Creation of Novel Methylating Conjugal Donor Strains for Genetically Recalcitrant Bacteria

Abstract A methodology is presented to integrate DNA methyltransferase genes of interest into the single copy, IncPβ ‘R’ Factor conjugal plasmid, R702. This is to enable DNA transfer, or improved frequency thereof, by mirroring the recipient native DNA methylation signature prior to transfer of plasmid DNA by conjugation, evading native restriction modification systems in the organism of study, without target strain modification. As proof of concept, the bacteriophage derived methyltransferase Ф3TI, known to facilitate DNA transfer into the industrially important model solventogenic organism, Clostridium acetobutylicum ATCC 824, was inserted into the single copy R702 to create an in vivo methylating strain. Plasmids extracted from this strain were transferred by electroporation at a comparable frequency to the established method which uses the multi-copy accessory plasmids pAN1 or pAN2, harbouring Ф3TI. The methodology was further exemplified and employed to enable high frequency of DNA transfer into the clinically relevant Clostridioides difficile ribotype 027 outbreak strain, R20291. The modification ( hsdM ) and specificity ( hsdS ) components of the native Type I restriction modification system of R20291 were inserted into R702 to create a functional methylating conjugal donor Escherichia coli strain. This approach of coupling methylation and the native transfer functions of R702 creates a system which can easily be mobilised into a genotypically appropriate E. coli strain; creating an in vivo methylating donor strain which may be utilized to protect and transfer DNA to the organism of study by conjugation.