Bacterial DNA methylases as novel molecular and synthetic biology tools: recent developments

Abstract

Bacterial DNA methylases are a diverse group of enzymes which have been pivotal in the development of technologies with applications including genetic engineering, bacteriology, biotechnology and agriculture. This review describes bacterial DNA methylase types, the main technologies for targeted methylation or demethylation and the recent roles of these enzymes in molecular and synthetic biology. Bacterial methylases can be exocyclic or endocyclic and can exist as orphan enzymes or as a part of the restriction-modifications (R-M) systems. As a group, they display a rich diversity of sequence-specificity. Additional technologies for targeting methylation involve using fusion proteins combining a methylase and a DNA-binding protein (DNBP) such as a zinc-finger (ZF), transcription activator-like effector (TALE) or CRISPR/dCas9. Bacterial methylases have contributed significantly to the creation of novel DNA assembly techniques, to the improvement of bacterial transformation and to crop plant engineering. Future studies to define the characteristics of more bacterial methylases have potential to identify new tools of value in synthetic and molecular biology and with widespread applications. KEY POINTS: • Bacterial methylases can be used to direct methylation to specific sequences in target DNA • DNA methylation using bacterial methylases has been applied to improve DNA assembly and to increase the efficiency of bacterial transformation • Site-selective methylation using bacterial methylases can alter plant gene expression and phenotype.

Keywords: Bacterial transformation; Crop engineering; DCas9/gRNA; DNA assembly; Restriction-modification systems; Targeted methylation.

Fig. 1

Bacterial DNA methylases classified according to their mode of action

Fig. 2

Restriction-modification (R-M) systems: methylation and restriction features

Fig. 3

Technologies for targeted DNA methylation. a Fusion proteins formed by DNA-binding proteins (DNBP) such as zinc finger (ZF), transcription activator-like effector (TALE) and CRISPR/Cas9 (dCas9) combined with a methylase. b Steric hindrance by dCas9/gRNA complex. Methylation by the methylase is blocked when the dCas9/gRNA complex is bound to the target sequence, but methylation can occur when it is not bound. A demethylase could be blocked in a similar manner

Fig. 4

Uses of methylases in DNA assembly. a Pairwise Selection Assembly (PSA), green bars represent potential sites of methylation and these are blocked in the flanking restriction sites (pink and light blue) by the oligonucleotide polymers (dark blue). b Methylation-Assisted Tailorable Ends Rational (MASTER) ligation, the MspJI restriction enzyme only cuts methylated recognition sites so leaves unmethylated internal sites uncut. c 2ab assembly, different site-specific methylases are used to inactivate a BglII site in plasmid 1 and a BamHI site in plasmid 2 respectively. BglII and BamHI produce compatible overhangs. d Three Nucleotides (TNT) cloning system, a methylase is used to inactivate type IIS restriction enzyme sites. e Methylase-assisted Cloning (MetClo), a switch methylase is used to methylate and block the pair of outer type IIS restriction enzyme sites in the acceptor plasmid. This outer restriction enzyme site is engineered to partially overlap with a recognition site for the methylase which does not recognise the restriction enzyme site alone

Fig. 5

Effect of pre-methylation of transforming DNA to increase transformation efficiency

Fig. 6

DNA methylation tools in plants using bacterial methylases and their effects. a Methylation using the methylase M.SssI can be targeted using zinc finger. b A mutated form of M.SssI with reduced off-target activity and targeted by dCas9/gRNA. c Recruitment of multiple methylase molecules to the target site using a SunTag. d Heritable transactivation of M.SssI methylation. e Persistent methylation induced by plant-grown promoting bacteria