DNA cytosine methylation at the lexA promoter of Escherichia coli is stationary phase specific

The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon.

Structural basis of ATP-dependent high-fidelity epigenome maintenance

Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance-type cytosine methyltransferase identified in the fungal or protist kingdoms. DNMT5 requires ATP and displays exquisite hemimethyl-DNA specificity. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out the DNA duplex. ATP binding then triggers a striking structural reconfiguration of the methyltransferase catalytic pocket that enables cofactor binding, completion of base-flipping, and catalysis. Unmethylated DNA binding fails to open cofactor pocket and subsequent ATP binding triggers its ejection to ensure fidelity. This chaperone-like, enzyme-remodeling role of the SNF2 domain illuminates how energy can be used to enable faithful epigenetic memory.

DNA Methyltransferases Contribute to Cold Tolerance in Ticks Dermacentor silvarum and Haemaphysalis longicornis (Acari: Ixodidae)

DNA methylation, mediated by DNA methyltransferases (Dnmts), is a typical epigenetic process that plays an important role in affecting organism acclimatization and adaptation to environmental changes. However, information about Dnmts and their associations with the cold tolerance of ticks remains meager. Hence, in the present study, the Dnmts in important vector ticks Dermacentor silvarum and Haemaphysalis longicornis were cloned and identified, and their functions in cold response were further explored. Results showed that the length of DsDnmt and DsDnmt1 in D. silvarum, and HlDnmt1 and HlDnmt in H. longicornis were 1,284, 549, 1,500, and 1,613 bp, respectively. Bioinformatics in protein analysis revealed that they were all unstable hydrophilic proteins and were mainly characterized with Dcm (DNA cytosine methyltransferase domain), Dnmt1-RFD (DNA methyltransferase replication foci domain), zf-CXXC (zinc finger-CXXC domain), and BAH (Bromo adjacent homology domain). The relative expression of these Dnmts was reduced after cold treatment for 3 days (P < 0.05), and increased with the extension of treatment. Western blot revealed that Dnmt1 decreased first and then increased significantly (P < 0.05) in both tick species, whereas other Dnmts fluctuated at varying degrees. RNA interference significantly silenced the genes Dnmts (P < 0.01), and mortality increased significantly (P < 0.05), when exposed to sub-lethal temperature, underscoring the important roles of Dnmts during the cold response of D. silvarum and H. longicornis. The above results lay the foundation for further understanding of the epigenetic regulation of DNA methylation in cold acclimatization and adaptation of ticks.

A functional bacteria-derived restriction modification system in the mitochondrion of a heterotrophic protist

The overarching trend in mitochondrial genome evolution is functional streamlining coupled with gene loss; therefore, gene acquisition by mitochondria is considered to be exceedingly rare. Selfish elements in the form of self-splicing introns occur in many organellar genomes, but the wider diversity of selfish elements, and how they persist in the DNA of organelles, has not been explored. In the mitochondrial genome of a marine heterotrophic katablepharid protist, we identify a functional type II restriction modification (RM) system originating from a horizontal gene transfer (HGT) event involving bacteria related to flavobacteria. This RM system consists of an HpaII-like endonuclease and a cognate cytosine methyltransferase (CM). We demonstrate that these proteins are functional by heterologous expression in both bacterial and eukaryotic cells. These results suggest that a mitochondrial-encoded RM system can function as a toxin–antitoxin selfish element and that such elements could be co-opted by eukaryotic genomes to drive biased organellar inheritance.

Regulation of DIM-2-dependent repeat-induced point mutation (RIP) by the recombination-independent homologous DNA pairing in Neurospora crassa

ABSTRACTRepeat-induced point mutation (RIP) is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. RIP detects duplications irrespective of their origin, particular sequence, coding capacity, or genomic positions. Previous studies suggested that RIP involves a cardinally new mechanism of sequence recognition that operates on intact double-stranded DNAs. In the fungus Neurospora crassa, RIP can be mediated by a putative C5-cytosine methyltransferase (CMT) RID or/and a canonical CMT DIM-2. These distinct RIP pathways feature opposite substrate preferences: RID-dependent RIP is largely limited to the duplicated sequences, whereas DIM-2-dependent RIP preferentially mutates adjacent non-repetitive regions. Using DIM-2-dependent RIP as a principal readout of repeat recognition, here we show that GC-rich repeats promote stronger RIP compared to AT-rich repeats (independently of their intrinsic propensities to become mutated), with the relative contribution of AT base-pairs being close to zero. We also show that direct repeats promote much more efficient DIM-2-dependent RIP than inverted repeats; both the spacer DNA between the repeat units (the linker) and the flanking regions are similarly affected by this process. These and other results support the idea that repeat recognition for RIP involves formation of many short interspersed quadruplexes between homologous double-stranded DNAs, which need to undergo concomitant changes in their linking number to accommodate pairing.SUMMARYDuring repeat-induced point mutation (RIP) gene-sized duplications of genomic DNA are detected by a mechanism that likely involves direct pairing of homologous double-stranded DNAs. We show that DIM-2-dependent RIP, triggered by closely-positioned duplications, is strongly affected by their relative orientations (direct versus inverted). We also show that GC-rich repeats promote RIP more effectively than AT-rich repeats. These results support a model in which homologous dsDNAs can pair by establishing interspersed quadruplex-based contacts with concomitant changes in their supercoiling status.

Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropod

Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy aimed at reducing the spread of mosquito-borne RNA viruses such as alphaviruses. Our prior work examining Wolbachia-mediated pathogen blocking has demonstrated (i) the importance of a host cytosine methyltransferase, DNMT2, in Drosophila, and (ii) viral RNA as a target through which pathogen-blocking is mediated. Here we report on the role of DNMT2 in Wolbachia induced virus inhibition of alphaviruses in Aedes sp.. Mosquito DNMT2 levels were altered in the presence of both viruses and Wolbachia, albeit in opposite directions. Elevated levels of DNMT2 in mosquito salivary glands induced by virus infection were suppressed in Wolbachia colonized animals coincident with a reduction of virus replication, and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells was proviral increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity was dependent on its methyltransferase activity. Finally, examination of the effects of Wolbachia on modifications of viral RNA by LC-MS showed a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is an important mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental processes of virus replication and transmission.

A functional bacterial-derived restriction modification system in the mitochondrion of a heterotrophic protist

The overarching trend in mitochondrial evolution is functional streamlining coupled with gene loss; therefore, gene acquisition by mitochondria is considered to be exceedingly rare. Selfish elements in the form of self-splicing introns occur in many organellar genomes, but the wider diversity of selfish elements, and how they persist in organellar genomes, has not been explored. In the mitochondrial genome of a marine heterotrophic katablepharid protist, we identify a functional type II restriction modification system originating from a horizontal gene transfer event involving bacteria related to flavobacteria. This restriction modification system consists of an HpaII-like endonuclease and a cognate cytosine methyltransferase. We demonstrate that these proteins are functional by heterologous expression in both bacterial and eukaryotic cells. These results suggest that toxin-antitoxin selfish elements, such as restriction modification systems, could be co-opted by eukaryotic genomes to drive uniparental organellar inheritance.

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1

Background Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism. Results We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA. Conclusions Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1

Background: Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism.Results: We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124 %). The introduced gene increased the production of carotenoid by 26 %. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70 % as well. The production of carotenoid was decreased by 40 % when the psy gene was translationally repressed by anti-psy sRNA. Conclusions: Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.