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

Tomas Aparicio, Akos Nyerges, István Nagy, Csaba Pal, Esteban Martínez-García, Víctor de Lorenzo

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
JournalEnvironmental Microbiology
DOIs
Publication statusAccepted/In press - Jan 1 2019

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Pseudomonas putida
single-stranded DNA
DNA Mismatch Repair
repair
Nucleotides
nucleotides
gene
Prokaryotic Cells
Genes
Bacteria
DNA
bacterium
genes
Single-Stranded DNA
soil bacteria
DNA replication
prokaryotic cells
DNA Replication
yeast
clone

ASJC Scopus subject areas

  • Microbiology
  • Ecology, Evolution, Behavior and Systematics

Cite this

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene. / Aparicio, Tomas; Nyerges, Akos; Nagy, István; Pal, Csaba; Martínez-García, Esteban; de Lorenzo, Víctor.

In: Environmental Microbiology, 01.01.2019.

Research output: Contribution to journalArticle

Aparicio, Tomas ; Nyerges, Akos ; Nagy, István ; Pal, Csaba ; Martínez-García, Esteban ; de Lorenzo, Víctor. / Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene. In: Environmental Microbiology. 2019.
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