RESUMEN
Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.
Asunto(s)
ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Especificidad por SustratoRESUMEN
Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.
Asunto(s)
Acinetobacter , Proteasa La , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Archaea/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , ADN/metabolismo , ADN Helicasas/metabolismo , Unión Proteica , Acinetobacter/enzimología , Acinetobacter/virología , Proteasa La/ultraestructuraRESUMEN
Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.
Asunto(s)
ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Secuencia de Bases , Multimerización de Proteína , Especificidad por Sustrato , Regulación Alostérica , ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/genéticaRESUMEN
Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into 'defence islands', providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterize a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR-family homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localized with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.
Asunto(s)
Bacterias , Factores de Transcripción , Bacterias/genética , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/genética , Islas Genómicas/genética , Plásmidos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.
Asunto(s)
Acinetobacter/fisiología , Factores de Restricción Antivirales , Bacteriófagos , Acinetobacter/genética , Acinetobacter/virología , Factores de Restricción Antivirales/genética , Bacteriófagos/fisiología , ADN/metabolismo , Metiltransferasas/genética , OperónRESUMEN
BACKGROUND: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. RESULTS: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. CONCLUSIONS: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.
Asunto(s)
Secuenciación de Nanoporos , Xanthomonas , Efectores Tipo Activadores de la Transcripción/genética , Xanthomonas/genética , GenomaRESUMEN
Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.
Asunto(s)
Bacteriófagos , Bacteriófagos/fisiología , Filogenia , Evolución Biológica , Bacterias , InglaterraRESUMEN
Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.
Asunto(s)
Proteínas Bacterianas/química , Colifagos/genética , Enzimas de Restricción-Modificación del ADN/química , Escherichia coli/genética , Escherichia/genética , Plásmidos/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Colifagos/metabolismo , Cristalografía por Rayos X , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , ADN Viral/química , ADN Viral/genética , ADN Viral/metabolismo , Escherichia/metabolismo , Escherichia/virología , Escherichia coli/metabolismo , Escherichia coli/virología , Expresión Génica , Islas Genómicas , Genómica/métodos , Modelos Moleculares , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.
Asunto(s)
Bacteriófago lambda/genética , Metilación de ADN/genética , Escherichia coli/genética , Motivos de Nucleótidos/genética , Adenina/metabolismo , Bacillus cereus/genética , Sistemas CRISPR-Cas/genética , Metiltransferasas/genética , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Many bacterial genomes exclusively display an N4-methyl cytosine base (m4C), whose physiological significance is not yet clear. Helicobacter pylori is a carcinogenic bacterium and the leading cause of gastric cancer in humans. Helicobacter pylori strain 26695 harbors a single m4C cytosine methyltransferase, M2.HpyAII which recognizes 5' TCTTC 3' sequence and methylates the first cytosine residue. To understand the role of m4C modification, M2.hpyAII deletion strain was constructed. Deletion strain displayed lower adherence to host AGS cells and reduced potential to induce inflammation and apoptosis. M2.hpyAII gene deletion strain exhibited reduced capacity for natural transformation, which was rescued in the complemented strain carrying an active copy of M2.hpyAII gene in the genome. Genome-wide gene expression and proteomic analysis were carried out to discern the possible reasons behind the altered phenotype of the M2.hpyAII gene deletion strain. Upon the loss of m4C modification a total of 102 genes belonging to virulence, ribosome assembly and cellular components were differentially expressed. The present study adds a functional role for the presence of m4C modification in H. pylori and provides the first evidence that m4C signal acts as a global epigenetic regulator in H. pylori.
Asunto(s)
Metilación de ADN/genética , Genoma Bacteriano/genética , Helicobacter pylori/genética , Proteómica , Citosina/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , HumanosRESUMEN
Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Emparejamiento Base , ADN/química , División del ADN , Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Edición Génica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Recombinasas/química , Recombinasas/genética , Recombinasas/metabolismo , Efectores Tipo Activadores de la Transcripción/química , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Dedos de ZincRESUMEN
We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo - using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Escherichia coli/genética , Profagos/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Adenina/metabolismo , Secuencia de Bases , Metilación de ADN , Enzimas de Restricción del ADN/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/virología , Profagos/genética , Unión Proteica , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Especificidad por SustratoRESUMEN
Restriction Modification (RM) systems prevent the invasion of foreign genetic material into bacterial cells by restriction and protect the host's genetic material by methylation. They are therefore important in maintaining the integrity of the host genome. RM systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Comparing the structures of the different types, similarities can be observed suggesting an evolutionary link between these different types. This work describes the 'deconstruction' of a large Type I RM enzyme into forms structurally similar to smaller Type II RM enzymes in an effort to elucidate the pathway taken by Nature to form these different RM enzymes. Based upon the ability to engineer new enzymes from the Type I 'scaffold', an evolutionary pathway and the evolutionary pressures required to move along the pathway from Type I RM systems to Type II RM systems are proposed. Experiments to test the evolutionary model are discussed.
Asunto(s)
ADN Bacteriano/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Modelos Genéticos , Secuencia de Aminoácidos , Sitios de Unión , ADN Bacteriano/química , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo I/química , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5'-TCCRAC-3'; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5'-TCCRAC-3'). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5'-TCCRAC-3'). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.
Asunto(s)
ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/química , Secuencia de Bases , Metilación de ADN , Hidrólisis , Datos de Secuencia MolecularRESUMEN
Staphylococcus aureus displays a clonal population structure in which horizontal gene transfer between different lineages is extremely rare. This is due, in part, to the presence of a Type I DNA restriction-modification (RM) system given the generic name of Sau1, which maintains different patterns of methylation on specific target sequences on the genomes of different lineages. We have determined the target sequences recognized by the Sau1 Type I RM systems present in a wide range of the most prevalent S. aureus lineages and assigned the sequences recognized to particular target recognition domains within the RM enzymes. We used a range of biochemical assays on purified enzymes and single molecule real-time sequencing on genomic DNA to determine these target sequences and their patterns of methylation. Knowledge of the main target sequences for Sau1 will facilitate the synthesis of new vectors for transformation of the most prevalent lineages of this 'untransformable' bacterium.
Asunto(s)
Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/química , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , ADN/química , ADN/metabolismo , Dominios Proteicos , Análisis de Secuencia de ADN , Staphylococcus aureus/genética , Transformación BacterianaRESUMEN
Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5'-GACCGA-3' and 5'-CACCCA-3'. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5'-GACCGA-3' sequence only. Here, we report the discovery, isolation, and characterization of TaqIII, the third R-M system from T. aquaticus YT-1. TaqIII is a 1101-aa Type IIC/IIL enzyme and recognizes the 5'-CACCCA-3' sequence previously attributed to TaqII. The cleavage site is 11/9 nucleotides downstream of the A residue. The enzyme exhibits striking biochemical similarity to TaqII. The 93% identity between their aa sequences suggests that they have a common evolutionary origin. The genes are located on two separate plasmids, and are probably paralogs or pseudoparalogs. Putative positions and aa that specify DNA recognition were identified and recognition motifs for 6 uncharacterized Thermus-family enzymes were predicted.
Asunto(s)
Proteínas Bacterianas/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Motivos de Nucleótidos , Plásmidos/metabolismo , Thermus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Clonación Molecular , División del ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Isoenzimas/genética , Isoenzimas/metabolismo , Peso Molecular , Plásmidos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Thermus/genéticaRESUMEN
DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.
Asunto(s)
Epigenómica , Células Procariotas/metabolismo , Secuencia Conservada , Metilación de ADN/genética , Replicación del ADN/genética , Enzimas de Restricción-Modificación del ADN/clasificación , Enzimas de Restricción-Modificación del ADN/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , Genoma , Metiltransferasas/metabolismo , Anotación de Secuencia Molecular , Familia de Multigenes , Motivos de Nucleótidos/genética , Filogenia , Especificidad por SustratoRESUMEN
We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.
Asunto(s)
Citosina/metabolismo , Enzimas de Restricción-Modificación del ADN/metabolismo , ADN/metabolismo , Adenina/metabolismo , Secuencia de Aminoácidos , Catálisis , Biología Computacional/métodos , ADN/química , Enzimas de Restricción-Modificación del ADN/química , Enzimas de Restricción-Modificación del ADN/genética , Metilación , Metiltransferasas/química , Metiltransferasas/metabolismo , Mutación , Motivos de Nucleótidos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Neisseria meningitidis/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Epigénesis Genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Metilación , Datos de Secuencia Molecular , Neisseria meningitidis/genéticaRESUMEN
The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.