Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing

Abstract

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeon Haloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes in H. volcanii and sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genes HVO_0794, HVO_A0006, and HVO_A0237 in a single strain abolished methylation of the sole cytosine motif in the genome (Cm4TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for Cm4TAG methylation in H. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome of H. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operon rmeRMS (HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCAm6BN6VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that the rmeRMS operon is responsible for adenine methylation in H. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation in H. volcanii, even though other putative MTases (HVO_C0040, HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (ΔRM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation in H. volcanii.

DNA methylation epigenetically regulates gene expression inBurkholderia cenocepacia

“…MTase BCAL3494 was specifically linked to methylation of the CACAG motif, MTase BCAM0992 to methylation of the GTWWAC motif. This strategy of DNA methylation analysis, in which the methylome of strains lacking MTases is determined, has been used in various bacteria, as it is an effective way to find associations between predicted MTases and genome-wide methylation motifs (34,35). For example, several methylation motifs were identified in Burkholderia pseudomallei , including motifs CACAG and GTWWAC (36).…”

The Impact of Restriction-Modification Systems on Mating inHaloferax volcanii

“…In H. volcanii, there are two major methylated motifs throughout the genome: the m4C motif C m4 TAG and the m6A motif GCA m6 BN6VTGC (Ouellette et al, 2015). The C m4 TAG motif is methylated by the orphan MTase HVO_0794 and the GCA m6 BN6VTGC motif is methylated by the Type I RM system RmeRMS (Ouellette et al, 2018). Since HVO_0794 is an orphan MTase and is not associated with a RM system, it is unlikely to have an impact on mating efficiency and recombination.…”

“…The most likely candidate for reducing mating between RMincompatible strains in H. volcanii is the RmeRMS system. In the ΔRM strain and its derivatives, the genome is missing the operon that encodes the RmeRMS system, and is unmethylated at its target sites (Ouellette et al, 2018). Therefore, the target sites of RmeRMS would be exposed to cleavage when ΔRM derivative strains are mated with strains H53 or H98, which have the RmeRMS system.…”

“…Another possible barrier to mating in the H. volcanii might be RM systems. Studies have characterized a few of these systems in H. volcanii, including a Type I RM system which targets the motif GCABN6VTGC (Ouellette et al, 2015;Ouellette et al, 2018). A Type IV REase known as Mrr has also been characterized in H. volcanii, and has been observed to reduce transformation efficiency on GATC-methylated plasmids (Holmes et al, 1991;Allers et al, 2010).…”

“…However, the overall role of these systems on cell-to-cell mating and recombination has not been examined in detail. In this study, derivatives of the H. volcanii RM system deletion strain from Ouellette et al (2018) were used in mating experiments to determine the impact of RM systems on cell-to-cell mating in H. volcanii.…”