RESUMO
PCR detection and quantification of vaginal lactobacilli remains problematic because of the high level of genetic heterogeneity and taxonomic complexity within the genus Lactobacillus. The aim of the present study was to identify conserved sequences among the genomes of major species of vaginal lactobacilli that could be used for the development of a PCR-based method for quantitative determination of vaginal microbiota-specific lactobacilli. Comparative analysis of the genomes of several species of vaginal lactobacilli allowed us to identify conserved regions in the rplK gene, which encodes ribosomal protein L11, and to design group-specific PCR primers and a probe for selected species from the L. acidophilus complex, including major vaginal lactobacilli Lactobacillus crispatus, L. gasseri, L. iners and L. jensenii as well as other species that are less common in vaginal microbiota. The applicability of the new assay in routine diagnostic testing was evaluated using a set of clinical samples. The assay was able to detect and quantify vagina-associated lactobacilli within a wide range of initial DNA template concentrations, indicating promising potential for clinical applications.
Assuntos
Lactobacillus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vagina/microbiologia , Sequência de Bases , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Genômica , Humanos , Lactobacillus/genética , Limite de Detecção , Sensibilidade e EspecificidadeRESUMO
We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , DNA/biossíntese , DNA/metabolismo , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Cinética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Taq Polimerase/química , Taq Polimerase/metabolismo , Temperatura , TermodinâmicaRESUMO
Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.
Assuntos
Edição de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Células CHO , Sistemas CRISPR-Cas , Cricetulus , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase/métodosRESUMO
Since evidence indicates that the Last Universal Common Ancestor (LUCA) was phylogenetically closest to Methanopyrus kandleri among living organisms with elucidated genomes, this study has been directed to a search for the most primitive Methanopyrus lineage. For this purpose, the divergence of valyl-tRNA synthetase (ValRS) and isoleucyl-tRNA synthetase (IleRS) was employed as a measure of primitivity. Comparison of Methanopyrus kandleri and the Methanopyrus isolates GC34 and GC37 from the Pacific Ocean and KOL6, TAG1, TAG11, and SNP6 from the Atlantic Ocean established that the Pacific lineages are more primitive than the Atlantic lineages. Both the groups, however, are younger than environmental genomes from the Kairei Field of Central Indian Ridge in the Indian Ocean. These results showed that different Methanopyrus isolates differ significantly with respect to ValRS-IleRS divergence. On this basis, genomes giving rise to the ValRS and IleRS gene fragments from the Central Indian Ridge represent the most primitive Methanopyrus, phylogenetically the oldest living lineage closest to LUCA.
Assuntos
Archaea/enzimologia , Archaea/genética , Variação Genética , Isoleucina-tRNA Ligase/genética , Valina-tRNA Ligase/genética , Oceano Atlântico , Sequência de Bases , Células Clonais , DNA Ribossômico/genética , Oceano Índico , Dados de Sequência Molecular , Oceano Pacífico , Homologia de Sequência do Ácido NucleicoRESUMO
The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mamíferos/genética , Animais , Células CHO , Mapeamento Cromossômico , Cricetinae , CricetulusRESUMO
Xanthan gum, a complex polysaccharide comprising glucose, mannose and glucuronic acid residues, is involved in numerous biotechnological applications in cosmetics, agriculture, pharmaceuticals, food and petroleum industries. Additionally, its oligosaccharides were shown to possess antimicrobial, antioxidant, and few other properties. Yet, despite its extensive usage, little is known about xanthan gum degradation pathways and mechanisms. Thermogutta terrifontis, isolated from a sample of microbial mat developed in a terrestrial hot spring of Kunashir island (Far-East of Russia), was described as the first thermophilic representative of the Planctomycetes phylum. It grows well on xanthan gum either at aerobic or anaerobic conditions. Genomic analysis unraveled the pathways of oligo- and polysaccharides utilization, as well as the mechanisms of aerobic and anaerobic respiration. The combination of genomic and transcriptomic approaches suggested a novel xanthan gum degradation pathway which involves novel glycosidase(s) of DUF1080 family, hydrolyzing xanthan gum backbone beta-glucosidic linkages and beta-mannosidases instead of xanthan lyases, catalyzing cleavage of terminal beta-mannosidic linkages. Surprisingly, the genes coding DUF1080 proteins were abundant in T. terrifontis and in many other Planctomycetes genomes, which, together with our observation that xanthan gum being a selective substrate for many planctomycetes, suggest crucial role of DUF1080 in xanthan gum degradation. Our findings shed light on the metabolism of the first thermophilic planctomycete, capable to degrade a number of polysaccharides, either aerobically or anaerobically, including the biotechnologically important bacterial polysaccharide xanthan gum.
RESUMO
Ferroplasmaceae represent ubiquitous iron-oxidising extreme acidophiles with a number of unique physiological traits. In a genome-based study of Ferroplasma acidiphilum YT, the only species of the genus Ferroplasma with a validly published name, we assessed its central metabolism and genome stability during a long-term cultivation experiment. Consistently with physiology, the genome analysis points to F. acidiphilum YT having an obligate peptidolytic oligotrophic lifestyle alongside with anaplerotic carbon assimilation. This narrow trophic specialisation abridges the sugar uptake, although all genes for glycolysis and gluconeogenesis, including bifunctional unidirectional fructose 1,6-bisphosphate aldolase/phosphatase, have been identified. Pyruvate and 2-oxoglutarate dehydrogenases are substituted by 'ancient' CoA-dependent pyruvate and alpha-ketoglutarate ferredoxin oxidoreductases. In the lab culture, after ~550 generations, the strain exhibited the mutation rate of ≥1.3 × 10-8 single nucleotide substitutions per site per generation, which is among the highest values recorded for unicellular organisms. All but one base substitutions were G:C to A:T, their distribution between coding and non-coding regions and synonymous-to-non-synonymous mutation ratios suggest the neutral drift being a prevalent mode in genome evolution in the lab culture. Mutations in nature seem to occur with lower frequencies, as suggested by a remarkable genomic conservation in F. acidiphilum YT variants from geographically distant populations.
Assuntos
Archaea/metabolismo , Evolução Biológica , Metabolismo Energético , Aminoácidos/metabolismo , Archaea/genética , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo de Erro de Pareamento de DNA , Evolução Molecular , Regulação da Expressão Gênica , Genoma Arqueal , Genômica/métodos , Redes e Vias Metabólicas , Mutação , Recombinação GenéticaRESUMO
Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.
Assuntos
DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Euryarchaeota/genética , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Reagentes de Ligações Cruzadas , DNA Topoisomerases Tipo I/metabolismo , DNA Arqueal/química , DNA Arqueal/ultraestrutura , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Euryarchaeota/química , Microscopia Eletrônica , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Concentração Osmolar , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Alinhamento de Sequência , Análise de Sequência , SoftwareRESUMO
Enzymes from (hyper)thermophiles "Thermozymes" offer a great potential for biotechnological applications. Thermophilic adaptation does not only provide stability toward high temperature but is also often accompanied by a higher resistance to other harsh physicochemical conditions, which are also frequently employed in industrial processes, such as the presence of, e.g., denaturing agents as well as low or high pH of the medium. In order to find new thermostable, xylan degrading hydrolases with potential for biotechnological application we used an in situ enrichment strategy incubating Hungate tubes with xylan as the energy substrate in a hot vent located in the tidal zone of Kunashir Island (Kuril archipelago). Using this approach a hyperthermophilic euryarchaeon, designated Thermococcus sp. strain 2319x1, growing on xylan as sole energy and carbon source was isolated. The organism grows optimally at 85°C and pH 7.0 on a variety of natural polysaccharides including xylan, carboxymethyl cellulose (CMC), amorphous cellulose (AMC), xyloglucan, and chitin. The protein fraction extracted from the cells surface with Tween 80 exhibited endoxylanase, endoglucanase and xyloglucanase activities. The genome of Thermococcus sp. strain 2319x1 was sequenced and assembled into one circular chromosome. Within the newly sequenced genome, a gene, encoding a novel type of glycosidase (143 kDa) with a unique five-domain structure, was identified. It consists of three glycoside hydrolase (GH) domains and two carbohydrate-binding modules (CBM) with the domain order GH5-12-12-CBM2-2 (N- to C-terminal direction). The full length protein, as well as truncated versions, were heterologously expressed in Escherichia coli and their activity was analyzed. The full length multidomain glycosidase (MDG) was able to hydrolyze various polysaccharides, with the highest activity for barley ß-glucan (ß- 1,3/1,4-glucoside), followed by that for CMC (ß-1,4-glucoside), cellooligosaccharides and galactomannan. The results reported here indicate that the modular MDG structure with multiple glycosidase and carbohydrate-binding domains not only extends the substrate spectrum, but also seems to allow the degradation of partially soluble and insoluble polymers in a processive manner. This report highlights the great potential in a multi-pronged approach consisting of a combined in situ enrichment, (comparative) genomics, and biochemistry strategy for the screening for novel enzymes of biotechnological relevance.
RESUMO
Fimers are specifically modified primers selected to inhibit nonspecific interactions occurring in cycle sequencing. They are postsynthetically derived from 2'-methoxyoxalamido or 2'-succinimido precursor oligonucleotides by treatment with appropriate small molecular weight modifiers (a primary aliphatic amine or hydroxide anion). We describe design, synthesis, and purification of fimers, and their use in protocols for direct sequencing of genomic deoxyribonucleic acid (DNA). Protocol for isolation of microbial genomic DNA is also reported.
Assuntos
Primers do DNA , Genoma , Sequência de BasesRESUMO
A new Lactobacillus acidophilus strain, FSI4, isolated from yogurt, was isolated and sequenced in our laboratory. Our data, although supportive of previous conclusions regarding the remarkable stability of L. acidophilus species, indicate accumulating mutations in commercial L. acidophilus strains that warrant further study of the effect of damaged genes on the competitiveness of these bacteria in gut microbiota.
RESUMO
A standard draft genome sequence of the white rot saprotrophic fungus Trametes hirsuta 072 (Basidiomycota, Polyporales) is presented. The genome sequence contains about 33.6 Mb assembled in 141 scaffolds with a G+C content of ~57.6%. The draft genome annotation predicts 14,598 putative protein-coding open reading frames (ORFs).
RESUMO
The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (R(w)) of approximately 3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw
Assuntos
Histonas/química , Methanobacteriales/química , Nucleossomos/química , Nucleossomos/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Sequência de Bases , Cristalografia por Raios X , DNA Arqueal/química , DNA Arqueal/metabolismo , DNA Arqueal/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Dimerização , Histonas/metabolismo , Histonas/ultraestrutura , Cinética , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/ultraestrutura , Nucleossomos/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Thermococcus nautili 30-1 (formerly Thermococcus nautilus), an anaerobic hyperthermophilic marine archaeon, was isolated in 1999 from a deep-sea hydrothermal vent during the Amistad campaign. Here, we present the complete sequence of T. nautili, which is able to produce membrane vesicles containing plasmid DNA. This property makes T. nautili a model organism to study horizontal gene transfer.
Assuntos
Archaea/enzimologia , Proteínas Arqueais/química , Bactérias/enzimologia , Proteínas de Bactérias/química , DNA Polimerase I/química , Archaea/genética , Proteínas Arqueais/genética , Bactérias/genética , Proteínas de Bactérias/genética , DNA Polimerase I/genética , Primers do DNA , RNA Polimerases Dirigidas por DNA/química , Desoxirribonucleotídeos/química , Estabilidade Enzimática/genética , Temperatura Alta , Cinética , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Proteínas ViraisRESUMO
The genomes of two closely related lytic Thermus thermophilus siphoviruses with exceptionally long (approximately 800 nm) tails, bacteriophages P23-45 and P74-26, were sequenced completely. The P23-45 genome consists of 84,201 bp with 117 putative open reading frames (ORFs), and the P74-26 genome has 83,319 bp and 116 putative ORFs. The two genomes are 92% identical with 113 ORFs shared. Only 25% of phage gene product functions can be predicted from similarities to proteins and protein domains with known functions. The structural genes of P23-45, most of which have no similarity to sequences from public databases, were identified by mass spectrometric analysis of virions. An unusual feature of the P23-45 and P74-26 genomes is the presence, in their largest intergenic regions, of long polypurine-polypyrimidine (R-Y) sequences with mirror repeat symmetry. Such sequences, abundant in eukaryotic genomes but rare in prokaryotes, are known to form stable triple helices that block replication and transcription and induce genetic instability. Comparative analysis of the two phage genomes shows that the area around the triplex-forming elements is enriched in mutational variations. In vitro, phage R-Y sequences form triplexes and block DNA synthesis by Taq DNA polymerase in orientation-dependent manner, suggesting that they may play a regulatory role during P23-45 and P74-26 development.
Assuntos
DNA Viral/química , Genoma Viral , Siphoviridae/genética , Thermus thermophilus/virologia , Sequência de Aminoácidos , Sequência de Bases , DNA/química , Replicação do DNA/genética , DNA Complementar/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteômica , Recombinação Genética/genética , Siphoviridae/química , Siphoviridae/metabolismo , Proteínas Virais/análise , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismoRESUMO
Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Calibragem , DNA Topoisomerases Tipo I/genética , Deinococcus/enzimologia , Euryarchaeota/enzimologia , Funções Verossimilhança , Magnetismo , Modelos Moleculares , Pinças Ópticas , Conformação Proteica , Vacínia/enzimologiaRESUMO
Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.