@article{MTMT:30927710,
	title = {Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene},
	url = {https://m2.mtmt.hu/api/publication/30927710},
	author = {Aparicio, Tomas and Nyerges, Ákos and Nagy, István and Pál, Csaba and Martinez-Garcia, Esteban and de, Lorenzo Victor},
	doi = {10.1111/1462-2920.14814},
	journal-iso = {ENVIRON MICROBIOL},
	journal = {ENVIRONMENTAL MICROBIOLOGY},
	volume = {22},
	unique-id = {30927710},
	issn = {1462-2912},
	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 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.},
	year = {2020},
	eissn = {1462-2920},
	pages = {45-58},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; de, Lorenzo Victor/0000-0002-6041-2731}
}

@article{MTMT:31139979,
	title = {Multiple-Site Diversification of Regulatory Sequences Enables Interspecies Operability of Genetic Devices.},
	url = {https://m2.mtmt.hu/api/publication/31139979},
	author = {Hueso-Gil, Angeles and Nyerges, Ákos and Pál, Csaba and Calles, Belén and de Lorenzo, Víctor},
	doi = {10.1021/acssynbio.9b00375},
	journal-iso = {ACS SYNTH BIOL},
	journal = {ACS SYNTHETIC BIOLOGY},
	volume = {9},
	unique-id = {31139979},
	issn = {2161-5063},
	abstract = {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.},
	keywords = {PSEUDOMONAS; Interoperability; optogenetics; recombineering; CcaSR},
	year = {2020},
	pages = {104-114},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X}
}

@article{MTMT:30777054,
	title = {Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations.},
	url = {https://m2.mtmt.hu/api/publication/30777054},
	author = {Szili, Petra and Draskovits, Gábor and Révész, Tamás and Bogár, Ferenc and Balogh, Dávid and Martinek, Tamás and Daruka, Lejla and Spohn, Réka and Vásárhelyi, Bálint Márk and Czikkely, Márton Simon and Kintses, Bálint and Grézal, Gábor and Ferenc, Györgyi and Pál, Csaba and Nyerges, Ákos},
	doi = {10.1128/AAC.00207-19},
	journal-iso = {ANTIMICROB AGENTS CH},
	journal = {ANTIMICROBIAL AGENTS AND CHEMOTHERAPY},
	volume = {63},
	unique-id = {30777054},
	issn = {0066-4804},
	abstract = {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.},
	keywords = {Antibiotic resistance; genome engineering; gepotidacin},
	year = {2019},
	eissn = {1098-6596},
	orcid-numbers = {Bogár, Ferenc/0000-0002-0611-1452; Martinek, Tamás/0000-0003-3168-8066; Vásárhelyi, Bálint Márk/0000-0003-1782-8691; Grézal, Gábor/0000-0003-1685-4791; Ferenc, Györgyi/0000-0002-3456-319X; Nyerges, Ákos/0000-0002-1581-490X}
}

@article{MTMT:3390047,
	title = {Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.},
	url = {https://m2.mtmt.hu/api/publication/3390047},
	author = {Nyerges, Ákos and Csörgő, Bálint and Draskovits, Gábor and Kintses, Bálint and Szili, Petra and Ferenc, Györgyi and Révész, Tamás and Ari, Eszter and Nagy, István and Bálint, Balázs and Vásárhelyi, Bálint Márk and Bihari, Péter and Számel, Mónika and Balogh, Dávid and Papp, Henrietta and Kalapis, Dorottya and Papp, Balázs and Pál, Csaba},
	doi = {10.1073/pnas.1801646115},
	journal-iso = {P NATL ACAD SCI USA},
	journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
	volume = {115},
	unique-id = {3390047},
	issn = {0027-8424},
	abstract = {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.},
	keywords = {ESCHERICHIA-COLI; DRUG-RESISTANCE; URINARY-TRACT-INFECTIONS; QUINOLONE RESISTANCE; ANTIMICROBIAL RESISTANCE; Synthetic biology; RANDOM MUTAGENESIS; Homologous recombination; DIRECTED EVOLUTION; GENE MODIFICATION; COLI DIHYDROFOLATE-REDUCTASE; STRANDED-DNA OLIGONUCLEOTIDES; multiplex automated genome engineering; high-throughput mutagenesis},
	year = {2018},
	eissn = {1091-6490},
	pages = {E5726-E5735},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; Csörgő, Bálint/0000-0003-0397-6845; Ferenc, Györgyi/0000-0002-3456-319X; Ari, Eszter/0000-0001-7774-1067; Vásárhelyi, Bálint Márk/0000-0003-1782-8691; Papp, Henrietta/0000-0003-3887-5657}
}

@article{MTMT:3315234,
	title = {A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.},
	url = {https://m2.mtmt.hu/api/publication/3315234},
	author = {Ricaurte, DE and Martinez-Garcia, E and Nyerges, Ákos and Pál, Csaba and de Lorenzo, V and Aparicio, T},
	doi = {10.1111/1751-7915.12846},
	journal-iso = {MICROB BIOTECHNOL},
	journal = {MICROBIAL BIOTECHNOLOGY},
	volume = {11},
	unique-id = {3315234},
	issn = {1751-7907},
	abstract = {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.},
	year = {2018},
	eissn = {1751-7915},
	pages = {176-188},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X}
}

@article{MTMT:3038670,
	title = {A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species},
	url = {https://m2.mtmt.hu/api/publication/3038670},
	author = {Nyerges, Ákos and Csörgő, Bálint and Nagy, István and Bálint, Balázs and Bihari, Péter and Lázár, Viktória and Apjok, Gábor and Umenhoffer, Kinga and Bogos, Balázs and Pósfai, György and Pál, Csaba},
	doi = {10.1073/pnas.1520040113},
	journal-iso = {P NATL ACAD SCI USA},
	journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
	volume = {113},
	unique-id = {3038670},
	issn = {0027-8424},
	abstract = {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.},
	keywords = {EXPRESSION; IN-VIVO; RESISTANCE; GENES; ESCHERICHIA-COLI; EVOLUTION; DNA-REPAIR; Synthetic biology; MUTL; REPAIR MUTANTS; methyl-directed mismatch repair; off-target effects; recombineering; genome engineering},
	year = {2016},
	eissn = {1091-6490},
	pages = {2502-2507},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; Csörgő, Bálint/0000-0003-0397-6845}
}

@article{MTMT:2591076,
	title = {Conditional DNA repair mutants enable highly precise genome engineering.},
	url = {https://m2.mtmt.hu/api/publication/2591076},
	author = {Nyerges, Ákos and Csörgő, Bálint and Nagy, István and Latinovics, Dóra and Szamecz, Béla and Pósfai, György and Pál, Csaba},
	doi = {10.1093/nar/gku105},
	journal-iso = {NUCLEIC ACIDS RES},
	journal = {NUCLEIC ACIDS RESEARCH},
	volume = {42},
	unique-id = {2591076},
	issn = {0305-1048},
	abstract = {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.},
	year = {2014},
	eissn = {1362-4962},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; Csörgő, Bálint/0000-0003-0397-6845}
}