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The evolution, survival, and adaptation of microbes are consequences of gene duplication, acquisition, and divergence in response to environmental challenges. In this context, enzymes play a central role in the evolution of organisms, because they are fundamental in cell metabolism. Here, we analyzed the enzymatic repertoire in 6,467 microbial genomes, including their abundances, and their associations with metabolic maps. We found that the enzymes follow a power-law distribution, in relation to the genome sizes. Therefore, we evaluated the total proportion enzymatic classes in relation to the genomes, identifying a descending-order proportion: transferases (EC:2.-), hydrolases (EC:3.-), oxidoreductases (EC:1.-), ligases (EC:6.-), lyases (EC:4.-), isomerases (EC:5.-), and translocases (EC:7-.). In addition, we identified a preferential use of enzymatic classes in metabolism pathways for xenobiotics, cofactors and vitamins, carbohydrates, amino acids, glycans, and energy. Therefore, this analysis provides clues about the functional constraints associated with the enzymatic repertoire of functions in Bacteria and Archaea.
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In recent years, the fermented milk product kefir has been intensively studied because of its health benefits. Here, we evaluated the microbial consortia of two kefir samples, from Escarcega, Campeche, and Campeche (México). We considered a functional comparison between both samples, including fungal and bacterial inhibition; second, we applied shotgun metagenomics to assess the structure and functional diversity of the communities of microorganisms. These two samples exhibited antagonisms against bacterial and fungal pathogens. Bioactive polyketides and nonribosomal peptides were identified by LC-HRMS analysis. We also observed a high bacterial diversity and an abundance of Actinobacteria in both kefir samples, and a greater abundance of Saccharomyces species in kefir of Escarcega than in the Campeche kefir. When the prophage compositions were evaluated, the Campeche sample showed a higher diversity of prophage sequences. In Escarcega, we observed a prevalence of prophage families that infect Enterobacteria and Lactobacillus. The sequences associated with secondary metabolites, such as plipastatin, fengycin, and bacillaene, and also bacteriocins like helveticin and zoocin, were also found in different proportions, with greater diversity in the Escarcega sample. The analyses described in this work open the opportunity to understand the microbial diversity in kefir samples from two distant localities.
Assuntos
Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Fungos/efeitos dos fármacos , Kefir/microbiologia , Metagenoma , Animais , Bactérias/classificação , Biodiversidade , Produtos Fermentados do Leite/microbiologia , DNA Bacteriano , DNA Fúngico , Fermentação , Microbiologia de Alimentos , Fungos/classificação , Metagenômica/métodos , México , Microbiota , Leite/microbiologia , Peptídeos/farmacologia , Policetídeos/farmacologia , Prófagos/genética , Metabolismo SecundárioRESUMO
BACKGROUND: The European Commission (EC) Horizon 2020 (H2020)-funded ZIKAlliance Consortium designed a multicentre study including pregnant women (PW), children (CH) and natural history (NH) cohorts. Clinical sites were selected over a wide geographic range within Latin America and the Caribbean, taking into account the dynamic course of the ZIKV epidemic. METHODS: Recruitment to the PW cohort will take place in antenatal care clinics. PW will be enrolled regardless of symptoms and followed over the course of pregnancy, approximately every 4 weeks. PW will be revisited at delivery (or after miscarriage/abortion) to assess birth outcomes, including microcephaly and other congenital abnormalities according to the evolving definition of congenital Zika syndrome (CZS). After birth, children will be followed for 2 years in the CH cohort. Follow-up visits are scheduled at ages 1-3, 4-6, 12, and 24 months to assess neurocognitive and developmental milestones. In addition, a NH cohort for the characterization of symptomatic rash/fever illness was designed, including follow-up to capture persisting health problems. Blood, urine, and other biological materials will be collected, and tested for ZIKV and other relevant arboviral diseases (dengue, chikungunya, yellow fever) using RT-PCR or serological methods. A virtual, decentralized biobank will be created. Reciprocal clinical monitoring has been established between partner sites. Substudies of ZIKV seroprevalence, transmission clustering, disabilities and health economics, viral kinetics, the potential role of antibody enhancement, and co-infections will be linked to the cohort studies. DISCUSSION: Results of these large cohort studies will provide better risk estimates for birth defects and other developmental abnormalities associated with ZIKV infection including possible co-factors for the variability of risk estimates between other countries and regions. Additional outcomes include incidence and transmission estimates of ZIKV during and after pregnancy, characterization of short and long-term clinical course following infection and viral kinetics of ZIKV. STUDY REGISTRATIONS: clinicaltrials.gov NCT03188731 (PW cohort), June 15, 2017; clinicaltrials.gov NCT03393286 (CH cohort), January 8, 2018; clinicaltrials.gov NCT03204409 (NH cohort), July 2, 2017.
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Arbovírus/isolamento & purificação , Microcefalia/complicações , Complicações Infecciosas na Gravidez/epidemiologia , Infecção por Zika virus/epidemiologia , Zika virus/imunologia , Adulto , Arbovírus/genética , Região do Caribe/epidemiologia , Criança , Pré-Escolar , Estudos de Coortes , Coinfecção , Feminino , Seguimentos , Humanos , Lactente , América Latina/epidemiologia , Microcefalia/epidemiologia , Microcefalia/virologia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Cuidado Pré-Natal , Estudos Prospectivos , Risco , Estudos Soroepidemiológicos , Zika virus/isolamento & purificação , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologiaRESUMO
Intermediate-salinity environments are distributed around the world. Here, we present a snapshot characterization of two Peruvian thalassohaline environments at high altitude, Maras and Acos, which provide an excellent opportunity to increase our understanding of these ecosystems. The main goal of this study was to assess the structure and functional diversity of the communities of microorganisms in an intermediate-salinity environment, and we used a metagenomic shotgun approach for this analysis. These Andean hypersaline systems exhibited high bacterial diversity and abundance of the phyla Proteobacteria, Bacteroidetes, Balneolaeota, and Actinobacteria; in contrast, Archaea from the phyla Euryarchaeota, Thaumarchaeota, and Crenarchaeota were identified in low abundance. Acos harbored a more diverse prokaryotic community and a higher number of unique species compared with Maras. In addition, we obtained the draft genomes of two bacteria, Halomonas elongata and Idiomarina loihiensis, as well as the viral genomes of Enterobacteria lambda-like phage and Halomonas elongata-like phage and 27 partial novel viral halophilic genomes. The functional metagenome annotation showed a high abundance of sequences associated with detoxification, DNA repair, cell wall and capsule formation, and nucleotide metabolism; sequences for these functions were overexpressed mainly in bacteria and also in some archaea and viruses. Thus, their metabolic profiles afford a decrease in oxidative stress as well as the assimilation of nitrogen, a critical energy source for survival. Our work represents the first microbial characterization of a community structure in samples collected from Peruvian hypersaline systems.
Assuntos
Metagenômica/métodos , Microbiota/genética , Microbiota/fisiologia , Tolerância ao Sal/genética , Actinobacteria/genética , Altitude , Archaea/genética , Bactérias/genética , Bacteroidetes/genética , Biodiversidade , Euryarchaeota/genética , Peru , Filogenia , Proteobactérias/genética , RNA Ribossômico 16S/genética , Salinidade , Vírus/genéticaRESUMO
In recent years, there has been a large increase in the amount of experimental evidence for diverse archaeal organisms, and these findings allow for a comprehensive analysis of archaeal genetic organization. However, studies about regulatory mechanisms in this cellular domain are still limited. In this context, we identified a repertoire of 86 DNA-binding transcription factors (TFs) in the archaeon Pyrococcus furiosus DSM 3638, that are clustered into 32 evolutionary families. In structural terms, 45% of these proteins are composed of one structural domain, 41% have two domains, and 14% have three structural domains. The most abundant DNA-binding domain corresponds to the winged helix-turn-helix domain; with few alternative DNA-binding domains. We also identified seven regulons, which represent 13.5% (279 genes) of the total genes in this archaeon. These analyses increase our knowledge about gene regulation in P. furiosus DSM 3638 and provide additional clues for comprehensive modeling of transcriptional regulatory networks in the Archaea cellular domain.
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We describe here the enzymatic production of levan type-fructooligosaccharides (L-FOS) with a DP from 2 to 10, through simultaneous synthesis and hydrolysis reactions. This was accomplished by LevB1SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from Bacillus subtilis and LevB1, an endolevanase from B. licheniformis. In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s-1. LevB1 in LevB1SacB kinetic behavior improves considerably reaching saturation with levan and following Michaelis-Menten kinetics, quite differently from the previously reported first order kinetic behavior. We also report that LevB1SacB or both enzymes (LevB1 & SacB) at equimolar concentrations in simultaneous reactions result in an optimal, wide and diverse L-FOS profile, including 6-kestose, levanbiose and blastose among other L-FOS and 1-kestose, which accumulates as by-product of SacB levan synthesis. Yields of around 40% (w/w) were obtained from 600g/l sucrose with either LevB1SacB or LevB1 & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.
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Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Oligossacarídeos/síntese química , Frutanos/química , Oligossacarídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sacarose/metabolismoRESUMO
It has been documented that bacteria from the Burkholderia genera produce different kinds of compounds that inhibit plant pathogens, however in Burkholderia tropica, an endophytic diazotrophic and phosphate-solubilizing bacterium isolated from a wide diversity of plants, the capacity to produce antifungal compounds has not been evaluated. In order to expand our knowledge about Burkholderia tropica as a potential biological control agent, we analyzed 15 different strains of this bacterium to evaluate their capacities to inhibit the growth of four phytopathogenic fungi, Colletotrichum gloeosporioides, Fusarium culmorum, Fusarium oxysporum and Sclerotium rolffsi. Diverse analytical techniques, including plant root protection and dish plate growth assays and gas chromatography-mass spectroscopy showed that the fungal growth inhibition was intimately associated with the volatile compounds produced by B. tropica and, in particular, two bacterial strains (MTo293 and TTe203) exhibited the highest radial mycelial growth inhibition. Morphological changes associated with these compounds, such as disruption of fungal hyphae, were identified by using photomicrographic analysis. By using gas chromatography-mass spectroscopy technique, 18 volatile compounds involved in the growth inhibition mechanism were identified, including α-pinene and limonene. In addition, we found a high proportion of bacterial strains that produced siderophores during growth with different carbon sources, such as alanine and glutamic acid; however, their roles in the antagonism mechanism remain unclear.
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Burkholderia/química , Fungos/efeitos dos fármacos , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/farmacologia , Agentes de Controle Biológico , Colletotrichum/efeitos dos fármacos , Fusarium/efeitos dos fármacosRESUMO
Archaea represent an important and vast domain of life. This cellular domain includes a large diversity of organisms characterized as prokaryotes with basal transcriptional machinery similar to eukarya. In this work we explore the most recent findings concerning the transcriptional regulatory organization in archaeal genomes since the perspective of the DNA-binding transcription factors (TFs), such as the high proportion of archaeal TFs homologous to bacteria, the apparent deficit of TFs, only comparable to the proportion of TFs in parasites or intracellular pathogenic bacteria, suggesting a deficit in this class of proteins. We discuss an appealing hypothesis to explain the apparent deficit of TFs in archaea, based on their characteristics, such as their small length sizes. The hypothesis suggests that a large fraction of these small-sized TFs could supply the deficit of TFs in archaea, by forming different combinations of monomers similar to that observed in eukaryotic transcriptional machinery, where a wide diversity of protein-protein interactions could act as mediators of regulatory feedback, indicating a chimera of bacterial and eukaryotic TFs' functionality. Finally, we discuss how global experiments can help to understand in a global context the role of TFs in these organisms.
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Archaea/genética , Regulação da Expressão Gênica em Archaea/genética , Genoma ArquealRESUMO
BACKGROUND & AIMS: An extrahepatic human neutrophil collagenase complementary DNA (matrix metalloprotease-8) cloned in an adenovirus vector was used as a therapeutic agent in cirrhosis. METHODS: A high titer of clinical-grade AdMMP8 was obtained. RESULTS: HeLa cells transduced with AdMMP8 expressed recombinant matrix metalloprotease-8 messenger RNA and matrix metalloprotease-8 protein. Matrix metalloprotease-8 in culture sups showed enzymatic activity against native collagen type I, which was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline, and tissue inhibitor of metalloprotease-1. In vivo transduction showed matrix metalloprotease-8 activity, and studies to establish the efficacy of this characterized vector were performed in CCl(4) and bile duct-ligated cirrhotic rats. Transduction with 3 x 10(11) viral particles per kilogram resulted in hepatic detection of both messenger RNA and protein matrix metalloprotease-8. A consistent response in fibrosis reversal was observed in CCl(4) rats. Liver fibrosis in bile duct-ligated cirrhotic animals was decreased in 45%, along with diminished hydroxyproline content, after AdMMP8 treatment. The expression of matrix metalloprotease-2 and matrix metalloprotease-3 was up-regulated in AdMMP8 rats. Free tissue inhibitor of metalloprotease-1, as an indirect measurement of active uncomplexed matrix metalloproteases, was also increased in the AdMMP8 groups. Transforming growth factor-beta messenger RNA was diminished, and matrix metalloprotease-9 and hepatocyte growth factor increased. Treatment in both models correlated with improvements in ascites, functional hepatic tests, and gastric varices, indicating diminished intrahepatic blood pressure in animals injected with AdMMP8. CONCLUSIONS: Therefore, therapy with the matrix metalloprotease-8 gene is promising for use in a clinical setting.
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Terapia Genética , Cirrose Hepática Experimental/terapia , Metaloproteinase 8 da Matriz/genética , Adenoviridae/genética , Animais , Tetracloreto de Carbono , Colágeno/metabolismo , Meios de Cultura , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Veia Ilíaca , Cirrose Hepática Experimental/induzido quimicamente , Masculino , Metaloproteinase 8 da Matriz/metabolismo , RNA Mensageiro/genética , Ratos , Ratos WistarRESUMO
Gene therapy may represent a new avenue for the development of multimodal treatment for diverse forms of cirrhosis. This study explores the potential benefits of combining adenovirus-mediated human urokinase-plasminogen activator (AdHuPA) gene delivery and biliodigestive anastomosis to enhance the therapeutic efficacy of each treatment alone for cholestatic disorders resulting in secondary biliary cirrhosis. In an experimental model of secondary biliary cirrhosis, application of 6 x 10(11) vp/kg AdHuPA adenovirus vector resulted in 25.8% liver fibrosis reduction and some improvement in liver histology. The relief of bile cholestasis by a surgical procedure (biliodigestive anastomosis) combined with AdHuPA hepatic gene delivery rendered a synergistic effect, with a substantial 56.9 to 42.9% fibrosis decrease. AdHuPA transduction resulted in clear-cut expression of human uPA protein detected by immunohistochemistry and induction of up-regulation in the expression of metalloproteinases MMP-3, MMP-9, and MMP-2. Importantly, functional hepatic tests, specifically direct bilirubin, were improved. Also, hepatic cell regeneration, rearrangement of hepatic architecture, ascites, and gastric varices improved in cirrhotic rats treated with AdHuPA but not in counterpart AdGFP cirrhotic animals. We believe this might represent a novel therapeutic strategy for human cholestatic diseases.
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Adenoviridae/genética , Coledocostomia , Terapia Genética , Cirrose Hepática Biliar/terapia , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Terapia Combinada , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Fígado/enzimologia , Cirrose Hepática Biliar/patologia , Cirrose Hepática Biliar/cirurgia , Regeneração Hepática , Metaloproteases/biossíntese , Ratos , Ratos Wistar , Regulação para CimaRESUMO
A questão do desenvolvimento econômico coloca-se para o município de maneira diferente, nesta década, implicando problemas e soluções muito diversos daqueles que foram discutidos há pouco tempo. Desenvolver o município é gerenciar os problemas fazendo frente à complexidade e à incerteza, é melhorar a qualidade dos serviços aos cidadãos e procurar o desenvolvimento humano ao mesmo tempo em que o econômico