Search Results (7)

Limited methyl-specific restriction of genomic DNA by endonuclease MAL1 revealed the changes in its methyl status caused by adenine modification in maize (Zea mays L.) leaves under different light conditions (dark, light, irradiation by red and far-red light). Incubation in the light and irradiation by red light exhibited an activating effect on DNA adenine methylase activity, which was reflected in an increase in the number of methylated adenines in GATC sites. Far-red light and darkness exhibited an opposite effect. The use of nitrite conversion of DNA followed by methyladenine-dependent restriction by MboI nuclease revealed a phytochrome B-dependent mechanism of regulation of the methyl status of adenine in the GATC sites in the promoter of the gene encoding the mitochondrial isoform of citrate synthase. Irradiation of plants with red light caused changes in the adenine methyl status of the analyzed amplicon, as evidenced by the presence of restriction products of 290, 254, and 121 nucleotides. Adenine methylation occurred at all three GATC sites in the analyzed DNA sequence. It is concluded that adenylate methylation is controlled by phytochrome B via the transcription factor PIF4 and represents an important mechanism for the tricarboxylic acid cycle regulation by light. Full article

In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria. Full article

In this study, a sensitive method for detecting DNA methyltransferase (MTase) activity was developed by combining the effective fluorescence resonance energy transfer (FRET) of cationic conjugated polymers and exonuclease (Exo) III–mediated signal amplification. DNA adenine MTase targets the GATC sequence within a substrate and converts the adenine in this sequence into N6-methyladenine. In the method developed in this study, the methylated substrate is cleaved using Dpn I, whereby a single-stranded oligodeoxynucleotide (oligo) is released. Afterward, the oligo is hybridized to the 3ʹ protruding end of the F-DNA probe to form a double-stranded DNA, which is then digested by Exo III. Subsequently, due to weak electrostatic interactions, only a weak FRET signal is observed. The introduction of the Exo-III–mediated target-recycling reaction improved the sensitivity for detecting MTase. This detection method was found to be sensitive for MTase detection, with the lowest detection limit of 0.045 U/mL, and was also suitable for MTase-inhibitor screening, whereby such inhibitors can be identified for disease treatment. Full article

Rhizobium leguminosarum (Rl) is a common name for several genospecies of rhizobia able to form nitrogen-fixing nodules on the roots of pea (Pisum sativum L.) while undergoing terminal differentiation into a symbiotic form called bacteroids. In this work, we used Oxford Nanopore sequencing to analyze the genome methylation states of the free-living and differentiated forms of the Rl strain RCAM1026. The complete genome was assembled; no significant genome rearrangements between the cell forms were observed, but the relative abundances of replicons were different. GANTC, GGCGCC, and GATC methylated motifs were found in the genome, along with genes encoding methyltransferases with matching predicted target motifs. The GGCGCC motif was completely methylated in both states, with two restriction–modification clusters on different replicons enforcing this specific pattern of methylation. Methylation patterns for the GANTC and GATC motifs differed significantly depending on the cell state, which indicates their possible connection to the regulation of symbiotic differentiation. Further investigation into the differences of methylation patterns in the bacterial genomes coupled with gene expression analysis is needed to elucidate the function of bacterial epigenetic regulation in nitrogen-fixing symbiosis. Full article

Restriction–modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase. Full article

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 (C^m4TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for C^m4TAG 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 (GCA^m6BN₆VTGC). 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. Full article

The sequence GCGATCGC (Highly Iterated Palindrome, HIP1) is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a G^meC-dependent mismatch repair system whose activity creates and maintains HIP sequences. Full article