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1.  Comparative study of esomeprazole and lansoprazole in triple therapy for eradication of Helicobacter pylori in Japan 
AIM: To evaluate the efficacy and safety of esomeprazole-based triple therapy compared with lansoprazole therapy as first-line eradication therapy for patients with Helicobacter pylori (H. pylori) in usual post-marketing use in Japan, where the clarithromycin (CAM) resistance rate is 30%.
METHODS: For this multicenter, randomized, open-label, non-inferiority trial, we recruited patients (≥ 20 years of age) with H. pylori infection from 20 hospitals in Japan. We randomly allocated patients to esomeprazole therapy (esomeprazole 20 mg, CAM 400 mg, amoxicillin (AC) 750 mg for the first 7 d, with all drugs given twice daily) or lansoprazole therapy (lansoprazole 30 mg, CAM 400 mg, AC 750 mg for the first 7 d, with all drugs given twice daily) using a minimization method with age, sex, and institution as adjustment factors. Our primary outcome was the eradication rate by intention-to-treat (ITT) and per-protocol (PP) analyses. H. pylori eradication was confirmed by a urea breath test from 4 to 8 wk after cessation of therapy.
RESULTS: ITT analysis revealed the eradication rates of 69.4% (95%CI: 61.2%-76.6%) for esomeprazole therapy and 73.9% (95%CI: 65.9%-80.6%) for lansoprazole therapy (P = 0.4982). PP analysis showed eradication rate of 76.9% (95%CI: 68.6%-83.5%) for esomeprazole therapy and 79.8% (95%CI: 71.9%-86.0%) for lansoprazole therapy (P = 0.6423). There were no differences in adverse effects between the two therapies.
CONCLUSION: Esomeprazole showed non-inferiority and safety in a 7 day-triple therapy for eradication of H. pylori compared with lansoprazole.
PMCID: PMC3989972  PMID: 24764674
Helicobacter pylori; Eradication; Esomeprazole; Lansoprazole; Proton pomp inhibitor
2.  Methylome Diversification through Changes in DNA Methyltransferase Sequence Specificity 
PLoS Genetics  2014;10(4):e1004272.
Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement—the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.
Author Summary
Living organisms are affected by epigenetic variation in addition to DNA sequence variation. DNA methylation is one of the most studied epigenetic modifications in both prokaryotes and eukaryotes. In prokaryotes, most DNA methylation is by DNA methyltransferases with high sequence specificity. Helicobacter pylori, a human stomach pathogen responsible for stomach cancer and other diseases, carries a large number of DNA methyltransferase genes that vary among strains. In this work, we examined the distribution of DNA methylation in multiple H. pylori genomes using single-molecule real-time sequencing technology, which detects DNA methylation with single-base resolution. Comparison of methylation motifs between closely related genomes allowed assignment of a recognition sequence to each DNA methylation specificity-determining gene. Highly methylated genes were detected, although the general DNA methylation pattern varied among strains. Knockout of a methylation specificity-determining gene led to changes in the transcriptome. These findings are consistent with our hypothesis that changes in the methylome lead to changes in the transcriptome and to changes in phenotypes, providing potential targets for natural and artificial selection in adaptive evolution.
PMCID: PMC3983042  PMID: 24722038
3.  To be or not to be: regulation of restriction–modification systems and other toxin–antitoxin systems 
Nucleic Acids Research  2013;42(1):70-86.
One of the simplest classes of genes involved in programmed death is that containing the toxin–antitoxin (TA) systems of prokaryotes. These systems are composed of an intracellular toxin and an antitoxin that neutralizes its effect. These systems, now classified into five types, were initially discovered because some of them allow the stable maintenance of mobile genetic elements in a microbial population through postsegregational killing or the death of cells that have lost these systems. Here, we demonstrate parallels between some TA systems and restriction–modification systems (RM systems). RM systems are composed of a restriction enzyme (toxin) and a modification enzyme (antitoxin) and limit the genetic flux between lineages with different epigenetic identities, as defined by sequence-specific DNA methylation. The similarities between these systems include their postsegregational killing and their effects on global gene expression. Both require the finely regulated expression of a toxin and antitoxin. The antitoxin (modification enzyme) or linked protein may act as a transcriptional regulator. A regulatory antisense RNA recently identified in an RM system can be compared with those RNAs in TA systems. This review is intended to generalize the concept of TA systems in studies of stress responses, programmed death, genetic conflict and epigenetics.
PMCID: PMC3874152  PMID: 23945938
4.  Chromosome Painting In Silico in a Bacterial Species Reveals Fine Population Structure 
Molecular Biology and Evolution  2013;30(6):1454-1464.
Identifying population structure forms an important basis for genetic and evolutionary studies. Most current methods to identify population structure have limitations in analyzing haplotypes and recombination across the genome. Recently, a method of chromosome painting in silico has been developed to overcome these shortcomings and has been applied to multiple human genome sequences. This method detects the genome-wide transfer of DNA sequence chunks through homologous recombination. Here, we apply it to the frequently recombining bacterial species Helicobacter pylori that has infected Homo sapiens since their birth in Africa and shows wide phylogeographic divergence. Multiple complete genome sequences were analyzed including sequences from Okinawa, Japan, that we recently sequenced. The newer method revealed a finer population structure than revealed by a previous method that examines only MLST housekeeping genes or a phylogenetic network analysis of the core genome. Novel subgroups were found in Europe, Amerind, and East Asia groups. Examination of genetic flux showed some singleton strains to be hybrids of subgroups and revealed evident signs of population admixture in Africa, Europe, and parts of Asia. We expect this approach to further our understanding of intraspecific bacterial evolution by revealing population structure at a finer scale.
PMCID: PMC3649679  PMID: 23505045
fineSTRUCTURE; homologous recombination; phylogenetic network; human evolution; Helicobacter pylori
5.  Unexpected variations in translation initiation machinery 
BMC Bioinformatics  2012;13(Suppl 18):A5.
PMCID: PMC3522020
6.  Mobility of DNA sequence recognition domains in DNA methyltransferases suggests epigenetics-driven adaptive evolution 
Mobile Genetic Elements  2012;2(6):292-296.
DNA methylation is one of the best studied epigenetic modifications observed in prokaryotes as well as eukaryotes. It affects nearby gene expression. Most DNA methylation reactions in prokaryotes are catalyzed by a DNA methyltransferase, the modification enzyme of a restriction-modification (RM) system. Its target recognition domain (TRD) recognizes a specific DNA sequence for methylation. In this commentary, we review recent evidence for movement of TRDs between non-orthologous genes and movement within a gene. These movements are likely mediated by DNA recombination machinery, and are expected to alter the methylation status of a genome. Such alterations potentially lead to changes in global gene expression pattern and various phenotypes. The targets of natural selection in adaptive evolution might be these diverse methylomes rather than diverse genome sequences, the target according to the current paradigm in biology. This “epigenetics-driven adaptive evolution” hypothesis can explain several observations in the evolution of prokaryotes and eukaryotes.
PMCID: PMC3575425  PMID: 23481556
DNA methylation; Helicobacter pylori; domain movement; epigenetics; evolution; genome evolution; methylome; protein evolution; restriction-modification
7.  Movement of DNA sequence recognition domains between non-orthologous proteins 
Nucleic Acids Research  2012;40(18):9218-9232.
Comparisons of proteins show that they evolve through the movement of domains. However, in many cases, the underlying mechanisms remain unclear. Here, we observed the movements of DNA recognition domains between non-orthologous proteins within a prokaryote genome. Restriction–modification (RM) systems, consisting of a sequence-specific DNA methyltransferase and a restriction enzyme, contribute to maintenance/evolution of genomes/epigenomes. RM systems limit horizontal gene transfer but are themselves mobile. We compared Type III RM systems in Helicobacter pylori genomes and found that target recognition domain (TRD) sequences are mobile, moving between different orthologous groups that occupy unique chromosomal locations. Sequence comparisons suggested that a likely underlying mechanism is movement through homologous recombination of similar DNA sequences that encode amino acid sequence motifs that are conserved among Type III DNA methyltransferases. Consistent with this movement, incongruence was observed between the phylogenetic trees of TRD regions and other regions in proteins. Horizontal acquisition of diverse TRD sequences was suggested by detection of homologs in other Helicobacter species and distantly related bacterial species. One of these RM systems in H. pylori was inactivated by insertion of another RM system that likely transferred from an oral bacterium. TRD movement represents a novel route for diversification of DNA-interacting proteins.
PMCID: PMC3467074  PMID: 22821560
8.  Genome-Wide Survey of Mutual Homologous Recombination in a Highly Sexual Bacterial Species 
Genome Biology and Evolution  2012;4(5):628-640.
The nature of a species remains a fundamental and controversial question. The era of genome/metagenome sequencing has intensified the debate in prokaryotes because of extensive horizontal gene transfer. In this study, we conducted a genome-wide survey of outcrossing homologous recombination in the highly sexual bacterial species Helicobacter pylori. We conducted multiple genome alignment and analyzed the entire data set of one-to-one orthologous genes for its global strains. We detected mosaic structures due to repeated recombination events and discordant phylogenies throughout the genomes of this species. Most of these genes including the “core” set of genes and horizontally transferred genes showed at least one recombination event. Taking into account the relationship between the nucleotide diversity and the minimum number of recombination events per nucleotide, we evaluated the recombination rate in every gene. The rate appears constant across the genome, but genes with a particularly high or low recombination rate were detected. Interestingly, genes with high recombination included those for DNA transformation and for basic cellular functions, such as biosynthesis and metabolism. Several highly divergent genes with a high recombination rate included those for host interaction, such as outer membrane proteins and lipopolysaccharide synthesis. These results provide a global picture of genome-wide distribution of outcrossing homologous recombination in a bacterial species for the first time, to our knowledge, and illustrate how a species can be shaped by mutual homologous recombination.
PMCID: PMC3381677  PMID: 22534164
homologous recombination; horizontal transfer; population genomics; species; Helicobacter pylori
9.  Large Variations in Bacterial Ribosomal RNA Genes 
Molecular Biology and Evolution  2012;29(10):2937-2948.
Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti–Shine-Dalgarno sequence (5′-CCTCC-3′). This loss was accompanied by elimination of Shine-Dalgarno–like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery.
PMCID: PMC3457768  PMID: 22446745
rRNA; Shine–Dalgarno sequence; symbiosis; Mycoplasma; Flavobacteria; genomic rearrangement
10.  Success of a suicidal defense strategy against infection in a structured habitat 
Scientific Reports  2012;2:238.
Pathogen infection often leads to the expression of virulence and host death when the host-pathogen symbiosis seems more beneficial for the pathogen. Previously proposed explanations have focused on the pathogen's side. In this work, we tested a hypothesis focused on the host strategy. If a member of a host population dies immediately upon infection aborting pathogen reproduction, it can protect the host population from secondary infections. We tested this "Suicidal Defense Against Infection" (SDAI) hypothesis by developing an experimental infection system that involves a huge number of bacteria as hosts and their virus as pathogen, which is linked to modeling and simulation. Our experiments and simulations demonstrate that a population with SDAI strategy is successful in the presence of spatial structure but fails in its absence. The infection results in emergence of pathogen mutants not inducing the host suicide in addition to host mutants resistant to the pathogen.
PMCID: PMC3268160  PMID: 22355751
12.  Evolution of cagA Oncogene of Helicobacter pylori through Recombination 
PLoS ONE  2011;6(8):e23499.
Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i) homologous recombination between DNA sequences for CagA multimerization (CM) sequence; (ii) recombination between DNA sequences for the EPIYA motif; and (iii) recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis.
PMCID: PMC3154945  PMID: 21853141
13.  Evolutionary genome engineering using a restriction–modification system 
Nucleic Acids Research  2011;39(20):9034-9046.
Modification of complex microbial cellular processes is often necessary to obtain organisms with particularly favorable characteristics, but such experiments can take many generations to achieve. In the present article, we accelerated the experimental evolution of Escherichia coli populations under selection for improved growth using one of the restriction–modification systems, which have shaped bacterial genomes. This resulted in faster evolutionary changes in both the genome and bacterial growth. Transcriptome/genome analysis at various stages enabled prompt identification of sequential genome rearrangements and dynamic gene-expression changes associated with growth improvement. The changes were related to cell-to-cell communication, the cell death program, as well as mass production and energy consumption. These observed changes imply that improvements in microorganism population growth can be achieved by inactivating the cellular mechanisms regulating fraction of active cells in a population. Some of the mutations were shown to have additive effects on growth. These results open the way for the application of evolutionary genome engineering to generate organisms with desirable properties.
PMCID: PMC3203608  PMID: 21785135
14.  Evolution in an oncogenic bacterial species with extreme genome plasticity: Helicobacter pylori East Asian genomes 
BMC Microbiology  2011;11:104.
The genome of Helicobacter pylori, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian H. pylori genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains.
A phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including oipA, hopMN, babABC, sabAB and vacA-2) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (tipα), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (miaA, tilS), a DNA recombinase/exonuclease that recognizes genome identity (addA), and DNA/RNA hybrid nucleases (rnhAB). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of cagA, vacA, homC (outer membrane protein), sotB (sugar transport), and a translation fidelity factor (miaA). Large divergence was seen in genes related to antibiotics: frxA (metronidazole resistance), def (peptide deformylase, drug target), and ftsA (actin-like, drug target).
These results demonstrate dramatic genome evolution within a species, especially in likely host interaction genes. The East Asian strains appear to differ greatly from the European strains in electron transfer and redox reactions. These findings also suggest a model of adaptive evolution through proteome diversification and selection through modulation of translational fidelity. The results define H. pylori East Asian lineages and provide essential information for understanding their pathogenesis and designing drugs and therapies that target them.
PMCID: PMC3120642  PMID: 21575176
15.  Domain Movement within a Gene: A Novel Evolutionary Mechanism for Protein Diversification 
PLoS ONE  2011;6(4):e18819.
A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.
PMCID: PMC3077401  PMID: 21533192
16.  Antisense RNA associated with biological regulation of a restriction–modification system 
Nucleic Acids Research  2011;39(13):5622-5632.
Restriction–modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (PREV0) at the 3′ end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the PM1,M2 promoter. PM1,M2 promoter-initiated transcription, in turn, appeared to be inhibited by PREV0. Mutational inactivation of PREV0 increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in PREV0 increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction–modification system.
PMCID: PMC3141266  PMID: 21459843
17.  Cleavage of a model DNA replication fork by a methyl-specific endonuclease 
Nucleic Acids Research  2011;39(13):5489-5498.
Epigenetic DNA methylation is involved in many biological processes. An epigenetic status can be altered by gain or loss of a DNA methyltransferase gene or its activity. Repair of DNA damage can also remove DNA methylation. In response to such alterations, DNA endonucleases that sense DNA methylation can act and may cause cell death. Here, we explored the possibility that McrBC, a methylation-dependent DNase of Escherichia coli, cleaves DNA at a replication fork. First, we found that in vivo restriction by McrBC of bacteriophage carrying a foreign DNA methyltransferase gene is increased in the absence of homologous recombination. This suggests that some cleavage events are repaired by recombination and must take place during or after replication. Next, we demonstrated that the enzyme can cleave a model DNA replication fork in vitro. Cleavage of a fork required methylation on both arms and removed one, the other or both of the arms. Most cleavage events removed the methylated sites from the fork. This result suggests that acquisition of even rarely occurring modification patterns will be recognized and rejected efficiently by modification-dependent restriction systems that recognize two sites. This process might serve to maintain an epigenetic status along the genome through programmed cell death.
PMCID: PMC3141261  PMID: 21441537
18.  IS-Linked Movement of a Restriction-Modification System 
PLoS ONE  2011;6(1):e16554.
Potential mobility of restriction-modification systems has been suggested by evolutionary/bioinformatic analysis of prokaryotic genomes. Here we demonstrate in vivo movement of a restriction-modification system within a genome under a laboratory condition. After blocking replication of a temperature-sensitive plasmid carrying a PaeR7I restriction-modification system in Escherichia coli cells, the plasmid was found integrated into the chromosome of the surviving cells. Sequence analysis revealed that, in the majority of products, the restriction-modification system was linked to chromosomal insertion sequences (ISs). Three types of products were: (I) apparent co-integration of the plasmid and the chromosome at a chromosomal IS1 or IS5 copy (24/28 analyzed); (II) de novo insertion of IS1 with the entire plasmid except for a 1–3 bp terminal deletion (2/28); and (III) reciprocal crossing-over between the plasmid and the chromosome involving 1–3 bp of sequence identity (2/28). An R-negative mutation apparently decreased the efficiency of successful integration by two orders of magnitude. Reconstruction experiments demonstrated that the restriction-dependence was mainly due to selection against cells without proper integration: their growth was inhibited by the restriction enzyme action. These results demonstrate collaboration of a mobile element and a restriction-modification system for successful joint migration. This collaboration may have promoted the spread and, therefore, the long-term persistence of these complexes and restriction-modification systems in a wide range of prokaryotes.
PMCID: PMC3031569  PMID: 21305031
19.  Conflicts Targeting Epigenetic Systems and Their Resolution by Cell Death: Novel Concepts for Methyl-Specific and Other Restriction Systems 
Epigenetic modification of genomic DNA by methylation is important for defining the epigenome and the transcriptome in eukaryotes as well as in prokaryotes. In prokaryotes, the DNA methyltransferase genes often vary, are mobile, and are paired with the gene for a restriction enzyme. Decrease in a certain epigenetic methylation may lead to chromosome cleavage by the partner restriction enzyme, leading to eventual cell death. Thus, the pairing of a DNA methyltransferase and a restriction enzyme forces an epigenetic state to be maintained within the genome. Although restriction enzymes were originally discovered for their ability to attack invading DNAs, it may be understood because such DNAs show deviation from this epigenetic status. DNAs with epigenetic methylation, by a methyltransferase linked or unlinked with a restriction enzyme, can also be the target of DNases, such as McrBC of Escherichia coli, which was discovered because of its methyl-specific restriction. McrBC responds to specific genome methylation systems by killing the host bacterial cell through chromosome cleavage. Evolutionary and genomic analysis of McrBC homologues revealed their mobility and wide distribution in prokaryotes similar to restriction–modification systems. These findings support the hypothesis that this family of methyl-specific DNases evolved as mobile elements competing with specific genome methylation systems through host killing. These restriction systems clearly demonstrate the presence of conflicts between epigenetic systems.
PMCID: PMC2993543  PMID: 21059708
intragenomic conflict; programmed cell death; epigenetic DNA methylation; restriction–modification system; McrBC
20.  Genome comparison and context analysis reveals putative mobile forms of restriction–modification systems and related rearrangements 
Nucleic Acids Research  2010;38(7):2428-2443.
The mobility of restriction–modification (RM) gene complexes and their association with genome rearrangements is a subject of active investigation. Here we conducted systematic genome comparisons and genome context analysis on fully sequenced prokaryotic genomes to detect RM-linked genome rearrangements. RM genes were frequently found to be linked to mobility-related genes such as integrase and transposase homologs. They were flanked by direct and inverted repeats at a significantly high frequency. Insertion by long target duplication was observed for I, II, III and IV restriction types. We found several RM genes flanked by long inverted repeats, some of which had apparently inserted into a genome with a short target duplication. In some cases, only a portion of an apparently complete RM system was flanked by inverted repeats. We also found a unit composed of RM genes and an integrase homolog that integrated into a tRNA gene. An allelic substitution of a Type III system with a linked Type I and IV system pair, and allelic diversity in the putative target recognition domain of Type IIG systems were observed. This study revealed the possible mobility of all types of RM systems, and the diversity in their mobility-related organization.
PMCID: PMC2853133  PMID: 20071371
21.  A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI) 
Nucleic Acids Research  2010;38(9):3019-3030.
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5′-GGCGC/C-3′ (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction–modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.
PMCID: PMC2875022  PMID: 20071747
22.  Cleavage of a model DNA replication fork by a Type I restriction endonuclease 
Nucleic Acids Research  2009;37(11):3531-3544.
Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction–modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various observations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from a recognition sequence. Here, we demonstrate that a Type I restriction enzyme preparation cleaves a model replication fork at its branch. The enzyme probably tracks along the DNA from an unmethylated recognition site on the daughter DNA and cuts the fork upon encountering the branch point. Our finding suggests that these restriction–modification systems contribute to genome maintenance through cell death and indicates that DNA replication fork cleavage represents a critical point in genome maintenance to choose between the restoration pathway and the destruction pathway.
PMCID: PMC2699502  PMID: 19357093
23.  From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction–modification gene complex 
Nucleic Acids Research  2009;37(9):3021-3031.
Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants’ deaths. With type II restriction–modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and σE-regulon was noticeable. With time, more SOS genes, stress-response genes (including σS-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of σE-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several σE-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes.
PMCID: PMC2685091  PMID: 19304752
24.  Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases 
Genome Biology  2008;9(11):R163.
The McrBC methyl-specific deoxyribonuclease from Escherichia coli can respond to genome methylation by host killing.
Alteration in epigenetic methylation can affect gene expression and other processes. In Prokaryota, DNA methyltransferase genes frequently move between genomes and present a potential threat. A methyl-specific deoxyribonuclease, McrBC, of Escherichia coli cuts invading methylated DNAs. Here we examined whether McrBC competes with genome methylation systems through host killing by chromosome cleavage.
McrBC inhibited the establishment of a plasmid carrying a PvuII methyltransferase gene but lacking its recognition sites, likely through the lethal cleavage of chromosomes that became methylated. Indeed, its phage-mediated transfer caused McrBC-dependent chromosome cleavage. Its induction led to cell death accompanied by chromosome methylation, cleavage and degradation. RecA/RecBCD functions affect chromosome processing and, together with the SOS response, reduce lethality. Our evolutionary/genomic analyses of McrBC homologs revealed: a wide distribution in Prokaryota; frequent distant horizontal transfer and linkage with mobility-related genes; and diversification in the DNA binding domain. In these features, McrBCs resemble type II restriction-modification systems, which behave as selfish mobile elements, maintaining their frequency by host killing. McrBCs are frequently found linked with a methyltransferase homolog, which suggests a functional association.
Our experiments indicate McrBC can respond to genome methylation systems by host killing. Combined with our evolutionary/genomic analyses, they support our hypothesis that McrBCs have evolved as mobile elements competing with specific genome methylation systems through host killing. To our knowledge, this represents the first report of a defense system against epigenetic systems through cell death.
PMCID: PMC2614495  PMID: 19025584
25.  Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site 
Catalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds.
R.Hpy188I is a Type II REase with unknown structure. PSI-BLAST searches of the non-redundant protein sequence database reveal only 1 homolog (R.HpyF17I, with nearly identical amino acid sequence and the same DNA sequence specificity). Standard application of state-of-the-art protein fold-recognition methods failed to predict the relationship of R.Hpy188I to proteins with known structure or to other protein families. In order to increase the amount of evolutionary information in the multiple sequence alignment, we have expanded our sequence database searches to include sequences from metagenomics projects. This search resulted in identification of 23 further members of R.Hpy188I family, both from metagenomics and the non-redundant database. Moreover, fold-recognition analysis of the extended R.Hpy188I family revealed its relationship to the GIY-YIG domain and allowed for computational modeling of the R.Hpy188I structure. Analysis of the R.Hpy188I model in the light of sequence conservation among its homologs revealed an unusual variant of the active site, in which the typical Tyr residue of the YIG half-motif had been substituted by a Lys residue. Moreover, some of its homologs have the otherwise invariant Arg residue in a non-homologous position in sequence that nonetheless allows for spatial conservation of the guanidino group potentially involved in phosphate binding.
The present study eliminates a significant "white spot" on the structural map of REases. It also provides important insight into sequence-structure-function relationships in the GIY-YIG nuclease superfamily. Our results reveal that in the case of proteins with no or few detectable homologs in the standard "non-redundant" database, it is useful to expand this database by adding the metagenomic sequences, which may provide evolutionary linkage to detect more remote homologs.
PMCID: PMC2630997  PMID: 19014591

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