Removal and bypass of restriction modification systems increases transformation efficiency of Thermococcus...

Archaea are increasingly recognized as key participants in global nutrient cycles, as idealized platforms for biotechnological applications, and as the progenitors of Eukarya. Despite such importance, limited genetic access to most archaeal taxa limits validation of key hypotheses, mandating a better understanding of archaeal transformation mechanisms and archaeal-encoding host-defense systems that typically hinder genetic competence. The hyperthermophilic marine species Thermococcus kodakarensis (T. kodakarensis) has emerged as a premier model archaeal species in large part due to natural competency and a wealth of genetic tools to manipulate the genome or ectopically complement strains with autonomously replicating plasmids. Despite continued successes, a limited number of genetic selections can be applied to specific type-strains of T. kodakarensis. Known restriction modification (RM) systems and other unknown host defense systems present in all strain variants reduce overall transformation efficiencies. We report here a unified T. kodakarensis strain (TS900) with a genotype that combines all the selective and counter-selective pressures currently available, that is compatible with all existing genetic procedures and plasmids.TS900-derived strains genetically deleted for two known Type II RM systems and a pair of McrBC endonucleases demonstrate dramatically enhanced transformation efficiency, opening T. kodakarensis to new approaches that demand efficient DNA uptake and genomic integration. Additionally, transformations with unmodified plasmid DNA likely bypasses host RM systems, further improving transformation efficiency. The unified genetic background, massively increased competence, and use of unmodified plasmid DNA in transformations will open new arenas of archaeal biology and the investigation of life in the extremes for experimental study.