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Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene
Author(s) -
Aparicio Tomas,
Nyerges Akos,
Nagy István,
Pal Csaba,
MartínezGarcía Esteban,
Lorenzo Víctor
Publication year - 2020
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/1462-2920.14814
Subject(s) - biology , recombineering , genetics , gene , pseudomonas putida , dna mismatch repair , dna repair , dna , computational biology , plasmid
Summary 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.