TY - JOUR AU - Aparicio, Tomas AU - Nyerges, Ákos AU - Nagy, István AU - Pál, Csaba AU - Martinez-Garcia, Esteban AU - de, Lorenzo Victor TI - Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene JF - ENVIRONMENTAL MICROBIOLOGY J2 - ENVIRON MICROBIOL VL - 22 PY - 2020 IS - 1 SP - 45 EP - 58 PG - 14 SN - 1462-2912 DO - 10.1111/1462-2920.14814 UR - https://m2.mtmt.hu/api/publication/30927710 ID - 30927710 N1 - Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid, 28049, Spain Synthetic and Systems Biology Unit, Institute of Biochemistry, Hungary Sequencing Platform, Biological Research Centre, Hungarian Academy of Sciences, Szeged, H-6726, Hungary Sequencing Laboratory, SeqOmics Biotechnology Ltd., Mórahalom, 6782, Hungary Cited By :5 Export Date: 8 December 2020 CODEN: ENMIF Correspondence Address: Martínez-García, E.; Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de CantoblancoSpain; email: emartinez@cnb.csic.es Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid, 28049, Spain Synthetic and Systems Biology Unit, Institute of Biochemistry, Hungary Sequencing Platform, Biological Research Centre, Hungarian Academy of Sciences, Szeged, H-6726, Hungary Sequencing Laboratory, SeqOmics Biotechnology Ltd., Mórahalom, 6782, Hungary Cited By :7 Export Date: 25 August 2021 CODEN: ENMIF Correspondence Address: Martínez-García, E.; Systems and Synthetic Biology Program, Spain; email: emartinez@cnb.csic.es AB - 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. LA - English DB - MTMT ER - TY - JOUR AU - Nyerges, Ákos AU - Csörgő, Bálint AU - Draskovits, Gábor AU - Kintses, Bálint AU - Szili, Petra AU - Ferenc, Györgyi AU - Révész, Tamás AU - Ari, Eszter AU - Nagy, István AU - Bálint, Balázs AU - Vásárhelyi, Bálint Márk AU - Bihari, Péter AU - Számel, Mónika AU - Balogh, Dávid AU - Papp, Henrietta AU - Kalapis, Dorottya AU - Papp, Balázs AU - Pál, Csaba TI - Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance. JF - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA J2 - P NATL ACAD SCI USA VL - 115 PY - 2018 IS - 25 SP - E5726 EP - E5735 PG - 10 SN - 0027-8424 DO - 10.1073/pnas.1801646115 UR - https://m2.mtmt.hu/api/publication/3390047 ID - 3390047 N1 - COIS Conflict of interest statement: A.N., B.C., B.K., and C.P. have filed a patent : application toward the European Patent Office. I.N., B.B., B.M.V., and P.B. had : consulting positions at SeqOmics Biotechnology Ltd. at the time the study was : conceived. SeqOmics Biotechnology Ltd. was not directly involved in the design : and execution of the experiments or in the writing of the manuscript. Hiányzó szerző: 'http' Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, 6726, Hungary Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged, 6720, Hungary Nucleic Acid Synthesis Laboratory, Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, 6726, Hungary Department of Genetics, Eötvös Loránd University, Budapest, 1053, Hungary Sequencing Laboratory, SeqOmics Biotechnology Ltd., Mórahalom, 6782, Hungary Sequencing Platform, Institute of Biochemistry, Biological Research Centre of the Hungarian, Academy of Sciences, Szeged, 6726, Hungary Department of Microbiology and Immunology, University of California, San Francisco, CA 94143 Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian, Academy of Sciences, Szeged, 6726, Hungary Cited By :20 Export Date: 8 December 2020 CODEN: PNASA Correspondence Address: Nyerges, Á.; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of SciencesHungary; email: nyerges.akos@brc.mta.hu AB - Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology. LA - English DB - MTMT ER -