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July 19, 2019

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.

Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

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July 19, 2019

One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly.

Like a jigsaw puzzle with large pieces, a genome sequenced with long reads is easier to assemble. However, recent sequencing technologies have favored lowering per-base cost at the expense of read length. This has dramatically reduced sequencing cost, but resulted in fragmented assemblies, which negatively affect downstream analyses and hinder the creation of finished (gapless, high-quality) genomes. In contrast, emerging long-read sequencing technologies can now produce reads tens of kilobases in length, enabling the automated finishing of microbial genomes for under $1000. This promises to improve the quality of reference databases and facilitate new studies of chromosomal structure and variation. We present an overview of these new technologies and the methods used to assemble long reads into complete genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 19, 2019

qDNAmod: a statistical model-based tool to reveal intercellular heterogeneity of DNA modification from SMRT sequencing data.

In an isogenic cell population, phenotypic heterogeneity among individual cells is common and critical for survival of the population under different environment conditions. DNA modification is an important epigenetic factor that can regulate phenotypic heterogeneity. The single molecule real-time (SMRT) sequencing technology provides a unique platform for detecting a wide range of DNA modifications, including N6-methyladenine (6-mA), N4-methylcytosine (4-mC) and 5-methylcytosine (5-mC). Here we present qDNAmod, a novel bioinformatic tool for genome-wide quantitative profiling of intercellular heterogeneity of DNA modification from SMRT sequencing data. It is capable of estimating proportion of isogenic haploid cells, in which the same loci of the genome are differentially modified. We tested the reliability of qDNAmod with the SMRT sequencing data of Streptococcus pneumoniae strain ST556. qDNAmod detected extensive intercellular heterogeneity of DNA methylation (6-mA) in a clonal population of ST556. Subsequent biochemical analyses revealed that the recognition sequences of two type I restriction–modification (R-M) systems are responsible for the intercellular heterogeneity of DNA methylation initially identified by qDNAmod. qDNAmod thus represents a valuable tool for studying intercellular phenotypic heterogeneity from genome-wide DNA modification.

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July 19, 2019

BREX is a novel phage resistance system widespread in microbial genomes.

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.© 2014 The Authors.

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July 19, 2019

ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media.

Moraxella catarrhalis is a significant cause of otitis media and exacerbations of chronic obstructive pulmonary disease. Here, we characterize a phase-variable DNA methyltransferase (ModM), which contains 5′-CAAC-3′ repeats in its open reading frame that mediate high-frequency mutation resulting in reversible on/off switching of ModM expression. Three modM alleles have been identified (modM1-3), with modM2 being the most commonly found allele. Using single-molecule, real-time (SMRT) genome sequencing and methylome analysis, we have determined that the ModM2 methylation target is 5′-GAR(m6)AC-3′, and 100% of these sites are methylated in the genome of the M. catarrhalis 25239 ModM2 on strain. Proteomic analysis of ModM2 on and off variants revealed that ModM2 regulates expression of multiple genes that have potential roles in colonization, infection, and protection against host defenses. Investigation of the distribution of modM alleles in a panel of M. catarrhalis strains, isolated from the nasopharynx of healthy children or middle ear effusions from patients with otitis media, revealed a statistically significant association of modM3 with otitis media isolates. The modulation of gene expression via the ModM phase-variable regulon (phasevarion), and the significant association of the modM3 allele with otitis media, suggests a key role for ModM phasevarions in the pathogenesis of this organism.-Blakeway, L. V., Power, P. M., Jen, F. E.-C., Worboys, S. R., Boitano, M., Clark, T. A., Korlach, J., Bakaletz, L. O., Jennings, M. P., Peak, I. R., Seib, K. L. ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media. © FASEB.

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July 19, 2019

Going beyond five bases in DNA sequencing.

DNA sequencing has provided a wealth of information about biological systems, but thus far has focused on the four canonical bases, and 5-methylcytosine through comparison of the genomic DNA sequence to a transformed four-base sequence obtained after treatment with bisulfite. However, numerous other chemical modifications to the nucleotides are known to control fundamental life functions, influence virulence of pathogens, and are associated with many diseases. These modifications cannot be accessed with traditional sequencing methods. In this opinion, we highlight several emerging single-molecule sequencing techniques that have the potential to directly detect many types of DNA modifications as an integral part of the sequencing protocol. Copyright © 2012 Elsevier Ltd. All rights reserved.

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July 19, 2019

Exploring bacterial epigenomics in the next-generation sequencing era: a new approach for an emerging frontier.

Epigenetics has an important role for the success of foodborne pathogen persistence in diverse host niches. Substantial challenges exist in determining DNA methylation to situation-specific phenotypic traits. DNA modification, mediated by restriction-modification systems, functions as an immune response against antagonistic external DNA, and bacteriophage-acquired methyltransferases (MTase) and orphan MTases – those lacking the cognate restriction endonuclease – facilitate evolution of new phenotypes via gene expression modulation via DNA and RNA modifications, including methylation and phosphorothioation. Recent establishment of large-scale genome sequencing projects will result in a significant increase in genome availability that will lead to new demands for data analysis including new predictive bioinformatics approaches that can be verified with traditional scientific rigor. Sequencing technologies that detect modification coupled with mass spectrometry to discover new adducts is a powerful tactic to study bacterial epigenetics, which is poised to make novel and far-reaching discoveries that link biological significance and the bacterial epigenome. Copyright © 2014 Elsevier Ltd. All rights reserved.

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July 19, 2019

A comparative analysis of methylome profiles of Campylobacter jejuni sheep abortion isolate and gastroenteric strains using PacBio data.

Campylobacter jejuni is a leading cause of human gastrointestinal disease and small ruminant abortions in the United States. The recent emergence of a highly virulent, tetracycline-resistant C. jejuni subsp. jejuni sheep abortion clone (clone SA) in the United States, and that strain’s association with human disease, has resulted in a heightened awareness of the zoonotic potential of this organism. Pacific Biosciences’ Single Molecule, Real-Time sequencing technology was used to explore the variation in the genome-wide methylation patterns of the abortifacient clone SA (IA3902) and phenotypically distinct gastrointestinal-specific C. jejuni strains (NCTC 11168 and 81-176). Several notable differences were discovered that distinguished the methylome of IA3902 from that of 11168 and 81-176: identification of motifs novel to IA3902, genome-specific hypo- and hypermethylated regions, strain level variability in genes methylated, and differences in the types of methylation motifs present in each strain. These observations suggest a possible role of methylation in the contrasting disease presentations of these three C. jejuni strains. In addition, the methylation profiles between IA3902 and a luxS mutant were explored to determine if variations in methylation patterns could be identified that might explain the role of LuxS-dependent methyl recycling in IA3902 abortifacient potential.

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July 19, 2019

Analysis of the Campylobacter jejuni genome by SMRT DNA Sequencing identifies restriction-modification motifs.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.

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July 19, 2019

Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.

Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: C(m4)TAG and GCA(m6)BN6VTGC. Analysis of the ?HVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

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July 19, 2019

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Sequence data for Clostridium autoethanogenum using three generations of sequencing technologies.

During the past decade, DNA sequencing output has been mostly dominated by the second generation sequencing platforms which are characterized by low cost, high throughput and shorter read lengths for example, Illumina. The emergence and development of so called third generation sequencing platforms such as PacBio has permitted exceptionally long reads (over 20?kb) to be generated. Due to read length increases, algorithm improvements and hybrid assembly approaches, the concept of one chromosome, one contig and automated finishing of microbial genomes is now a realistic and achievable task for many microbial laboratories. In this paper, we describe high quality sequence datasets which span three generations of sequencing technologies, containing six types of data from four NGS platforms and originating from a single microorganism, Clostridium autoethanogenum. The dataset reported here will be useful for the scientific community to evaluate upcoming NGS platforms, enabling comparison of existing and novel bioinformatics approaches and will encourage interest in the development of innovative experimental and computational methods for NGS data.

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July 19, 2019

Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome.

One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility.Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length?=?1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing.Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

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July 19, 2019

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis.

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGY M6A: G-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-AC M6A: CC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CC M6A: GC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGY M6A: G-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGC M6A: GG-3′ sites, to 100% at 5′-ACGT M6A: GG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

An adenine code for DNA: A second life for N6-methyladenine.

DNA N6-methyladenine (6mA) protects against restriction enzymes in bacteria. However, isolated reports have suggested additional activities and its presence in other organisms, such as unicellular eukaryotes. New data now find that 6mA may have a gene regulatory function in green alga, worm, and fly, suggesting m6A as a potential “epigenetic” mark. Copyright © 2015 Elsevier Inc. All rights reserved.

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