July 19, 2019  |  

Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes.

Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regulation of gene expression, virulence and antibiotic resistance. Bacterial cells in a clonal population can generate epigenetic heterogeneity to increase population-level phenotypic plasticity. Single molecule, real-time (SMRT) sequencing enables the detection of N6-methyladenine and N4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing SMRT sequencing-based methods for studying bacterial methylomes rely on a population-level consensus that lacks the single-cell resolution required to observe epigenetic heterogeneity. Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation. Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity. SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.


July 19, 2019  |  

Lineage-specific methyltransferases define the methylome of the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone. Copyright © 2015 Forde et al.


July 19, 2019  |  

DNA methylation assessed by SMRT Sequencing is linked to mutations in Neisseria meningitidis isolates.

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.


July 19, 2019  |  

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC’s genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 19, 2019  |  

The complete genome sequence of the murine pathobiont Helicobacter typhlonius.

Immuno-compromised mice infected with Helicobacter typhlonius are used to model microbially inducted inflammatory bowel disease (IBD). The specific mechanism through which H. typhlonius induces and promotes IBD is not fully understood. Access to the genome sequence is essential to examine emergent properties of this organism, such as its pathogenicity. To this end, we present the complete genome sequence of H. typhlonius MIT 97-6810, obtained through single-molecule real-time sequencing.The genome was assembled into a single circularized contig measuring 1.92 Mbp with an average GC content of 38.8%. In total 2,117 protein-encoding genes and 43 RNA genes were identified. Numerous pathogenic features were found, including a putative pathogenicity island (PAIs) containing components of type IV secretion system, virulence-associated proteins and cag PAI protein. We compared the genome of H. typhlonius to those of the murine pathobiont H. hepaticus and human pathobiont H. pylori. H. typhlonius resembles H. hepaticus most with 1,594 (75.3%) of its genes being orthologous to genes in H. hepaticus. Determination of the global methylation state revealed eight distinct recognition motifs for adenine and cytosine methylation. H. typhlonius shares four of its recognition motifs with H. pylori.The complete genome sequence of H. typhlonius MIT 97-6810 enabled us to identify many pathogenic features suggesting that H. typhlonius can act as a pathogen. Follow-up studies are necessary to evaluate the true nature of its pathogenic capabilities. We found many methylated sites and a plethora of restriction-modification systems. The genome, together with the methylome, will provide an essential resource for future studies investigating gene regulation, host interaction and pathogenicity of H. typhlonius. In turn, this work can contribute to unraveling the role of Helicobacter in enteric disease.


July 19, 2019  |  

DNA methylation on N(6)-adenine in mammalian embryonic stem cells.

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.


July 19, 2019  |  

Biosynthesis and function of modified bases in bacteria and their viruses.

Naturally occurring modification of the canonical A, G, C, and T bases can be found in the DNA of cellular organisms and viruses from all domains of life. Bacterial viruses (bacteriophages) are a particularly rich but still underexploited source of such modified variant nucleotides. The modifications conserve the coding and base-pairing functions of DNA, but add regulatory and protective functions. In prokaryotes, modified bases appear primarily to be part of an arms race between bacteriophages (and other genomic parasites) and their hosts, although, as in eukaryotes, some modifications have been adapted to convey epigenetic information. The first half of this review catalogs the identification and diversity of DNA modifications found in bacteria and bacteriophages. What is known about the biogenesis, context, and function of these modifications are also described. The second part of the review places these DNA modifications in the context of the arms race between bacteria and bacteriophages. It focuses particularly on the defense and counter-defense strategies that turn on direct recognition of the presence of a modified base. Where modification has been shown to affect other DNA transactions, such as expression and chromosome segregation, that is summarized, with reference to recent reviews.


July 19, 2019  |  

Methylome analysis of two Xanthomonas spp. using Single-Molecule Real-Time Sequencing.

Single-molecule real-time (SMRT) sequencing allows identification of methylated DNA bases and methylation patterns/motifs at the genome level. Using SMRT sequencing, diverse bacterial methylomes including those of Helicobacter pylori, Lactobacillus spp., and Escherichia coli have been determined, and previously unreported DNA methylation motifs have been identified. However, the methylomes of Xanthomonas species, which belong to the most important plant pathogenic bacterial genus, have not been documented. Here, we report the methylomes of Xanthomonas axonopodis pv. glycines (Xag) strain 8ra and X. campestris pv. vesicatoria (Xcv) strain 85-10. We identified N(6)-methyladenine (6mA) and N(4)-methylcytosine (4mC) modification in both genomes. In addition, we assigned putative DNA methylation motifs including previously unreported methylation motifs via REBASE and MotifMaker, and compared methylation patterns in both species. Although Xag and Xcv belong to the same genus, their methylation patterns were dramatically different. The number of 4mC DNA bases in Xag (66,682) was significantly higher (29 fold) than in Xcv (2,321). In contrast, the number of 6mA DNA bases (4,147) in Xag was comparable to the number in Xcv (5,491). Strikingly, there were no common or shared motifs in the 10 most frequently methylated motifs of both strains, indicating they possess unique species- or strain-specific methylation motifs. Among the 20 most frequent motifs from both strains, for 9 motifs at least 1% of the methylated bases were located in putative promoter regions. Methylome analysis by SMRT sequencing technology is the first step toward understanding the biology and functions of DNA methylation in this genus.


July 19, 2019  |  

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.


July 19, 2019  |  

Widespread adenine N6-methylation of active genes in fungi.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.


July 19, 2019  |  

Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally.

DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genome.


July 19, 2019  |  

Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing.

Anaerobic ammonium-oxidizing (anammox) bacteria are a group of strictly anaerobic chemolithoautotrophic microorganisms. They are capable of oxidizing ammonium to nitrogen gas using nitrite as a terminal electron acceptor, thereby facilitating the release of fixed nitrogen into the atmosphere. The anammox process is thought to exert a profound impact on the global nitrogen cycle and has been harnessed as an environment-friendly method for nitrogen removal from wastewater. In this study, we present the first closed genome sequence of an anammox bacterium, Kuenenia stuttgartiensis MBR1. It was obtained through Single-Molecule Real-Time (SMRT) sequencing of an enrichment culture constituting a mixture of at least two highly similar Kuenenia strains. The genome of the novel MBR1 strain is different from the previously reported Kuenenia KUST reference genome as it contains numerous structural variations and unique genomic regions. We find new proteins, such as a type 3b (sulf)hydrogenase and an additional copy of the hydrazine synthase gene cluster. Moreover, multiple copies of ammonium transporters and proteins regulating nitrogen uptake were identified, suggesting functional differences in metabolism. This assembly, including the genome-wide methylation profile, provides a new foundation for comparative and functional studies aiming to elucidate the biochemical and metabolic processes of these organisms.


July 19, 2019  |  

The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis.

The occurrence of harmful Microcystis blooms is increasing in frequency in a myriad of freshwater ecosystems. Despite considerable research pertaining to the cause and nature of these blooms, the molecular mechanisms behind the cosmopolitan distribution and phenotypic diversity in Microcystis are still unclear. We compared the patterns and extent of DNA methylation in three strains of Microcystis, PCC 7806SL, NIES-2549 and FACHB-1757, using Single Molecule Real-Time (SMRT) sequencing technology. Intact restriction-modification (R-M) systems were identified from the genomes of these strains, and from two previously sequenced strains of Microcystis, NIES-843 and TAIHU98. A large number of methylation motifs and R-M genes were identified in these strains, which differ substantially among different strains. Of the 35 motifs identified, eighteen had not previously been reported. Strain NIES-843 contains a larger number of total putative methyltransferase genes than have been reported previously from any bacterial genome. Genomic comparisons reveal that methyltransferases (some partial) may have been acquired from the environment through horizontal gene transfer. Copyright © 2018 Elsevier B.V. All rights reserved.


July 19, 2019  |  

Identification and analysis of adenine N6-methylation sites in the rice genome.

DNA N6-methyladenine (6mA) is a non-canonical DNA modification that is present at low levels in different eukaryotes1-8, but its prevalence and genomic function in higher plants are unclear. Using mass spectrometry, immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements. In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylcytosine (5mC) at CG sites in gene bodies and is complementary to 5mC at CHH sites in transposable elements. We show that OsALKBH1 may be potentially involved in 6mA demethylation in rice. The results suggest that 6mA is complementary to 5mC as an epigenomic mark in rice and reinforce a distinct role for 6mA as a gene expression-associated epigenomic mark in eukaryotes.


July 7, 2019  |  

Complete genome sequence of the Clostridium difficile laboratory strain 630¿ erm reveals differences from strain 630, including translocation of the mobile element CTn 5.

Background Clostridium difficile strain 630¿erm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns.ResultsIn addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain.ConclusionsTogether, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.


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