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 - Hueso-Gil, Angeles AU - Nyerges, Ákos AU - Pál, Csaba AU - Calles, Belén AU - de Lorenzo, Víctor TI - Multiple-Site Diversification of Regulatory Sequences Enables Interspecies Operability of Genetic Devices. JF - ACS SYNTHETIC BIOLOGY J2 - ACS SYNTH BIOL VL - 9 PY - 2020 IS - 1 SP - 104 EP - 114 PG - 11 SN - 2161-5063 DO - 10.1021/acssynbio.9b00375 UR - https://m2.mtmt.hu/api/publication/31139979 ID - 31139979 N1 - Systems Biology Program, Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, 28049, Spain Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, H-6726, Hungary Cited By :5 Export Date: 25 August 2021 Correspondence Address: De Lorenzo, V.; Systems Biology Program, Spain; email: vdlorenzo@cnb.csic.es AB - The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e., ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis, and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by (i) reassembling them together in a single broad host range, standardized vector and (ii) subjecting the noncoding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness, and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species, i.e., optimization of relative expression levels and upturning of subcomplex stoichiometry. LA - English DB - MTMT ER - TY - JOUR AU - Szili, Petra AU - Draskovits, Gábor AU - Révész, Tamás AU - Bogár, Ferenc AU - Balogh, Dávid AU - Martinek, Tamás AU - Daruka, Lejla AU - Spohn, Réka AU - Vásárhelyi, Bálint Márk AU - Czikkely, Márton Simon AU - Kintses, Bálint AU - Grézal, Gábor AU - Ferenc, Györgyi AU - Pál, Csaba AU - Nyerges, Ákos TI - Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations. JF - ANTIMICROBIAL AGENTS AND CHEMOTHERAPY J2 - ANTIMICROB AGENTS CH VL - 63 PY - 2019 IS - 9 PG - 15 SN - 0066-4804 DO - 10.1128/AAC.00207-19 UR - https://m2.mtmt.hu/api/publication/30777054 ID - 30777054 N1 - Funding Agency and Grant Number: European Research Council [H2020-ERC-2014-CoG 648364]; Wellcome Trust; GINOP (EVOMER); New National Excellence Program of the Ministry of Human Capacities [UNKP-18-3, UNKP-18-4]; "Lendulet" Program of the Hungarian Academy of Sciences; NKFIH [K120220]; Boehringer Ingelheim Fonds; Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences; Szeged Scientists Academy under Hungarian Ministry of Human Capacities [EMMI: 13725-2/2018/INTFIN]; GINOP (MolMedEx TUMORDNS) [GINOP-2.3.2-15-2016-00020]; [GINOP-2.3.2-15-2016-00014]; [EFOP 3.6.3-VEKOP-16-2017-00009] Funding text: This work was supported by grants from the European Research Council (H2020-ERC-2014-CoG 648364 "Resistance Evolution" to C.P.), the Wellcome Trust (to C.P.), and GINOP (MolMedEx TUMORDNS) GINOP-2.3.2-15-2016-00020, GINOP (EVOMER), GINOP-2.3.2-15-2016-00014 (to C.P.), EFOP 3.6.3-VEKOP-16-2017-00009 (to P.S. and T.R.), and UNKP-18-3 New National Excellence Program of the Ministry of Human Capacities (to P.S.); the "Lendulet" Program of the Hungarian Academy of Sciences (to C.P.), an NKFIH grant K120220 (to B.K.), and a Ph.D. fellowship from the Boehringer Ingelheim Fonds (to A.N.). B.K. was supported by the UNKP-18-4 New National Excellence Program of the Ministry of Human Capacities, the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences, and M. C. was supported by the Szeged Scientists Academy under the sponsorship of the Hungarian Ministry of Human Capacities (EMMI: 13725-2/2018/INTFIN). Összes idézések száma a WoS-ban: 0 Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Szeged, Hungary Department of Medical Chemistry, University of Szeged, Szeged, Hungary Nucleic Acid Synthesis Laboratory, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary Doctoral School of Theoretical Medicine, University of Szeged, Szeged, Hungary Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary Szeged Scientists Academy, Szeged, Hungary Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary Cited By :2 Export Date: 24 August 2020 CODEN: AMACC Correspondence Address: Pál, C.; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of SciencesHungary; email: pal.csaba@brc.mta.hu Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Szeged, Hungary Department of Medical Chemistry, University of Szeged, Szeged, Hungary Nucleic Acid Synthesis Laboratory, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary Doctoral School of Theoretical Medicine, University of Szeged, Szeged, Hungary Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary Szeged Scientists Academy, Szeged, Hungary Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary Cited By :3 Export Date: 8 December 2020 CODEN: AMACC Correspondence Address: Pál, C.; Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of SciencesHungary; email: pal.csaba@brc.mta.hu Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Szeged, Hungary Department of Medical Chemistry, University of Szeged, Szeged, Hungary Nucleic Acid Synthesis Laboratory, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary Doctoral School of Theoretical Medicine, University of Szeged, Szeged, Hungary Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary Szeged Scientists Academy, Szeged, Hungary Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary Cited By :4 Export Date: 5 February 2021 CODEN: AMACC Correspondence Address: Pál, C.; Synthetic and Systems Biology Unit, Hungary; email: pal.csaba@brc.mta.hu Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Szeged, Hungary Department of Medical Chemistry, University of Szeged, Szeged, Hungary Nucleic Acid Synthesis Laboratory, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary Doctoral School of Theoretical Medicine, University of Szeged, Szeged, Hungary Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary Szeged Scientists Academy, Szeged, Hungary Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary Cited By :5 Export Date: 25 August 2021 CODEN: AMACC Correspondence Address: Pál, C.; Synthetic and Systems Biology Unit, Hungary; email: pal.csaba@brc.mta.hu AB - Multitargeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets, are generally considered to be a promising therapeutic strategy against resistance evolution. The rationale for this theory is that multitargeting antibiotics demand the simultaneous acquisition of multiple mutations at their respective target genes to achieve significant resistance. The theory presumes that individual mutations provide little or no benefit to the bacterial host. Here, we propose that such individual stepping-stone mutations can be prevalent in clinical bacterial isolates, as they provide significant resistance to other antimicrobial agents. To test this possibility, we focused on gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an >2,000-fold reduction in susceptibility, while individually, none of these mutations affect resistance significantly. Alarmingly, strains with decreased susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed gyrase inhibitor, coselected for reduced susceptibility against gepotidacin. We conclude that extensive antibiotic usage could select for mutations that serve as stepping-stones toward resistance against antimicrobial compounds still under development. Our research indicates that even balanced multitargeting antibiotics are prone to resistance evolution. 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 - 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 - TY - JOUR AU - Nyerges, Ákos AU - Csörgő, Bálint AU - Nagy, István AU - Latinovics, Dóra AU - Szamecz, Béla AU - Pósfai, György AU - Pál, Csaba TI - Conditional DNA repair mutants enable highly precise genome engineering. JF - NUCLEIC ACIDS RESEARCH J2 - NUCLEIC ACIDS RES VL - 42 PY - 2014 IS - 8 PG - 10 SN - 0305-1048 DO - 10.1093/nar/gku105 UR - https://m2.mtmt.hu/api/publication/2591076 ID - 2591076 N1 - PMC PMC4005651 AB - Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement. LA - English DB - MTMT ER -