RESUMO
Germline pathogenic variants in two genes encoding the lysine-specific histone methyltransferase genes SETD1A and SETD2 are associated with neurodevelopmental disorders (NDDs) characterized by developmental delay and congenital anomalies. The SETD1A and SETD2 gene products play a critical role in chromatin-mediated regulation of gene expression. Specific methylation episignatures have been detected for a range of chromatin gene-related NDDs and have impacted clinical practice by improving the interpretation of variant pathogenicity. To investigate if SETD1A and/or SETD2-related NDDs are associated with a detectable episignature, we undertook targeted genome-wide methylation profiling of > 2 M CpGs using a next-generation sequencing-based assay. A comparison of methylation profiles in patients with SETD1A variants (n = 6) did not reveal evidence of a strong methylation episignature. A review of the clinical and genetic features of the SETD2 patient group revealed that, as reported previously, there were phenotypic differences between patients with truncating mutations (n = 4, Luscan-Lumish syndrome; MIM:616831) and those with missense codon 1740 variants [p.Arg1740Trp (n = 4) and p.Arg1740Gln (n = 2)]. Both SETD2 subgroups demonstrated a methylation episignature, which was characterized by hypomethylation and hypermethylation events, respectively. Within the codon 1740 subgroup, both the methylation changes and clinical phenotype were more severe in those with p.Arg1740Trp variants. We also noted that two of 10 cases with a SETD2-NDD had developed a neoplasm. These findings reveal novel epigenotype-genotype-phenotype correlations in SETD2-NDDs and predict a gain-of-function mechanism for SETD2 codon 1740 pathogenic variants.
Assuntos
Cromatina , Transtornos do Neurodesenvolvimento , Humanos , Cromatina/genética , Metilação de DNA/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Estudos de Associação Genética , CódonRESUMO
The European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena), provided from EMBL-EBI, has for more than three decades been responsible for archiving the world's public sequencing data and presenting this important resource to the scientific community to support and accelerate the global research effort. Here, we outline ENA services and content in 2018 and provide an overview of a selection of focus areas of development work: extending data coordination services around ENA, sequence submissions through template expansion, early pre-submission validation tools and our move towards a new browser and retrieval infrastructure.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Genômica/métodos , Europa (Continente) , Genoma , Humanos , Anotação de Sequência Molecular , Ferramenta de Busca , Software , Transcriptoma , Interface Usuário-Computador , NavegadorRESUMO
For 35 years the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) has been responsible for making the world's public sequencing data available to the scientific community. Advances in sequencing technology have driven exponential growth in the volume of data to be processed and stored and a substantial broadening of the user community. Here, we outline ENA services and content in 2017 and provide insight into a selection of current key areas of development in ENA driven by challenges arising from the above growth.
Assuntos
Bases de Dados de Ácidos Nucleicos , Biologia Computacional , Bases de Dados de Ácidos Nucleicos/tendências , Europa (Continente) , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Armazenamento e Recuperação da Informação , Internet , Anotação de Sequência MolecularRESUMO
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) offers a rich platform for data sharing, publishing and archiving and a globally comprehensive data set for onward use by the scientific community. With a broad scope spanning raw sequencing reads, genome assemblies and functional annotation, the resource provides extensive data submission, search and download facilities across web and programmatic interfaces. Here, we outline ENA content and major access modalities, highlight major developments in 2016 and outline a number of examples of data reuse from ENA.
Assuntos
Bases de Dados de Ácidos Nucleicos , Análise de Sequência de DNA , Análise de Sequência de RNA , Genômica , Internet , Anotação de Sequência MolecularRESUMO
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is a repository for the submission, maintenance and presentation of nucleotide sequence data and related sample and experimental information. In this article we report on ENA in 2015 regarding general activity, notable published data sets and major achievements. This is followed by a focus on sustainable biocuration of functional annotation, an area which has particularly felt the pressure of sequencing growth. The importance of functional annotation, how it can be submitted and the shifting role of the biocurator in the context of increasing volumes of data are all discussed.
Assuntos
Bases de Dados de Ácidos Nucleicos , Anotação de Sequência Molecular , Análise de Sequência de DNA , Análise de Sequência de RNA , Curadoria de DadosRESUMO
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe's primary resource for nucleotide sequence information. With the growing volume and diversity of public sequencing data comes the need for increased sophistication in data organisation, presentation and search services so as to maximise its discoverability and usability. In response to this, ENA has been introducing and improving checklists for use during submission and expanding its search facilities to provide targeted search results. Here, we give a brief update on ENA content and some major developments undertaken in data submission services during 2014. We then describe in more detail the services we offer for data discovery and retrieval.
Assuntos
Bases de Dados de Ácidos Nucleicos , Sequência de Bases , Genômica , Anotação de Sequência Molecular , Análise de SequênciaRESUMO
We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management.
Assuntos
Lipopolissacarídeos/farmacologia , Proteína Adaptadora de Sinalização NOD1/genética , Peptidoglicano/farmacologia , Pericitos/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/metabolismo , Artérias Cerebrais/citologia , Artérias Cerebrais/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação , Interleucina-6/biossíntese , Interleucina-6/metabolismo , Interleucina-8/biossíntese , Interleucina-8/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Pericitos/citologia , Pericitos/metabolismo , Cultura Primária de Células , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is a repository for the world public domain nucleotide sequence data output. ENA content covers a spectrum of data types including raw reads, assembly data and functional annotation. ENA has faced a dramatic growth in genome assembly submission rates, data volumes and complexity of datasets. This has prompted a broad reworking of assembly submission services, for which we now reach the end of a major programme of work and many enhancements have already been made available over the year to components of the submission service. In this article, we briefly review ENA content and growth over 2013, describe our rapidly developing services for genome assembly information and outline further major developments over the last year.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genômica , Europa (Continente) , InternetRESUMO
Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αß complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.
Assuntos
Anticorpos Antineoplásicos/imunologia , Biomarcadores Tumorais/imunologia , Imunoterapia/métodos , Proteínas Musculares/metabolismo , Neoplasias da Próstata/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Anticorpos Antineoplásicos/metabolismo , Especificidade de Anticorpos , Western Blotting , Técnicas de Visualização da Superfície Celular , Cromatografia de Afinidade , Cromatografia Líquida , Sistemas de Liberação de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/terapia , Estatísticas não Paramétricas , Espectrometria de Massas em TandemRESUMO
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/) collects, maintains and presents comprehensive nucleic acid sequence and related information as part of the permanent public scientific record. Here, we provide brief updates on ENA content developments and major service enhancements in 2012 and describe in more detail two important areas of development and policy that are driven by ongoing growth in sequencing technologies. First, we describe the ENA data warehouse, a resource for which we provide a programmatic entry point to integrated content across the breadth of ENA. Second, we detail our plans for the deployment of CRAM data compression technology in ENA.
Assuntos
Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Compressão de Dados , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Internet , Interface Usuário-ComputadorRESUMO
BACKGROUND: Recombinant antibodies are highly successful in many different pathological conditions and currently enjoy overwhelming recognition of their potential. There are a wide variety of protein expression systems available, but almost all therapeutic antibodies are produced in mammalian cell lines, which mimic human glycosylation. The production of clinical-grade antibodies in mammalian cells is, however, extremely expensive. Compared to mammalian systems, protein production in yeast strains such as Pichia pastoris, is simpler, faster and usually results in higher yields. RESULTS: In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for the human carcinoembryonic antigen (CEA), was expressed in human embryonic kidney 293 cells and in Pichia pastoris. Mammalian- and yeast-produced anti-CEA trimerbody molecules display similar functional and structural properties, yet, the yield of trimerbody expressed in P. pastoris is about 20-fold higher than in human cells. CONCLUSIONS: P. pastoris is an efficient expression system for multivalent trimerbody molecules, suitable for their commercial production.
Assuntos
Biotecnologia/métodos , Pichia/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Antígeno Carcinoembrionário/imunologia , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Células HEK293 , Humanos , Proteínas Imobilizadas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Soro/metabolismo , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Over the last decade, cholera outbreaks have become common in some parts of Kenya. The most recent cholera outbreak occurred in Coastal and Lake Victoria region during January 2009 and May 2010, where a total of 11,769 cases and 274 deaths were reported by the Ministry of Public Health and Sanitation. The objective of this study is to isolate Vibrio cholerae bacteriophages from the environmental waters of the Lake Victoria region of Kenya with potential for use as a biocontrol for cholera outbreaks. Water samples from wells, ponds, sewage effluent, boreholes, rivers, and lakes of the Lake Victoria region of Kenya were enriched for 48 h at 37 °C in broth containing a an environmental strain of V. cholerae. Bacteriophages were isolated from 5 out of the 42 environmental water samples taken. Isolated phages produced tiny, round, and clear plaques suggesting that these phages were lytic to V. cholerae. Transmission electron microscope examination revealed that all the nine phages belonged to the family Myoviridae, with typical icosahedral heads, long contractile tails, and fibers. Head had an average diameter of 88.3 nm and tail of length and width 84.9 and 16.1 nm, respectively. Vibriophages isolated from the Lake Victoria region of Kenya have been characterized and the isolated phages may have a potential to be used as antibacterial agents to control pathogenic V. cholerae bacteria in water reservoirs.
Assuntos
Bacteriófagos/isolamento & purificação , Lagos/microbiologia , Vibrio cholerae/virologia , Microbiologia da Água , Bacteriófagos/patogenicidade , Bacteriófagos/ultraestrutura , QuêniaRESUMO
Microorganisms from L. terrestris gut previously exposed to different types of plastic (PET, LDPE, LLDPE, and PS) were studied to be used as probiotics of earthworms in plastic-contaminated soils (LDPE, LLDPE and recycled mulching film) at mesocosm-scale trials. The most abundant morphotypes with enzymatic capacities of interest were identified. Pseudomonas alkylphenolica (PL4) and Pseudomonas putida (PL5) strains were selected to be used as inoculants using Morus alba leaves as carriers to strengthen the intestinal microbiota of earthworms. Culture (selective cetrimide agar medium) and molecular (qPCR) techniques were used to trace the presence of the inoculum in the intestine of the earthworms. Additionally, a metataxonomic analysis was carried out to study the biodiversity and functionality of the earthworm microbiome, and their measure of survival and weight. Probiotics improved the survival rates of earthworms exposed to plastics, which also increased the abundance of microbial groups of interest in plastic bioremediation tasks.
Assuntos
Microbioma Gastrointestinal , Oligoquetos , Animais , Polietileno , Biodiversidade , SoloRESUMO
Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen. In this study, through PFGE, sequencing of isolates showing variation, whole genome transcriptome analysis and examination of the mobile genetic elements, we found that, consistent with our previous hypothesis, the genome of C. rodentium is unstable as a result of repeat-mediated, large-scale genome recombination and because of active transposition of mobile genetic elements such as the prophages. We sequenced an additional C. rodentium strain, EX-33, to reveal that the reference strain ICC168 is representative of the species and that most of the inactivating mutations were common to both isolates and likely to have occurred early on in the evolution of this pathogen. We draw parallels with the evolution of other bacterial pathogens and conclude that C. rodentium is a recently evolved pathogen that may have emerged alongside the development of inbred mice as a model for human disease.
Assuntos
Citrobacter rodentium/genética , Citrobacter rodentium/patogenicidade , Genoma Bacteriano , Animais , Citrobacter rodentium/classificação , DNA Bacteriano/genética , Modelos Animais de Doenças , Eletroforese em Gel de Campo Pulsado , Infecções por Enterobacteriaceae/patologia , Feminino , Perfilação da Expressão Gênica , Rearranjo Gênico , Instabilidade Genômica , Humanos , Sequências Repetitivas Dispersas , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Prófagos/genética , Análise de Sequência de DNA , VirulênciaRESUMO
The increase in the production of highly recalcitrant plastic materials, and their accumulation in ecosystems, generates the need to investigate new sustainable strategies to reduce this type of pollution. Based on recent works, the use of microbial consortia could contribute to improving plastic biodegradation performance. This work deals with the selection and characterization of plastic-degrading microbial consortia using a sequential and induced enrichment technique from artificially contaminated microcosms. The microcosm consisted of a soil sample in which LLDPE (linear low-density polyethylene) was buried. Consortia were obtained from the initial sample by sequential enrichment in a culture medium with LLDPE-type plastic material (in film or powder format) as the sole carbon source. Enrichment cultures were incubated for 105 days with monthly transfer to fresh medium. The abundance and diversity of total bacteria and fungi were monitored. Like LLDPE, lignin is a very complex polymer, so its biodegradation is closely linked to that of some recalcitrant plastics. For this reason, counting of ligninolytic microorganisms from the different enrichments was also performed. Additionally, the consortium members were isolated, molecularly identified and enzymatically characterized. The results revealed a loss of microbial diversity at each culture transfer at the end of the induced selection process. The consortium selected from selective enrichment in cultures with LLDPE in powder form was more effective compared to the consortium selected in cultures with LLDPE in film form, resulting in a reduction of microplastic weight between 2.5 and 5.5%. Some members of the consortia showed a wide range of enzymatic activities related to the degradation of recalcitrant plastic polymers, with Pseudomonas aeruginosa REBP5 or Pseudomonas alloputida REBP7 strains standing out. The strains identified as Castellaniella denitrificans REBF6 and Debaryomyces hansenii RELF8 were also considered relevant members of the consortia although they showed more discrete enzymatic profiles. Other consortium members could collaborate in the prior degradation of additives accompanying the LLDPE polymer, facilitating the subsequent access of other real degraders of the plastic structure. Although preliminary, the microbial consortia selected in this work contribute to the current knowledge of the degradation of recalcitrant plastics of anthropogenic origin accumulated in natural environments.
RESUMO
BACKGROUND: Imprinting disorders (ImpDis) comprise diseases which are caused by aberrant regulation of monoallelically and parent-of-origin-dependent expressed genes. A characteristic molecular change in ImpDis patients is aberrant methylation signatures at disease-specific loci, without an obvious DNA change at the specific differentially methylated region (DMR). However, there is a growing number of reports on multilocus imprinting disturbances (MLIDs), i.e. aberrant methylation at different DMRs in the same patient. These MLIDs account for a significant number of patients with specific ImpDis, and several reports indicate a central role of pathogenic maternal effect variants in their aetiology by affecting the maturation of the oocyte and the early embryo. Though several studies on the prevalence and the molecular causes of MLID have been conducted, homogeneous datasets comprising both genomic and methylation data are still lacking. RESULTS: Based on a cohort of 36 MLID patients, we here present both methylation data obtained from next-generation sequencing (NGS, ImprintSeq) approaches and whole-exome sequencing (WES). The compilation of methylation data did not reveal a disease-specific MLID episignature, and a predisposition for the phenotypic modification was not obvious as well. In fact, this lack of epigenotype-phenotype correlation might be related to the mosaic distribution of imprinting defects and their functional relevance in specific tissues. CONCLUSIONS: Due to the higher sensitivity of NGS-based approaches, we suggest that ImprintSeq might be offered at reference centres in case of ImpDis patients with unusual phenotypes but MLID negative by conventional tests. By WES, additional MLID causes than the already known maternal effect variants could not be identified, neither in the patients nor in the maternal exomes. In cases with negative WES results, it is currently unclear to what extent either environmental factors or undetected genetic variants contribute to MLID.
Assuntos
Metilação de DNA , Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
HNRNPU encodes a multifunctional RNA-binding protein that plays critical roles in regulating pre-mRNA splicing, mRNA stability, and translation. Aberrant expression and dysregulation of HNRNPU have been implicated in various human diseases, including cancers and neurological disorders. We applied a next generation sequencing based assay (EPIC-NGS) to investigate genome-wide methylation profiling for >2 M CpGs for 7 individuals with a neurodevelopmental disorder associated with HNRNPU germline pathogenic loss-of-function variants. Compared to healthy individuals, 227 HNRNPU-associated differentially methylated positions were detected. Both hyper- and hypomethylation alterations were identified but the former predominated. The identification of a methylation episignature for HNRNPU-associated neurodevelopmental disorder (NDD) implicates HNPRNPU-related chromatin alterations in the aetiopathogenesis of this disorder and suggests that episignature profiling should have clinical utility as a predictor for the pathogenicity of HNRNPU variants of uncertain significance. The detection of a methylation episignaure for HNRNPU-associated NDD is consistent with a recent report of a methylation episignature for HNRNPK-associated NDD.
Assuntos
Epigenoma , Transtornos do Neurodesenvolvimento , Humanos , Metilação de DNA , Células Germinativas , Mutação em Linhagem Germinativa , Transtornos do Neurodesenvolvimento/genéticaRESUMO
We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.
Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Myoviridae/classificação , Myoviridae/genética , Bacteriófagos/ultraestrutura , Colífagos/classificação , Colífagos/genética , Colífagos/ultraestrutura , Genoma Viral , Glicosídeo Hidrolases , Myoviridae/ultraestrutura , Filogenia , Fagos de Salmonella/classificação , Fagos de Salmonella/genética , Fagos de Salmonella/ultraestrutura , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas Virais/química , Proteínas Virais/genética , Proteínas da Cauda Viral/química , Proteínas da Cauda Viral/genéticaRESUMO
Phage vB_EcoM_CBA120 (CBA120), isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd) into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra).
Assuntos
Colífagos , Escherichia coli O157/virologia , Genoma Viral , Proteínas Virais/genética , Animais , Evolução Biológica , Bovinos , Colífagos/química , Colífagos/classificação , Colífagos/genética , Colífagos/metabolismo , Biologia Computacional , Impressões Digitais de DNA , Escherichia coli O157/fisiologia , Microscopia Eletrônica de Transmissão , Filogenia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Análise de Sequência de DNA , Timidina/análise , Timidina/metabolismo , Trítio/análise , Trítio/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/fisiologiaRESUMO
Composting involves the selection of a microbiota capable of resisting the high temperatures generated during the process and degrading the lignocellulose. A deep understanding of the thermophilic microbial community involved in such biotransformation is valuable to improve composting efficiency and to provide thermostable biomass-degrading enzymes for biorefinery. This study investigated the lignocellulose-degrading thermophilic microbial culturome at all the stages of plant waste composting, focusing on the dynamics, enzymes, and thermotolerance of each member of such a community. The results revealed that 58% of holocellulose (cellulose plus hemicellulose) and 7% of lignin were degraded at the end of composting. The whole fungal thermophilic population exhibited lignocellulose-degrading activity, whereas roughly 8-10% of thermophilic bacteria had this trait, although exclusively for hemicellulose degradation (xylan-degrading). Because of the prevalence of both groups, their enzymatic activity, and the wide spectrum of thermotolerance, they play a key role in the breakdown of hemicellulose during the entire process, whereas the degradation of cellulose and lignin is restricted to the activity of a few thermophilic fungi that persists at the end of the process. The xylanolytic bacterial isolates (159 strains) included mostly members of Firmicutes (96%) as well as a few representatives of Actinobacteria (2%) and Proteobacteria (2%). The most prevalent species were Bacillus licheniformis and Aeribacillus pallidus. Thermophilic fungi (27 strains) comprised only four species, namely Thermomyces lanuginosus, Talaromyces thermophilus, Aspergillus fumigatus, and Gibellulopsis nigrescens, of whom A. fumigatus and T. lanuginosus dominated. Several strains of the same species evolved distinctly at the stages of composting showing phenotypes with different thermotolerance and new enzyme expression, even not previously described for the species, as a response to the changing composting environment. Strains of Bacillus thermoamylovorans, Geobacillus thermodenitrificans, T. lanuginosus, and A. fumigatus exhibiting considerable enzyme activities were selected as potential candidates for the production of thermozymes. This study lays a foundation to further investigate the mechanisms of adaptation and acquisition of new traits among thermophilic lignocellulolytic microorganisms during composting as well as their potential utility in biotechnological processing.