RESUMO
Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.
Assuntos
Beauveria , Técnicas de Inativação de Genes , Beauveria/genética , Animais , Deleção de Genes , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Insetos/microbiologia , Genética Microbiana/métodosRESUMO
Deep-sea ecosystems are home to a diverse community of microorganisms. These microbes are not only fundamental to ecological processes but also a treasure trove of natural products and enzymes with significant scientific and industrial applications. This forum focuses on the vast diversity of deep-sea microbes and their potential for bioprospecting. It also discusses threats posed by climate change and deep-sea mining to deep-sea microbial genetic resources, and proposes future research directions.
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Produtos Biológicos , Bioprospecção , Ecossistema , Genética Microbiana , Mudança ClimáticaRESUMO
Introducción: El síndrome de Behcet, o enfermedad de Behcet, es un proceso autoinflamatorio crónico, poco frecuente, de etiología desconocida. Es una vasculitis que afecta arterias y venas de todos los calibres, con alteración de la función endotelial, que se expresa clínicamente con lesiones orgánicas variadas. En su fisiopatogenia intervienen factores genéticos, microbianos e inmunológicos. Los síntomas más comunes son las úlceras orales y genitales, inflamaciones oculares (uveítis, retinitis e iritis), lesiones de piel y artritis. Objetivo: Evaluar diversos marcadores de la respuesta inmune en paciente con síndrome de Behcet. Presentación del caso: Paciente masculino. 39 años de edad, con diagnóstico clínico de enfermedad de Behcet con reactantes de fase aguda y marcadores serológicos de autoinmunidad negativa. Las subpoblaciones linfocitarias están dentro de los valores referenciales, sin evidencias de activación linfocitaria. La presencia de una doble población de linfocitos B y los antecedentes familiares, sugieren la existencia de una población de linfocitos B de autoreconocimiento y la posible presencia de factores genéticos, respectivamente. El paciente respondió favorablemente a la terapia con esteroides. Conclusiones: El estudio apoya el criterio de que, en condiciones basales, no se detectan marcadores humorales de autoinmunidad, alteraciones de los valores de las subpoblaciones linfocitarias, ni evidencias de activación linfocitaria, pero no se puede excluir la presencia de una población de linfocitos B de autoreconocimiento(AU)
Introduction: Behcet's syndrome, also known as Behcet's disease is a chronic autoinflammatory process of low frequency and unknown etiology. It's an all sizes arteries and veins affecting vasculitis that causes an alteration of endothelial function and is expressed clinically by organ damage at various levels. Its pathogenesis involves genetic, microbial and immunological factors. The most common symptoms are oral and genital ulcers, eye inflammation (uveitis, iritis and retinitis), skin lesions and arthritis. Objective: to evaluate several inmunological markers in a patient with Behcet syndrome. Case presentation: 39 years old masculine patientwith clinical diagnosis of Behcet disease with negative acute phase reactants and serological authoinmunity markers and lymphocyte populations within referential range, without evidences of lymphocyte activation. The presence of a double B lymphocyte population and familial background, suggest the presence of a self recognitionB lymphocyte population and the probable presence of genetic factors, respectively. There was a good response to steroids treatment. Conclusions: The study supports the idea that at baseline, not humoral autoimmunity markers, changes in the values of lymphocyte subpopulations, and evidence of lymphocyte activation is detected, but can not exclude the presence of a population of B lymphocytes self-recognition(AU)
Assuntos
Humanos , Masculino , Pessoa de Meia-Idade , Artrite , Uveíte , Vasculite , Autoimunidade , Síndrome de Behçet , Genética Microbiana , Fatores Imunológicos , Diagnóstico ClínicoRESUMO
Genomic islands are related to microbial adaptation and carry different genomic characteristics from the host. Therefore, many methods have been proposed to detect genomic islands from the rest of the genome by evaluating its sequence composition. Many sequence features have been proposed, but many of them have not been applied to the identification of genomic islands. In this paper, we present a scheme to predict genomic islands using the chi-square test and random forest algorithm. We extract seven kinds of sequence features and select the important features with the chi-square test. All the selected features are then input into the random forest to predict the genome islands. Three experiments and comparison show that the proposed method achieves the best performance. This understanding can be useful to design more powerful method for the genomic island prediction.
Assuntos
Ilhas Genômicas , Genômica/métodos , Algoritmos , Distribuição de Qui-Quadrado , Biologia Computacional , Bases de Dados Genéticas/estatística & dados numéricos , Genética Microbiana/métodos , Genética Microbiana/estatística & dados numéricos , Genoma Bacteriano , Genômica/estatística & dados numéricos , Modelos GenéticosRESUMO
Specialised metabolites from microbial sources are well-known for their wide range of biomedical applications, particularly as antibiotics. When mining paired genomic and metabolomic data sets for novel specialised metabolites, establishing links between Biosynthetic Gene Clusters (BGCs) and metabolites represents a promising way of finding such novel chemistry. However, due to the lack of detailed biosynthetic knowledge for the majority of predicted BGCs, and the large number of possible combinations, this is not a simple task. This problem is becoming ever more pressing with the increased availability of paired omics data sets. Current tools are not effective at identifying valid links automatically, and manual verification is a considerable bottleneck in natural product research. We demonstrate that using multiple link-scoring functions together makes it easier to prioritise true links relative to others. Based on standardising a commonly used score, we introduce a new, more effective score, and introduce a novel score using an Input-Output Kernel Regression approach. Finally, we present NPLinker, a software framework to link genomic and metabolomic data. Results are verified using publicly available data sets that include validated links.
Assuntos
Genética Microbiana/estatística & dados numéricos , Genômica/estatística & dados numéricos , Metabolômica/estatística & dados numéricos , Software , Vias Biossintéticas/genética , Biologia Computacional , Mineração de Dados , Bases de Dados Factuais , Bases de Dados Genéticas , Genoma Microbiano , Fenômenos Microbiológicos , Família Multigênica , Análise de RegressãoRESUMO
DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or "haplotypes." However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.
Assuntos
Técnicas Genéticas , Genética Microbiana/métodos , Haplótipos , Software , Algoritmos , Evolução Biológica , HIV/genética , Humanos , Plasmodium vivax/genéticaRESUMO
Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.
Assuntos
Genética Microbiana , Proteínas de Protozoários/genética , Transgenes , Trypanosoma congolense/genética , Trypanosoma congolense/patogenicidade , Tripanossomíase Africana/parasitologia , Animais , Feminino , Genoma de Protozoário , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Tripanossomíase Africana/genéticaRESUMO
Climate change is strongly affecting high-mountain soils and warming in particular is associated with pronounced changes in microbe-mediated C and N cycling, affecting plant-soil interactions and greenhouse gas balances and therefore feedbacks to global warming. We used shotgun metagenomics to assess changes in microbial community structures, as well as changes in microbial C- and N-cycling potential and stress response genes and we linked these data with changes in soil C and N pools and temperature-dependent measurements of bacterial growth rates. We did so by incubating high-elevation soil from the Swiss Alps at 4°C, 15°C, 25°C, or 35°C for 1 month. We found no shift with increasing temperature in the C-substrate-degrader community towards taxa more capable of degrading recalcitrant organic matter. Conversely, at 35°C, we found an increase in genes associated with the degradation and modification of microbial cell walls, together with high bacterial growth rates. Together, these findings suggest that the rapidly growing high-temperature community is fueled by necromass from heat-sensitive taxa. This interpretation was further supported by a shift in the microbial N-cycling potential towards N mineralization and assimilation under higher temperatures, along with reduced potential for conversions among inorganic N forms. Microbial stress-response genes reacted inconsistently to increasing temperature, suggesting that the high-temperature community was not severely stressed by these conditions. Rather, soil microbes were able to acclimate by changing the thermal properties of membranes and cell walls as indicated by an increase in genes involved in membrane and cell wall modifications as well as a shift in the optimum temperature for bacterial growth towards the treatment temperature. Overall, our results suggest that high temperatures, as they may occur with heat waves under global warming, promote a highly active microbial community capable of rapid mineralization of microbial necromass, which may transiently amplify warming effects.
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Microbiologia do Solo , Solo , Genética Microbiana , Temperatura Alta , TemperaturaRESUMO
Microbes constitute the very core of our existence. Long believed to be a nuisance and proponents of various disease, latest research point toward their functions in processes that can prove beneficial for human survival and afford long-term protection from disease. The wide range of functions exhibited by a host of microbes implies diversity and heterogeneity at the level of the molecular machinery, thus stressing the need to take a closer look at the molecular underpinnings that dictate distinct outcomes.
Assuntos
Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interação Gene-Ambiente , Genética Microbiana , Interações Hospedeiro-Patógeno , Imunidade/genética , Estresse Fisiológico , Bactérias/classificação , Evolução Molecular , Genoma Bacteriano , HumanosRESUMO
Water infiltration into the subsurface can result in pronounced biogeochemical depth gradients. In this study, we assess metabolic potential and properties of the subsurface microbiome during water infiltration by analyzing sediments from spatially-segmented columns. Past work in these laboratory set-ups demonstrated that removal efficiencies of trace organic pollutants were enhanced by limited availability of biodegradable dissolved organic carbon (BDOC) associated with higher humic ratios and deeper sediment regions. Distinct differences were observed in the microbial community when contrasting shallow versus deeper profile sediments. Metagenomic analyses revealed that shallow sediments contained an enriched potential for bacterial growth and division processes. In contrast, deeper sediments harbored a significant increase in genes associated with the metabolism of secondary metabolites and the biotransformation of xenobiotic water pollutants. Metatranscripts further supported this trend, with increased potential for metabolic attributes associated with the biotransformation of xenobiotics and antibiotic resistance within deeper sediments. Furthermore, increasing ratios of humics in feed solutions correlated to enhanced expression of genes associated with xenobiotic biodegradation. These results provide genetic support for the interplay of dissolved organic carbon limitation and enhanced trace organic biotransformation by the subsurface microbiome.
Assuntos
Carbono , Genética Microbiana , Biodegradação Ambiental , Biotransformação , Sedimentos Geológicos , Microbiologia da ÁguaRESUMO
Streptococcus pneumoniae is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are available to manipulate this pathogen. In this study, we attempted to utilize one such tool, the Sweet Janus cassette, to replace the capsule locus with other capsule loci in our strain background and found that the efficiency of allelic replacement was low and the number of revertant false-positive colonies was high. We determined that the capacity to recombine capsule varied by the initial isolated colony, suggesting that frequency of reversion is dependent on the bacterial clone. Alternative selection markers may further expand the application of Sweet Janus. We created novel cassettes that utilized chlorinated phenylalanine as an alternative counter-selection agent in conjunction with the Janus or Sweet Janus cassette, providing a new dual or triple selection marker. Moreover, we created cassettes that do not require engineered resistance in the background strain, including both single and dual selection markers. We were able to utilize all constructs in allelic replacement of the capsule loci. These novel constructs provide a new means for generating gene deletions in S. pneumoniae that expand experimental applications.
Assuntos
Cápsulas Bacterianas/genética , Farmacorresistência Bacteriana/genética , Genética Microbiana , Recombinação Genética , Seleção Genética , Streptococcus pneumoniae/genética , Alelos , Conversão Gênica , Deleção de Genes , PlasmídeosRESUMO
Microbial genetics and breeding is a compulsory course for "Bioengineering Excellence Talents Experimental Class" and "Bioengineering International Student Class". However, the traditional teaching model has many deficiencies in terms of content selection, teaching methods and examination forms. At Tianjin University of Science and Technology, to improve the quality and effectiveness of teaching, especially in the field of microbiology, innovative leaders who meet the needs of national and international communities are highly needed. This article describes the reformed teaching content, teaching methods, and curriculum assessment methods of microbial genetics and breeding. With the help of the latest scientific research progress, pre-class preview system, video display, and diversified assessment methods, teaching mode has been innovatively reformed. As such, students not only mastered the relevant professional knowledge of microbial genetics and breeding, but also exercised their subjective initiative, teamwork consciousness, professional foreign language expression level, and cultivated their interest in scientific knowledge related to microbial genetics.
Assuntos
Cruzamento , Currículo , Genética Microbiana , Bioengenharia/educação , Currículo/normas , Genética Microbiana/educação , Genética Microbiana/tendências , Humanos , EstudantesRESUMO
The impact of modern agriculture on the evolutionary trajectory of plant pathogens is a central question for crop sustainability. The Green Revolution replaced traditional rice landraces with high-yielding varieties, creating a uniform selection pressure that allows measuring the effect of such intervention. In this study, we analyzed a unique historical pathogen record to assess the impact of a major resistance gene, Xa4, in the population structure of Xanthomonas oryzae pv. oryzae (Xoo) collected in the Philippines in a span of 40 years. After the deployment of Xa4 in the early 1960s, the emergence of virulent pathogen groups was associated with the increasing adoption of rice varieties carrying Xa4, which reached 80% of the total planted area. Whole genomes analysis of a representative sample suggested six major pathogen groups with distinctive signatures of selection in genes related to secretion system, cell-wall degradation, lipopolysaccharide production, and detoxification of host defense components. Association genetics also suggested that each population might evolve different mechanisms to adapt to Xa4. Interestingly, we found evidence of strong selective sweep affecting several populations in the mid-1980s, suggesting a major bottleneck that coincides with the peak of Xa4 deployment in the archipelago. Our study highlights how modern agricultural practices facilitate the adaptation of pathogens to overcome the effects of standard crop improvement efforts.
Assuntos
Resistência à Doença/genética , Genética Microbiana , Oryza/microbiologia , Seleção Artificial/genética , Xanthomonas/genética , Genes de Plantas , Genética Populacional , Genoma Bacteriano , Oryza/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Patologia Vegetal , Proteínas de Plantas/genética , Xanthomonas/patogenicidadeRESUMO
Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation.IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.
Assuntos
Clostridiales/genética , Edição de Genes/métodos , Genética Microbiana/métodos , Biologia Molecular/métodos , Carbono/metabolismo , Clostridiales/crescimento & desenvolvimento , Clostridiales/metabolismo , Clostridiales/virologia , Deleção de Genes , Aptidão Genética , Integrases , Íntrons , Prófagos/genéticaRESUMO
The evolutionary fate of mutator mutations - genetic variants that raise the genome-wide mutation rate - in asexual populations is often described as being frequency (or number) dependent. Mutators can invade a population by hitchhiking with a sweeping beneficial mutation, but motivated by earlier experiments results, it has been repeatedly suggested that mutators must be sufficiently frequent to produce such a driver mutation before non-mutators do. Here, we use stochastic, agent-based simulations to show that neither the strength nor the sign of selection on mutators depend on their initial frequency, and while the overall probability of hitchhiking increases predictably with frequency, the per-capita probability of fixation remains unchanged.
Assuntos
Evolução Molecular , Modelos Genéticos , Taxa de Mutação , Seleção Genética , Simulação por Computador , Genética MicrobianaRESUMO
Synthetic promoters are considered ideal candidates in driving robust gene expression. Most of the available synthetic promoters are minimal promoters, for which the upstream sequence of the 5' end of the core region is usually excluded. Although the upstream sequence has been shown to mediate transcription of natural promoters, its impact on synthetic promoters has not been widely studied. Here, a library of chromosomal DNA fragments is randomly fused with the 5' end of the J23119 synthetic promoter, and the transcriptional performance of the promoter is evaluated through ß-galactosidase assay, fluorescence intensity and chemical biosynthesis. Results show that changes in the upstream sequence can induce significant variation in the promoter strength of up to 5.8-fold. The effect is independent of the length of the insertions and the number of potential transcription factor binding sites. Several DNA fragments that are able to enhance the transcription of both the natural and the synthetic promoters are identified. This study indicates that the synthetic minimal promoters are susceptible to the surrounding sequence context. Therefore, the upstream sequence should be treated as an indispensable component in the design and application of synthetic promoters, or as an independent genetic part for the fine-tuning of gene expression.