Methylation Motifs Research Articles

The DNA cytosine methylome revealed two methylation motifs in the upstream regions of genes related to morphological and physiological differentiation in Streptomyces coelicolor A(3)2 M145

DNA methylation is an epigenetic modification detected in both prokaryotic and eukaryotic genomic DNAs. In bacteria, the importance of 5-methylcytosine (m5C) in gene expression has been less investigated than in eukaryotic systems. Through dot-blot analysis employing m5C antibodies against chromosomal DNA, we have previously demonstrated that m5C influences the differentiation of Streptomyces coelicolor A(3)2 M145 in solid sporulating and liquid non-sporulating complex media. Here, we mapped the methylated cytosines of the M145 strain growing in the defined Maltose Glutamate (MG) liquid medium. Sequencing of the M145 genome after bisulfite treatment (BS-sequencing) evidenced 3360 methylated cytosines and the two methylation motifs, GGCmCGG and GCCmCG, in the upstream regions of 321 genes. Besides, the role of cytosine methylation was investigated using the hypo-methylating agent 5′-aza-2′-deoxycytidine (5-aza-dC) in S. coelicolor cultures, demonstrating that m5C affects both growth and antibiotic biosynthesis. Finally, quantitative reverse-transcription polymerase-chain-reaction (RT-qPCR) analysis of genes containing the methylation motifs in the upstream regions showed that 5-aza-dC treatment influenced their transcriptional levels and those of the regulatory genes for two antibiotics. To the best of our knowledge, this is the first study that reports the cytosine methylome of S. coelicolor M145, supporting the crucial role ascribed to cytosine methylation in controlling bacterial gene expression.

Extension of bacterial rDNA sequencing for simultaneous methylation detection and its application in microflora analysis

Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing.

Analysis of Whole-Genome Sequences of Pathogenic Gram-Positive and Gram-Negative Isolates from the Same Hospital Environment to Investigate Common Evolutionary Trends Associated with Horizontal Gene Exchange, Mutations and DNA Methylation Patterning.

Hospital-acquired infections are a generally recognized problem for healthcare professionals. Clinical variants of Gram-negative and Gram-positive pathogens are characterized with enhanced antibiotic resistance and virulence due to mutations and the horizontal acquisition of respective genetic determinants. In this study, two Escherichia coli, two Klebsiella pneumoniae, three Pseudomonas aeruginosa, two Staphylococcus aureus, one Staphylococcus epidermidis and one Streptococcus pneumoniae showing broad spectra of antibiotic resistance were isolated from patients suffering from nosocomial infections in a local hospital in Almaty, Kazakhstan. The aim of the study was to compare general and species-specific pathways of the development of virulence and antibiotic resistance through opportunistic pathogens causing hospital-acquired infections. The whole-genome PacBio sequencing of the isolates allowed for the genotyping and identification of antibiotic resistance and virulence genetic determinants located in the chromosomes, plasmids and genomic islands. It was concluded that long-read sequencing is a useful tool for monitoring the epidemiological situation in hospitals. Marker antibiotic resistance mutations common for different microorganisms were identified, which were acquired due to antibiotic-selective pressure in the same clinical environment. The genotyping and identification of strain-specific DNA methylation motifs were found to be promising in estimating the risks associated with hospital infection outbreaks and monitoring the distribution and evolution of nosocomial pathogens.

Restriction Endonuclease-Based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing.

Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus enriching for undigested DNA from the organism of interest. As a proof of concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. IMPORTANCE Pathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA, which may escape unnoticed in metagenomic samples.

Role of DNA modifications in Mycoplasma gallisepticum.

The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene.

DNA Methyltransferase Regulates Nitric Oxide Homeostasis and Virulence in a Chronically Adapted Pseudomonas aeruginosa Strain.

ABSTRACTOpportunistic pathogens such as Pseudomonas aeruginosa adapt their genomes rapidly during chronic infections. Understanding their epigenetic regulation may provide biomarkers for diagnosis and reveal novel regulatory mechanisms. We performed single-molecule real-time sequencing (SMRT-seq) to characterize the methylome of a chronically adapted P. aeruginosa clinical strain, TBCF10839. Two N6-methyladenine (6mA) methylation recognition motifs (RCCANNNNNNNTGAR and TRGANNNNNNTGC [modification sites are in bold]) were identified and predicted as new type I methylation sites using REBASE analysis. We confirmed that the motif TRGANNNNNNTGC was methylated by the methyltransferase (MTase) M.PaeTBCFII, according to methylation sensitivity assays in vivo and vitro. Transcriptomic analysis showed that a ΔpaeTBCFIIM knockout mutant significantly downregulated nitric oxide reductase (NOR) regulation and expression of coding genes such as nosR and norB, which contain methylated motifs in their promoters or coding regions. The ΔpaeTBCFIIM strain exhibited reduced intercellular survival capacity in NO-producing RAW264.7 macrophages and attenuated virulence in a Galleria mellonella infection model; the complemented strain recovered these defective phenotypes. Further phylogenetic analysis demonstrated that homologs of M.PaeTBCFII occur frequently in P. aeruginosa as well as other bacterial species. Our work therefore provided new insights into the relationship between DNA methylation, NO detoxification, and bacterial virulence, laying a foundation for further exploring the molecular mechanism of DNA methyltransferase in regulating the pathogenicity of P. aeruginosa.IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed broad genome diversity among P. aeruginosa clinical strains and revealed their different regulatory traits compared to the laboratory strains. While current investigation of the epigenetics of P. aeruginosa is still lacking, understanding epigenetic regulation may provide biomarkers for diagnosis and facilitate development of novel therapies. Denitrification capability is critical for microbial versatility in response to different environmental stress conditions, including the bacterial infection process, where nitric oxide (NO) can be generated by phagocytic cells. The denitrification regulation mechanisms have been studied intensively at genetic and biochemical levels. However, there is very little evidence about the epigenetic regulation of bacterial denitrification mechanism. P. aeruginosa TBCF10839 is a chronically host-adapted strain isolated from a cystic fibrosis (CF) patient with special antiphagocytosis characteristics. Here, we investigated the regulatory effect of an orphan DNA MTase, M.PaeTBCFII, in P. aeruginosa TBCF10839. We demonstrated that the DNA MTase regulates the transcription of denitrification genes represented by NOR and affects antiphagocytic ability in bacteria. In silico analysis suggested that DNA methylation modification may enhance gene expression by affecting the binding of transacting factors such as DNR and RpoN. Our findings not only deepen the understanding of the role of DNA MTase in transcriptional regulation in P. aeruginosa but also provide a theoretical foundation for the in-depth study of the molecular mechanism of the epigenetic regulation on denitrification, virulence, and host-pathogen interaction.

Changes in the genomes and methylomes of three Salmonella enterica serovars after long-term storage in ground black pepper.

Low moisture foods (LMFs) have traditionally been recognized as safe for consumption, as most bacteria require higher water content to grow. However, outbreaks due to LMF foods are increasing, and the microbial pathogen Salmonella enterica is frequently implicated. S. enterica can survive in LMFs for years, but few serovars have been studied, and the mechanisms which underlie this longevity are not well understood. Here, we determine that S. enterica serovars S. Tennessee, S. Anatum, and S. Reading but not S. Oranienburg can survive in the ground black pepper for 6 years. S. Reading was not previously associated with any LMF. Using both Illumina and Pacific Biosciences sequencing technologies, we also document changes in the genomes and methylomes of the surviving serovars over this 6-year period. The three serovars acquired a small number of single nucleotide polymorphisms (SNPs) including seven substitutions (four synonymous, two non-synonymous, and one substitution in a non-coding region), and two insertion-deletions. Nine distinct N6-methyladenine (m6A) methylated motifs across the three serovars were identified including five which were previously known, Gm6ATC, CAGm6AG, BATGCm6AT, CRTm6AYN6CTC, and CCm6AN7TGAG, and four novel serovar-specific motifs, GRTm6AN8TTYG, GAm6ACN7GTA, GAA m6ACY, and CAAm6ANCC. Interestingly, the BATGCAT motif was incompletely methylated (35–64% sites across the genome methylated), suggesting a possible role in gene regulation. Furthermore, the number of methylated BATGCm6AT motifs increased after storage in ground black pepper for 6 years from 475 to 657 (S. Tennessee), 366 to 608 (S. Anatum), and 525 to 570 (S. Reading), thus warranting further study as an adaptive mechanism. This is the first long-term assessment of genomic changes in S. enterica in a low moisture environment, and the first study to examine the methylome of any bacteria over a period of years, to our knowledge. These data contribute to our understanding of S. enterica survival in LMFs, and coupled with further studies, will provide the information necessary to design effective interventions which reduce S. enterica in LMFs and maintain a healthy, safe food supply.

The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

BackgroundImprinting Control Regions (ICRs) are CpG-rich sequences acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected high mutation rate due to spontaneous deamination of methylated cytosines, ICRs show conservation of CpG-richness and CpG-containing transcription factor binding sites in mammalian species. However, little is known about the mechanisms contributing to the maintenance of a high density of methyl CpGs at these loci.ResultsTo gain functional insights into the mechanisms for maintaining CpG methylation, we sought to identify the proteins binding the methylated allele of the ICRs by determining the interactors of ZFP57 that recognizes a methylated hexanucleotide motif of these DNA regions in mouse ESCs. By using a tagged approach coupled to LC–MS/MS analysis, we identified several proteins, including factors involved in mRNA processing/splicing, chromosome organization, transcription and DNA repair processes. The presence of the post-replicative mismatch-repair (MMR) complex components MSH2 and MSH6 among the identified ZFP57 interactors prompted us to investigate their DNA binding profile by chromatin immunoprecipitation and sequencing. We demonstrated that MSH2 was enriched at gene promoters overlapping unmethylated CpG islands and at repeats. We also found that both MSH2 and MSH6 interacted with the methylated allele of the ICRs, where their binding to DNA was mediated by the ZFP57/KAP1 complex.ConclusionsOur findings show that the MMR complex is concentrated on gene promoters and repeats in mouse ESCs, suggesting that maintaining the integrity of these regions is a primary function of highly proliferating cells. Furthermore, the demonstration that MSH2/MSH6 are recruited to the methylated allele of the ICRs through interaction with ZFP57/KAP1 suggests a role of the MMR complex in the maintenance of the integrity of these regulatory regions and evolution of genomic imprinting in mammalian species.

Alphaherpesvirus US3 protein-mediated inhibition of the m6A mRNA methyltransferase complex.

SUMMARYChemical modifications of mRNA, the so-called epitranscriptome, represent an additional layer of post-transcriptional regulation of gene expression. The most common epitranscriptomic modification, N6-methyladenosine (m6A), is generated by a multi-subunit methyltransferase complex. We show that alphaherpesvirus kinases trigger phosphorylation of several components of the m6A methyltransferase complex, including METTL3, METTL14, and WTAP, which correlates with inhibition of the complex and a near complete loss of m6A levels in mRNA of virus-infected cells. Expression of the viral US3 protein is necessary and sufficient for phosphorylation and inhibition of the m6A methyltransferase complex. Although m6A methyltransferase complex inactivation is not essential for virus replication in cell culture, the consensus m6A methylation motif is under-represented in alphaherpesvirus genomes, suggesting evolutionary pressure against methylation of viral transcripts. Together, these findings reveal that phosphorylation can be associated with inactivation of the m6A methyltransferase complex, in this case mediated by the viral US3 protein.

NT-seq: a chemical-based sequencing method for genomic methylome profiling

DNA methylation plays vital roles in both prokaryotes and eukaryotes. There are three forms of DNA methylation in prokaryotes: N6-methyladenine (6mA), N4-methylcytosine (4mC), and 5-methylcytosine (5mC). Although many sequencing methods have been developed to sequence specific types of methylation, few technologies can be used for efficiently mapping multiple types of methylation. Here, we present NT-seq for mapping all three types of methylation simultaneously. NT-seq reliably detects all known methylation motifs in two bacterial genomes and can be used for identifying de novo methylation motifs. NT-seq provides a simple and efficient solution for detecting multiple types of DNA methylation.

Impacts of Mycoplasma agalactiae restriction-modification systems on pan-epigenome dynamics and genome plasticity.

DNA methylations play an important role in the biology of bacteria. Often associated with restriction modification (RM) systems, they are important drivers of bacterial evolution interfering in horizontal gene transfer events by providing a defence against foreign DNA invasion or by favouring genetic transfer through production of recombinogenic DNA ends. Little is known regarding the methylome of the Mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, genome-wide detection of DNA methylations was conducted using single molecule real-time (SMRT) and bisulphite sequencing in several strains of Mycoplasma agalactiae , an important ruminant pathogen and a model organism. Combined with whole-genome analysis, this allowed the identification of 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenomic landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.

Epigenome editing reveals core DNA methylation for imprinting control in the Dlk1-Dio3 imprinted domain.

The Dlk1-Dio3 imprinted domain is controlled by an imprinting control region (ICR) called IG-DMR that is hypomethylated on the maternal allele and hypermethylated on the paternal allele. Although several genetic mutation experiments have shown that IG-DMR is essential for imprinting control of the domain, how DNA methylation itself functions has not been elucidated. Here, we performed both gain and loss of DNA methylation experiments targeting IG-DMR by transiently introducing CRISPR/Cas9 based-targeted DNA methylation editing tools along with one guide RNA into mouse ES cells. Altered DNA methylation, particularly at IG-DMR-Rep, which is a tandem repeat containing ZFP57 methylated DNA-binding protein binding motifs, affected the imprinting state of the whole domain, including DNA methylation, imprinted gene expression, and histone modifications. Moreover, the altered imprinting states were persistent through neuronal differentiation. Our results suggest that the DNA methylation state at IG-DMR-Rep, but not other sites in IG-DMR, is a master element to determine whether the allele behaves as the intrinsic maternal or paternal allele. Meanwhile, this study provides a robust strategy and methodology to study core DNA methylation in cis-regulatory elements, such as ICRs and enhancers.

Pan-methylarginine antibody generation using PEG linked GAR motifs as antigens.

Arginine methylation is a prevalent posttranslational modification which is deposited by a family of protein arginine methyltransferases (PRMTs), and is found in three different forms in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Pan-methylarginine antibodies are critical for identifying proteins that are methylated on arginine residues, and are also used for evaluating signaling pathways that modulate this methyltransferase activity. Although good pan-MMA, -ADMA and -SDMA antibodies have been developed over the years, there is still room for improvement. Here we use a novel antigen approach, which involves the separation of short methylated motifs with inert polyethylene glycol (PEG) linkers, to generate a set of pan antibodies to the full range of methylarginine marks. Using these antibodies, we observed substrate scavenging by PRMT1, when PRMT5 activity is blocked. Specifically, we find that the splicing factor SmD1 displays increased ADMA levels upon PRMT5 inhibitor treatment. Furthermore, when the catalysis of both SDMA and ADMA is blocked with small molecule inhibitors, we demonstrate that SmD1 and SMN no longer interact. This could partially explain the synergistic effect of PRMT5 and type I PRMT inhibition on RNA splicing and cancer cell growth.

A mobile restriction-modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease.

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction–modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

Proteome-wide identification of non-histone lysine methylation in tomato during fruit ripening

IntroductionHistone and non-histone methylations are important post-translational modifications in plants. Histone methylation plays a crucial role in regulating chromatin structure and gene expression. However, the involvement of non-histone methylation in plant biological processes remains largely unknown. MethodsThe methylated substrates and methylation sites during tomato fruit ripening were identified by LC-MS/MS. Bioinformatics of lysine methylated proteins was conducted to analyze the possible role of methylated proteins. The effects of methylation modification on protein functions were preliminarily investigated by site-directed mutation simulation. ResultsA total of 241 lysine methylation (mono-, di- and trimethylation) sites in 176 proteins were identified with two conserved methylation motifs: xxxxxxExxx_K_xxxExxxxxx and xxxxxxExxx_K_xxxxxxxxxx. These methylated proteins were mainly related to fruit ripening and senescence, oxidation reduction process, signal transduction, stimulus and stress responses, and energy metabolism. Three representative proteins, thioredoxin (Trx), glutathione S-transferase T1 (GST T1), and NADH dehydrogenase (NOX), were selected to investigate the effect of methylation modifications on protein activity. Mimicking demethylation led to decreased Trx activity but increased GST T1 and NOX activities. In addition, RT-qPCR exhibited that the expression of many genes that encode proteins subjected to methylation was upregulated during fruit ripening. ConclusionOur study suggests that tomato fruit ripening undergo non-histone lysine methylation, which may participate in the regulation of fruit ripening. It is the first report of methyl proteome profiling of non-histone lysine in horticultural crops.

Detecting DNA Methylations in the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius Using SMRT Sequencing.

DNA methylations are one of the most well-known epigenetic modifications along with histone modifications and noncoding RNAs. They are found at specific sites along the DNA in all domains of life, with 5-mC and 6-mA/4-mC being well-characterized in eukaryotes and bacteria respectively, and they have not only been described as contributing to the structure of the double helix itself but also as regulators of DNA-based processes such as replication, transcription, and recombination. Different methods have been developed to accurately identify and/or map methylated motifs to decipher the involvement of DNA methylations in regulatory networks that affect the cellular state.Although DNA methylations have been detected along archaeal genomes, their involvement as regulators of DNA-based processes remains the least known. To highlight the importance of DNA methylations in the control of key cellular mechanisms and their dynamics in archaea cells, we have used single-molecule real-time (SMRT) sequencing. This sequencing technology allows the identification and direct mapping of the methylated motifs along the genome of an organism. In this chapter, we present a step-by-step protocol for detecting DNA methylations in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius using SMRT sequencing. This protocol can easily be adapted to other prokaryotes.

Methylation Motifs in Promoter Sequences May Contribute to the Maintenance of a Conserved m5C Methyltransferase in Helicobacter pylori

DNA methylomes of Helicobacter pylori strains are complex due to the large number of DNA methyltransferases (MTases) they possess. H. pylori J99 M.Hpy99III is a 5-methylcytosine (m5C) MTase that converts GCGC motifs to Gm5CGC. Homologs of M.Hpy99III are found in essentially all H. pylori strains. Most of these homologs are orphan MTases that lack a cognate restriction endonuclease, and their retention in H. pylori strains suggest they have roles in gene regulation. To address this hypothesis, green fluorescent protein (GFP) reporter genes were constructed with six putative promoters that had a GCGC motif in the extended −10 region, and the expression of the reporter genes was compared in wild-type H. pylori G27 and a mutant lacking the M.Hpy99III homolog (M.HpyGIII). The expression of three of the GFP reporter genes was decreased significantly in the mutant lacking M.HpyGIII. In addition, the growth rate of the H. pylori G27 mutant lacking M.HpyGIII was reduced markedly compared to that of the wild type. These findings suggest that the methylation of the GCGC motif in many H. pylori GCGC-containing promoters is required for the robust expression of genes controlled by these promoters, which may account for the universal retention of M.Hpy99III homologs in H. pylori strains.

DNA Methylation Patterns Differ between Free-Living Rhizobium leguminosarum RCAM1026 and Bacteroids Formed in Symbiosis with Pea (Pisum sativum L.).

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.

Comparative methylation and RNA-seq expression analysis in CpG context to identify genes involved in Backfat vs. Liver diversification in Nanchukmacdon Pig

BackgroundDNA methylation and demethylation at CpG islands is one of the main regulatory factors that allow cells to respond to different stimuli. These regulatory mechanisms help in developing tissue without affecting the genomic composition or undergoing selection. Liver and backfat play important roles in regulating lipid metabolism and control various pathways involved in reproductive performance, meat quality, and immunity. Genes inside these tissue store a plethora of information and an understanding of these genes is required to enhance tissue characteristics in the future generation.ResultsA total of 16 CpG islands were identified, and they were involved in differentially methylation regions (DMRs) as well as differentially expressed genes (DEGs) of liver and backfat tissue samples. The genes C7orf50, ACTB and MLC1 in backfat and TNNT3, SIX2, SDK1, CLSTN3, LTBP4, CFAP74, SLC22A23, FOXC1, GMDS, GSC, GATA4, SEMA5A and HOXA5 in the liver, were categorized as differentially-methylated. Subsequently, Motif analysis for DMRs was performed to understand the role of the methylated motif for tissue-specific differentiation. Gene ontology studies revealed association with collagen fibril organization, the Bone Morphogenetic Proteins (BMP) signaling pathway in backfat and cholesterol biosynthesis, bile acid and bile salt transport, and immunity-related pathways in methylated genes expressed in the liver.ConclusionsIn this study, to understand the role of genes in the differentiation process, we have performed whole-genome bisulfite sequencing (WGBS) and RNA-seq analysis of Nanchukmacdon pigs. Methylation and motif analysis reveals the critical role of CpG islands and transcriptional factors binding site (TFBS) in guiding the differential patterns. Our findings could help in understanding how methylation of certain genes plays an important role and can be used as biomarkers to study tissue specific characteristics.

Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.