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 - TY - JOUR AU - Ricaurte, DE AU - Martinez-Garcia, E AU - Nyerges, Ákos AU - Pál, Csaba AU - de Lorenzo, V AU - Aparicio, T TI - A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities. JF - MICROBIAL BIOTECHNOLOGY J2 - MICROB BIOTECHNOL VL - 11 PY - 2018 IS - 1 SP - 176 EP - 188 PG - 13 SN - 1751-7907 DO - 10.1111/1751-7915.12846 UR - https://m2.mtmt.hu/api/publication/3315234 ID - 3315234 AB - Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli lambda phage recombinase beta - an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recbeta homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus-wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recbeta functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads - and beyond. LA - English DB - MTMT ER - TY - JOUR AU - Nyerges, Ákos AU - Csörgő, Bálint AU - Nagy, István AU - Bálint, Balázs AU - Bihari, Péter AU - Lázár, Viktória AU - Apjok, Gábor AU - Umenhoffer, Kinga AU - Bogos, Balázs AU - Pósfai, György AU - Pál, Csaba TI - A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species JF - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA J2 - P NATL ACAD SCI USA VL - 113 PY - 2016 IS - 9 SP - 2502 EP - 2507 PG - 6 SN - 0027-8424 DO - 10.1073/pnas.1520040113 UR - https://m2.mtmt.hu/api/publication/3038670 ID - 3038670 AB - Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species. LA - English DB - MTMT ER -