in the Delta ung Delta dut cells, as compared to the wild-type, many were involved in nucleotide metabolism. Unexpectedly, genome uracylation did not greatly change the gross environmental endurance profile of P. putida, increased spontaneous mutagenesis by only twofold and supported expression of heterologous proteins well. As U-enriched DNA is potentially degraded by the base excision repair of recipients encoding a uracil DNA glycosylase, we then tested the spread potential of genetic material originating in the Delta ung Delta dut cells either within the same species or in a commonly used Escherichia coli strain. Transformation and conjugation experiments revealed that horizontal gene transfer of U-containing plasmids fared worse than those made of standard DNA by two orders of magnitude. Although this figure does not guarantee the certainty of containment, it suggests a general strategy for curbing the dispersal of recombinant genetic constructs.