A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

Ákos Nyerges, Bálint Csörgo, István Nagy, Balázs Bálint, Péter Bihari, Viktória Lázár, Gábor Apjok, Kinga Umenhoffer, Balázs Bogos, György Pósfai, Csaba Pál

Research output: Article

58 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2502-2507
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number9
DOIs
Publication statusPublished - márc. 1 2016

ASJC Scopus subject areas

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