A Cell-Free Pipeline for Rapidly Imprinting Complex Methylation Patterns on DNA to Enhance Plasmid Transformation in Bacteria

There has been an increasing interest in the development of bacteria for use as next-generation industrial biocatalysts or as cell-based diagnostics and therapeutics. Bacterial genotype-phenotype relationships and microbe-host interactions are poorly characterized, partially due to formidable and strain-specific barriers to DNA transformation. One of the most common approaches to overcoming these barriers is to overcome restriction-modification (R-M) systems by mimicking the DNA methylation pattern which confers self-nonself recognition. However, this approach is limited by the required expression of a host’s numerous DNA methyltransferases by the cellular system. This occurrence often results in methyltransferase-induced toxicity and incomplete methylation, especially for complex Type I systems. Here we describe an alternative approach called IMPRINT, which involves the use of cell-free transcription-translation systems to co-express several methyltransferases. IMPRINT rapidly recreates the host’s DNA methylation pattern in vitro thereby boosting DNA transformation efficiencies. We demonstrate that IMPRINT can be used to readily reproduce DNA methylation patterns present in various hosts and we further optimized this approach to achieve complete multiplexed methylation when complex Type I R-M systems are present. We show that IMPRINT can achieve the same maximum transformation efficiency in strains with known R-M barriers and high transformation efficiencies in recalcitrant strains of probiotic Bifidobacteria. Finally, we demonstrate how barcoding shuttle vectors can rapidly reveal the minimal methylome to achieve maximum DNA transformation in a given strain when more than two R-M systems are present. IMPRINT offers a rapid means of enhancing DNA transformation across the bacterial world.