The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic
cells have for ensuring fidelity of DNA replication. While the canonical MMR of E.
coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads
putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this
abridged system to different types of mismatches is unknown. In this background, sensitivity
to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering
of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos
representative of every possible mispairing under either wild-type conditions, permanent
deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible
dominant negative allele mutL(E36K)). Analysis of single nucleotide mutations borne
by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed
prohibited and tolerated single-nucleotide replacements and exposed a clear grading
of mismatch recognition. The resulting data unequivocally established the hierarchy
A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent
in Pseudomonas putida. This information is vital for enabling recombineering strategies
aimed at single-nucleotide changes in this biotechnologically important species.
