Systematic evasion of the restriction-modification barrier in bacteria

Abstract

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation. Here, we describe a systematic approach to overcome RM systems. Our approach was inspired by a simple hypothesis: if a synthetic piece of DNA lacks the highly specific target recognition motifs for a host's RM systems, then it is invisible to these systems and will not be degraded during artificial transformation. Accordingly, in this process, we determine the genome and methylome of an individual bacterial strain and use this information to define the bacterium's RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent "SyngenicDNA" tool, and propagate the tool as minicircle plasmids, termed SyMPL (SyngenicDNA Minicircle Plasmid) tools, before transformation. In a proof-of-principle of our approach, we demonstrate a profound improvement (five orders of magnitude) in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus This stealth-by-engineering SyngenicDNA approach is effective, flexible, and we expect in future applications could enable microbial genetics free of the restraints of restriction-modification barriers.

Keywords: SyngenicDNA; genetic intractability; minicircle; restriction modification; transformation.

Fig. 1.

Schematic representation of the SyngenicDNA approach. (A) Identification of RM system target motifs by SMRTseq. Methylome analysis of polymerase kinetics during sequencing permits detection of methylated sites at single-nucleotide resolution across the genome, revealing the exact motifs targeted by innate RM systems (indicated by colored nucleotides; N is any nucleotide) (kinetic trace image adapted from www.pacb.com). (B) Assembly in silico of a genetic tool with a desired functionality, followed by screening for the presence of RM target sequences and sequence adaptation, using SNPs or synonymous codon substitutions in coding regions, to create an RM-silent template which is synthetized de novo to assemble a SyngenicDNA tool. (C) Artificial transformation of the bacterium of interest target bacterium. Inappropriately methylated target motifs of the original genetic tool are recognized as nonself-DNA and degraded by RM systems. In contrast, the SyngenicDNA variant retains the form and functionality of the genetic tool, but is uniquely designed at the nucleotide level to evade the RM systems and can operate as desired within the target bacterial host.

Fig. 2.

The SyngenicDNA approach applied to Staphylococcus aureus JE2. (A) JE2 maintains two type I RM systems and a type IV restriction system. REase (HsdR and SauUSI) and MTase (HsdM) genes are shown in red and blue, respectively. Specificity subunit (HsdS) genes are shown in yellow. RM systems and their corresponding target motifs were identified by SMRTseq and REBASE analysis. (B) Construction of pEPSA5SynJE2, an RM-silent variant of the pEPSA5 plasmid tailored to JE2. Six nucleotide substitutions (two synonymous codon substitutions and four SNPs) eliminated all type I RM system targets from pEPSA5 sequence. (C) Plasmid propagation scheme. E. coli host strains produce DNA susceptible (DH5α; Dcm+) or resistant (E. coli ER2796; Dcm-) to the JE2 type IV restriction system. (D) Comparison of plasmid transformation efficiency (cfu/µg DNA) with pEPSA5 and the SyngenicDNA variant pEPSA5SynJE2.

Fig. 3.

The SyMPL approach applied to Staphylococcus aureus JE2. (A) Propagation of minicircles (pEPSA5MC and pEPSA5SynJE2MC) lacking Dcm-methylated sites within SyMPL-producer strain E. coli JMC1. (B) Comparison of SyngenicDNA and pEPSA5-based SyMPL plasmid transformation efficiency (cfu/µg DNA) with JE2. (C) Secondary analysis of SyngenicDNA and pEPSA5-based SyMPL plasmid transformation efficiencies in cfu/pmol DNA. Data are means ± SEM from nine independent experiments (three biological replicates with three technical replicates each).