Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

Abstract

ABSTRACTIsolation ofClostridiummutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimizedmazFtoxin gene fromEscherichia coliunder the control of a lactose-inducible promoter fromClostridium perfringens. This system is potentially applicable to almost all members of the genusClostridiumdue to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167,sigF, andsigK) or disruptions (ca_p0157andsigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of aClostridium ljungdahlii2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into theClostridium acetobutylicumchromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from thefdhintegrant strain as well as thesigKdeletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.