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.

Plasmid Transfer by Conjugation in Gram-Negative Bacteria: From the Cellular to the Community Level

“…The newly acquired ssDNA conjugative plasmid might be considered as foreign DNA, against which host bacteria have developed defense mechanisms, such as restriction modification, exonucleases, and recombination system or adaptive immunity, such as the CRISPR-Cas system [ 161 ]. Despite these defense mechanisms, horizontal gene transfer plays an important role in genomic evolution (5–6% of bacterial genomes and up to 20% in some organisms) [ 6 , 162 , 163 ], implying that transferrable plasmids have evolved adaptive mechanisms to counteract these host defenses.…”

Toward a genetic system in the marine cyanobacterium Prochlorococcus

“…We are confident, however, that effective solutions can be found to overcome these hurdles. Solutions might come from new transposome systems [ 86 ], which could reveal more efficient means than the Tn5 transposition; from the delivery of Cpf1-RNA complexes inside cells [ 87, 88 ]; from using more systematic approaches to evade restriction-modification defences [ 15 ]; through the discovery of yet unknown defence systems [ 89 ] that are hampering transformation; or finally by harnessing endogenous mobile genetic elements, which have been tailored by evolution to work efficiently in these minimal cells. The latter is the direction we are currently exploring.…”

“…There is no single solution for all bacteria, and no way to predict which techniques will succeed [ 14 ]. Thus, there is a pressing need for broad-spectrum, automatized, and standardized genetic transformation procedures [ 15 ] that could enable downstream genetic screening methods such as transposon insertion sequencing [ 5, 16–18 ]. Such projects are not suitable for graduate students or post-docs because of the high risk involved, nor are they easily funded.…”

Approaches to genetic tool development for rapid domestication of non-model microorganisms

“…One approach is transforming DNA that lacks the identified motifs, or mutating DNA so that it no longer contains the motif [ 50 , 65 , 66 ]. Recently, software has been designed to aid in the process of eliminating restriction sites [ 67 ]. This can often be done for uncommon motifs, but it cannot always be used for shorter, more common motifs, like four base pair recognition sequences that may exist in plasmid origins of replication or other DNAs that require a specific sequence, such as those needed for homologous recombination.…”