Environmental Performance of Pseudomonas putida with a Uracylated Genome

• Published in: Chembiochem : a European journal of chemical biology 2020-11, Vol.21 (22), p.3255-3265
• Main Authors: Algar, Elena, Al‐Ramahi, Yamal, Lorenzo, Víctor, Martínez‐García, Esteban
• Format: Article
• Language: English
• Subjects: Base excision repair; biocontainment; Conjugation; Containment; Deoxyribonucleic acid; Dispersal; DNA; DNA glycosylase; E coli; Environmental management; Environmental performance; Gene transfer; Genomes; GMO escape; horizontal gene transfer; Horizontal transfer; Hydrolase; Mutagenesis; Nucleotides; Plasmids; Proteins; Pseudomonas putida; Soil bacteria; Soil microorganisms; Thymine; Uracil; Uracil-DNA glycosidase
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202000330

A variant of the soil bacterium Pseudomonas putida with a genome containing a ∼20 % replacement of the whole of thymine (T) by uracil (U) was made by deleting genes ung (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 Δung Δdut 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 Δung Δdut 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. Stay where you are! The Δung and Δdut mutations enable partial incorporation of U residues to DNA instead of T, thus making the helix vulnerable to the action of U‐DNA glycosylases. This could suggest a general strategy for curbing the dispersal of recombinant genetic constructs into the environment.