Methylation Motifs Research Articles

A Type I Restriction Modification System Influences Genomic Evolution Driven by Horizontal Gene Transfer in Paenibacillus polymyxa.

Considered a “Generally Recognized As Safe” (GRAS) bacterium, the plant growth–promoting rhizobacterium Paenibacillus polymyxa has been widely applied in agriculture and animal husbandry. It also produces valuable compounds that are used in medicine and industry. Our previous work showed the presence of restriction modification (RM) system in P. polymyxa ATCC 842. Here, we further analyzed its genome and methylome by using SMRT sequencing, which revealed the presence of a larger number of genes, as well as a plasmid documented as a genomic region in a previous report. A number of mobile genetic elements (MGEs), including 78 insertion sequences, six genomic islands, and six prophages, were identified in the genome. A putative lysozyme-encoding gene from prophage P6 was shown to express lysin which caused cell lysis. Analysis of the methylome and genome uncovered a pair of reverse-complementary DNA methylation motifs which were widespread in the genome, as well as genes potentially encoding their cognate type I restriction-modification system PpoAI. Further genetic analysis confirmed the function of PpoAI as a RM system in modifying and restricting DNA. The average frequency of the DNA methylation motifs in MGEs was lower than that in the genome, implicating a role of PpoAI in restricting MGEs during genomic evolution of P. polymyxa. Finally, comparative analysis of R, M, and S subunits of PpoAI showed that homologs of the PpoAI system were widely distributed in species belonging to other classes of Firmicute, implicating a role of the ancestor of PpoAI in the genomic evolution of species beyond Paenibacillus.

Precision Methylome and In Vivo Methylation Kinetics Characterization of Klebsiella pneumoniae

Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can cause severe hospital- and community-acquired infections. To systematically investigate its methylation features, we determined the whole-genome sequences of 14 K. pneumoniae strains covering varying serotypes, multilocus sequence types, clonal groups, viscosity/virulence, and drug resistance. Their methylomes were further characterized using Pacific Biosciences single-molecule real-time and bisulfite technologies. We identified 15 methylation motifs [13 N6-methyladenine (6mA) and two 5-methylcytosine (5mC) motifs], among which eight were novel. Their corresponding DNA methyltransferases were also validated. Additionally, we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains, and identified differential distribution patterns of some hemi-/un-methylated GATC motifs, which tend to be located within intergenic regions (IGRs). Specifically, we characterized the in vivo methylation kinetics at single-base resolution on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed re-methylation. The slow methylation of the GATC motifs in the replication origin (oriC) regions and IGRs implicates the epigenetic regulation of replication initiation and transcription. Our findings illustrate the first comprehensive dynamic methylome map of K. pneumoniae at single-base resolution, and provide a useful reference to better understand epigenetic regulation in this and other bacterial species.

Could Changing the DNA Methylation Landscape Promote the Destruction of Epstein-Barr Virus-Associated Cancers?

DNA methylation at CpG motifs provides an epigenetic route to regulate gene expression. In general, an inverse correlation between DNA hypermethylation at CpG motifs and gene expression is observed. Epstein Barr-virus (EBV) infects people and the EBV genome resides in the nucleus where either its replication cycle initiates or it enters a long-term latency state where the viral genome becomes hypermethylated at CpG motifs. Viral gene expression shows a largely inverse correlation with DNA hypermethylation. DNA methylation occurs through the action of DNA methyl transferase enzymes: writer DNA methyl transferases add methyl groups to specific regions of unmethylated DNA; maintenance DNA methyl transferases reproduce the pattern of DNA methylation during genome replication. The impact of DNA methylation is achieved through the association of various proteins specifically with methylated DNA and their influence on gene regulation. DNA methylation can be changed through altering DNA methyl transferase activity or through the action of enzymes that further modify methylated CpG motifs. Azacytidine prodrugs that are incorporated into CpG motifs during DNA replication are recognized by DNA methyl transferases and block their function resulting in hypomethylation of DNA. EBV-associated cancers have hypermethylated viral genomes and many carcinomas also have highly hypermethylated cellular genomes. Decitabine, a member of the azacytidine prodrug family, reactivates viral gene expression and promotes the recognition of lymphoma cells by virus-specific cytotoxic T-cells. For EBV-associated cancers, the impact of decitabine on the cellular genome and the prospect of combining decitabine with other therapeutic approaches is currently unknown but exciting.

Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments.

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

Methylation analysis of Klebsiella pneumoniae from Portuguese hospitals

Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, but also epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. Here, we apply the PacBio sequencing platform to eight Portuguese hospital isolates, including one carbapenemase producing isolate, to identify methylation motifs. The resulting assembled chromosomes were between 5.2 and 5.5Mbp in length, and twenty-six PEGEs were found. Four of our eight samples carry blaCTX-M-15, a dominant Extended Spectrum Beta Lactamase in Europe. We identified methylation motifs that control Restriction–Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. There was a consistent lack of methylation by Dam of the GATC motif downstream of two genes: fosA, a locus associated with low level fosfomycin resistance, and tnpB transposase on IncFIB(K) plasmids. Overall, we have constructed eight high quality reference genomes of K. pneumoniae, with insights into horizontal gene transfer and methylation m6A motifs.

Genome-wide methylome analysis of two strains belonging to the hypervirulent Neisseria meningitidis serogroup W ST-11 clonal complex

A rising incidence of meningococcal serogroup W disease has been evident in many countries worldwide. Serogroup W isolates belonging to the sequence type (ST)-11 clonal complex have been associated with atypical symptoms and increased case fatality rates. The continued expansion of this clonal complex in the later part of the 2010s has been largely due to a shift from the so-called original UK strain to the 2013 strain. Here we used single-molecule real-time (SMRT) sequencing to determine the methylomes of the two major serogroup W strains belonging to ST-11 clonal complex. Five methylated motifs were identified in this study, and three of the motifs, namely 5′-GATC-3′, 5′-GAAGG-3′, 5′-GCGCGC-3′, were found in all 13 isolates investigated. The results showed no strain-specific motifs or difference in active restriction modification systems between the two strains. Two phase variable methylases were identified and the enrichment or depletion of the methylation motifs generated by these methylases varied between the two strains. Results from this work give further insight into the low diversity of methylomes in highly related strains and encourage further research to decipher the role of regions with under- or overrepresented methylation motifs.

Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia, an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia.

A systematic survey of PRMT interactomes reveals the key roles of arginine methylation in the global control of RNA splicing and translation

Thousands of proteins undergo arginine methylation, a widespread post-translational modification catalyzed by several protein arginine methyltransferases (PRMTs). However, global understanding of their biological functions is limited due to the lack of a complete picture of the catalytic network for each PRMT. Here, we systematically identified interacting proteins for all human PRMTs and demonstrated their functional importance in mRNA splicing and translation. We demonstrated significant overlapping of interactomes of human PRMTs with the known methylarginine-containing proteins. Different PRMTs are functionally redundant with a high degree of overlap in their substrates and high similarities between their putative methylation motifs. Importantly, RNA-binding proteins involved in regulating RNA splicing and translation contain highly enriched arginine methylation regions. Moreover, inhibition of PRMTs globally alternates alternative splicing (AS) and suppresses translation. In particular, ribosomal proteins are extensively modified with methylarginine, and mutations in their methylation sites suppress ribosome assembly, translation, and eventually cell growth. Collectively, our study provides a global view of different PRMT networks and uncovers critical functions of arginine methylation in regulating mRNA splicing and translation.

Identification of New Helicobacter pylori Subpopulations in Native Americans and Mestizos From Peru.

Region-specific Helicobacter pylori subpopulations have been identified. It is proposed that the hspAmerind subpopulation is being displaced from the Americans by an hpEurope population following the conquest. Our study aimed to describe the genomes and methylomes of H. pylori isolates from distinct Peruvian communities: 23 strains collected from three groups of Native Americans (Asháninkas [ASHA, n = 9], Shimaas [SHIM, n = 5] from Amazonas, and Punos from the Andean highlands [PUNO, n = 9]) and 9 modern mestizos from Lima (LIM). Closed genomes and DNA modification calls were obtained using SMRT/PacBio sequencing. We performed evolutionary analyses and evaluated genomic/epigenomic differences among strain groups. We also evaluated human genome-wide data from 74 individuals from the selected Native communities (including the 23 H. pylori strains donors) to compare host and bacterial backgrounds. There were varying degrees of hspAmerind ancestry in all strains, ranging from 7% in LIM to 99% in SHIM. We identified three H. pylori subpopulations corresponding to each of the Native groups and a novel hspEuropePeru which evolved in the modern mestizos. The divergence of the indigenous H. pylori strains recapitulated the genetic structure of Native Americans. Phylogenetic profiling showed that Orthogroups in the indigenous strains seem to have evolved differentially toward epigenomic regulation and chromosome maintenance, whereas OGs in the modern mestizo (LIM) seem to have evolved toward virulence and adherence. The prevalence of cagA+/vacA s1i1m1 genotype was similar across populations (p = 0.32): 89% in ASHA, 67% in PUNO, 56% in LIM and 40% in SHIM. Both cagA and vacA sequences showed that LIM strains were genetically differentiated (p < 0.001) as compared to indigenous strains. We identified 642 R-M systems with 39% of the associated genes located in the core genome. We found 692 methylation motifs, including 254 population-specific sequences not previously described. In Peru, hspAmerind is not extinct, with traces found even in a heavily admixed mestizo population. Notably, our study identified three new hspAmerind subpopulations, one per Native group; and a new subpopulation among mestizos that we named hspEuropePeru. This subpopulation seems to have more virulence-related elements than hspAmerind. Purifying selection driven by variable host immune response may have shaped the evolution of Peruvian subpopulations, potentially impacting disease outcomes.

Treg-inducing capacity of genomic DNA of Bifidobacterium longum subsp. infantis.

Background: Allergic and autoimmune diseases comprise a group of inflammatory disorders caused by aberrant immune responses in which CD25+ forkhead box P3-positive regulatory T cells (Treg) cells that normally suppress inflammatory events are often poorly functioning. This has stimulated an intensive investigative effort to find ways of increasing Tregs as a method of therapy for these conditions. Commensal microbiota known to have health benefits in humans include the lactic acid-producing, probiotic bacteria B. longum subsp. infantis and Lactobacillus rhamnosus. Mechanistically, several mechanisms have been proposed to explain how probiotics may favorably affect host immunity, including the induction of Tregs. Analysis of emerging data from several laboratories, including our own, suggest that DNA methylation may be an important determinant of immune reactivity responsible for Treg induction. Although methylated CpG moieties in normal mammalian DNA are both noninflammatory and lack immunogenicity, unmethylated CpGs, found largely in microbial DNA, are immunostimulatory and display proinflammatory properties. Objective: We hypothesize that microbiota with more DNA methylation may potentiate Treg induction to a greater degree than microbiota with a lower content of methylation. The purpose of the present study was to test this hypothesis by studying the methylation status of whole genomic DNA (gDNA) and the Treg-inducing capacity of purified gDNA in each of the probiotic bacteria B. longum subsp. infantis and L. rhamnosus, and a pathogenic Escherichia coli strain B. Results: We showed that gDNA from B. longum subsp. infantis is a potent Treg inducer that displays a dose-dependent response pattern at a dose threshold of 20 µg of gDNA. No similar Treg-inducing responses were observed with the gDNA from L. rhamnosus or E. coli. We identified a unique CpG methylated motif in the gDNA sequencing of B. longum subsp. infantis which was not found in L. rhamnosus or E. coli strain B. Conclusion: Although the literature indicates that both B. longum subsp. infantis and L. rhamnosus strains contribute to health, our data suggest that they do so by different mechanisms. Further, because of its small molecular size, low cost, ease of synthesis, and unique Treg-inducing feature, this methylated CpG oligodeoxynucleotide (ODN) from B. longum would offer many attractive features for an ideal novel therapeutic vaccine candidate for the treatment of immunologic diseases, such as the allergic and autoimmune disorders, in which Treg populations are diminished.

Differential DNA methylomes of clinical MDR, XDR and XXDR Mycobacterium tuberculosis isolates revealed by using single-molecule real-time sequencing

Post-replicative DNA methylation is essential for diverse biological processes in both eukaryotes and prokaryotes. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, remains one of the most formidable threats worldwide. Although DNA methylation of M. tuberculosis has been documented, little information is available for clinical drug-resistant M. tuberculosis. Single-molecule real-time (SMRT) sequencing was used to profile the core methylome of three clinical isolates, namely multidrug-resistant (MDR), extensively drug-resistant (XDR) and extremely drug-resistant (XXDR) strains. 3812, 6808 and 6041 DNA methylated sites were identified in MDR-MTB, XDR-MTB and XXDR-MTB genome, respectively. There are two types of methylated motifs, namely N6-methyladenine (m6A) and N4-methylcytosine (m4C). A novel widespread 6 mA methylation motif 5′-CACGCAG-3′ was found in XDR-MTB and XXDR-MTB. The methylated genes are involved in multiple cellular processes, especially metabolic enzymes engaged in glucose metabolism, fatty acid and TCA cycle. Many methylated genes are involved in mycobacterial virulence, antibiotic resistance and tolerance. This provided a comprehensive list of methylated genes in drug-resistant clinical isolates and the basis for further functional elucidation.

DNA Methylation Epigenetically Regulates Gene Expression in Burkholderia cenocepacia and Controls Biofilm Formation, Cell Aggregation, and Motility.

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2 and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. B. cenocepacia wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation, cell aggregation, and motility.IMPORTANCE CF patients diagnosed with Burkholderia cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of B. cenocepacia cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle B. cenocepacia infections in CF patients.

The protective role of m1A during stress-induced granulation.

Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.

A Conformational Switch in the Zinc Finger Protein Kaiso Mediates Differential Readout of Specific and Methylated DNA Sequences.

Recognition of the epigenetic mark 5-methylcytosine (mC) at CpG sites in DNA has emerged as a novel function of many eukaryotic transcription factors (TFs). It remains unclear why the sequence specificity of these TFs differs for CpG-methylated motifs and consensus motifs. Here, we dissect the structural and dynamic basis for this differential DNA binding specificity in the human zinc finger TF Kaiso, which exhibits high affinity for two consecutive mCpG sites in variable contexts and also for a longer, sequence-specific Kaiso binding site (KBS). By integrating structural analysis and DNA binding studies with targeted protein mutagenesis and nucleotide substitutions, we identify distinct mechanisms for readout of methylated and KBS motifs by Kaiso. We show that a key glutamate residue (E535), critical for mCpG site recognition, adopts different conformations in complexes with specific and methylated DNA. These conformational differences, together with intrinsic variations in DNA flexibility and/or solvation at TpG versus mCpG sites, contribute to the different DNA affinity and sequence specificity. With methylated DNA, multiple direct contacts between E535 and the 5' mCpG site dominate the binding affinity, allowing for tolerance of different flanking DNA sequences. With KBS, Kaiso employs E535 as part of an indirect screen of the 5' flanking sequence, relying on key tyrosine-DNA interactions to stabilize an optimal DNA conformation and select against noncognate sites. These findings demonstrate how TFs use conformational adaptation and exploit variations in DNA flexibility to achieve distinct DNA readout outcomes and target a greater variety of regulatory and epigenetic sites than previously appreciated.

Complete genome and methylome analysis of Neisseria meningitidis associated with increased serogroup Y disease

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis emerged in Europe during the 2000s. Draft genomes of serogroup Y isolates in Sweden revealed that although the population structure of these isolates was similar to other serogroup Y isolates internationally, a distinct strain (YI) and more specifically a sublineage (1) of this strain was responsible for the increase of serogroup Y IMD in Sweden. We performed single molecule real-time (SMRT) sequencing on eight serogroup Y isolates from different sublineages to unravel the genetic and epigenetic factors delineating them, in order to understand the serogroup Y emergence. Extensive comparisons between the serogroup Y sublineages of all coding sequences, complex genomic regions, intergenic regions, and methylation motifs revealed small point mutations in genes mainly encoding hypothetical and metabolic proteins, and non-synonymous variants in genes involved in adhesion, iron acquisition, and endotoxin production. The methylation motif CACNNNNNTAC was only found in isolates of sublineage 2. Only seven genes were putatively differentially expressed, and another two genes encoding hypothetical proteins were only present in sublineage 2. These data suggest that the serogroup Y IMD increase in Sweden was most probably due to small changes in genes important for colonization and transmission.

A novel DNA methylation motif identified in Bacillus pumilus BA06 and possible roles in the regulation of gene expression.

Single-molecule real-time (SMRT) sequencing can be used to identify a wide variety of chemical modifications of the genome, such as methylation. Here, we applied this approach to identify N6-methyl-adenine (m6A) and N4-methyl-cytosine (m4C) modification in the genome of Bacillus pumilus BA06. A typical methylation recognition motif of the type I restriction-modification system (R-M), 5'-TCm6AN8TTGG-3'/3'-AGTN8m6AACC-5', was identified. We confirmed that this motif was a new type I methylation site using REBASE analysis and that it was recognized by a type I R-M system, Bpu6ORFCP, according to methylation sensitivity assays in vivo and vitro. Furthermore, we found that deletion of the R-M system Bpu6ORFCP induced transcriptional changes in many genes and led to increased gene expression in pathways related to ABC transporters, sulfur metabolism, ribosomes, cysteine and methionine metabolism and starch and sucrose metabolism, suggesting that the R-M system in B. pumilus BA06 has other significant biological functions beyond protecting the B. pumilus BA06 genome from foreign DNA.

Integrated Genome-Wide Analysis of an Isogenic Pair of Pseudomonas aeruginosa Clinical Isolates with Differential Antimicrobial Resistance to Ceftolozane/Tazobactam, Ceftazidime/Avibactam, and Piperacillin/Tazobactam.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the main causes of morbidity and mortality in hospitalized patients and the leading cause of nosocomial infections. We investigated, here, two MDR P. aeruginosa clinical isolates from a hospitalized patient with differential antimicrobial resistance to ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), and piperacillin/tazobactam (P/T). Their assembled complete genomes revealed they belonged to ST235, a widespread MDR clone; and were isogenic with only a single nucleotide variant, causing G183D mutation in AmpC β-lactamase, responsible for a phenotypic change from susceptible to resistant to CZA and C/T. Further epigenomic profiling uncovered two conserved DNA methylation motifs targeted by two distinct putative methyltransferase-containing restriction-modification systems, respectively; more intriguingly, there was a significant difference between the paired isolates in the pattern of genomic DNA methylation and modifications. Moreover, genome-wide gene expression profiling demonstrated the inheritable genomic methylation and modification induced 14 genes being differentially regulated, of which only toxR (downregulated), a regulatory transcription factor, had its promoter region differentially methylate and modified. Since highly expressed opdQ encodes an OprD porin family protein, therefore, we proposed an epigenetic regulation of opdQ expression pertinent to the phenotypic change of P. aeruginosa from resistant to susceptible to P/T. The disclosed epigenetic mechanism controlling phenotypic antimicrobial resistance deserves further experimental investigation.

Revealing mitogenome-wide DNA methylation and RNA editing of three Ascomycotina fungi using SMRT sequencing

Beauveria bassiana, Cordyceps militaris and Ophiocordyceps sinensis (Ascomycotina) are traditional Chinese medicines. Here, mitogenomes of these three Ascomycotina fungi were sequenced and de-novo assembled using single-molecule real-time sequencing. The results showed that their complete mitogenomes were 31,258, 31,854 and 157,584 bp, respectively, with sequencing depth approximately 278,760×, 326,283× and 69,385×. Types of repeat sequences were mainly (AA)n, (AAT)n, (TA)n and (TATT)n. DNA methylation motifs were revealed in DNA modifications of these three fungi. We discovered new models of RNA editing through analysis of transcriptomes from B. bassiana and C. militaris. These data lay a solid foundation for further genetic and biological studies about these three fungi, especially for elucidating the mitogenome evolution and exploring the regulatory mechanism of adapting environment.

PSVI-3 The effect of oligodeoxynucleotides on Toll-like receptor pathway genes in goat blood

Abstract The objective of this study was to investigate the effect of two classes of oligodeoxynucleotides (ODNs) on the expression of genes involved in Toll-like receptor signaling in goat blood. Toll-like receptors play a critical role in initiating innate immune responses. Select synthetic oligodeoxynucleotides containing methylated CpG motifs (CpG ODN) have immunologic effects similar to those seen with bacterial DNA. These short synthetic single-stranded DNA molecules are being explored as adjuvants to enhance animal health. Blood samples were collected from the jugular vein of BoerXSpanish goats (n = 3) into tubes containing an anticoagulant. Blood was treated with the 10 µg/ml CpG ODN (2216, 2006) class A and B, or 200µl of PBS which served as control. Cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 minutes. Total RNA was isolated using Trizol and then converted to cDNA using RETROscript kit (Qiagen). The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array was evaluated using real-time PCR. Fold change in gene expression was calculated using the 2−ΔΔCt method. The housekeeping genes GAPDH, ACTB, HPRT1, TBP, and YWHAZ was used to normalize the data. Of 84 genes tested, 74 genes were expressed in the control group. Following treatment with ODN 2006, 50 genes were expressed, 29 were up-regulated while 17 genes were down-regulated. The genes HSPD1, IFNG, MAPK8, and PTGS2 were induced by ODN 2006 treatment. Following treatment with ODN 2216, 52 genes were expressed. Treatment with ODN 2216, upregulated 26 genes and down-regulated the expression of 19 genes. The genes CLEC4E, HSPD1, IFNG, IRF1, MAPK8, PTGS2, and UBE2V1 were induced by ODN 2216. Thus specific patterns of TLR signaling may be involved in response to the immunostimulatory effect of CpG ODN in goats with implicates for their use as adjuvants to enhance animal health.

Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

Restriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAXII as 5′-GCAGA-3′, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae.