Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

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 homo...

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Bibliographic Details
Published in:Environmental microbiology 2020-01, Vol.22 (1), p.45-58
Main Authors: Aparicio, Tomas, Nyerges, Akos, Nagy, István, Pal, Csaba, Martínez‐García, Esteban, Lorenzo, Víctor
Format: Article
Language:English
Subjects:
Acid resistance
Clonal deletion
Clones
Deoxyribonucleic acid
DNA
DNA biosynthesis
DNA replication
E coli
Homology
Mismatch repair
Mutation
Nucleotides
Pseudomonas putida
PyrF gene
Repair
Sensitivity
Soil
Soil bacteria
Soil microorganisms
Yeast
Yeasts
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Summary: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 mutLE36K). 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.
ISSN:1462-2912
1462-2920
DOI:10.1111/1462-2920.14814