Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for <jats:italic>Clostridium thermocellum</jats:italic> DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform <jats:italic>C. thermocellum</jats:italic> at dramatically different efficiencies.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We report an increase in transformation efficiency of <jats:italic>C. thermocellum</jats:italic> for a variety of plasmids by using DNA that has been methylated by <jats:italic>Escherichia coli</jats:italic> Dam but not Dcm methylases. When isolated from a <jats:italic>dam</jats:italic>+<jats:italic>dcm</jats:italic>+<jats:italic>E. coli</jats:italic> strain, pAMG206 transforms <jats:italic>C. thermocellum</jats:italic> 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the <jats:italic>pyrF</jats:italic> gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 <jats:italic>pyrF</jats:italic> gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p> <jats:italic>E. coli</jats:italic> Dcm methylation decreases transformation efficiency in <jats:italic>C. thermocellum</jats:italic> DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.</jats:p> </jats:sec>