RESUMO
Extracellular vesicles (EVs) are small, cell-derived membranous particles containing various nucleic acids, proteins, and lipids that play essential roles in intercellular communication. Evidence indicating that part of the regenerative benefit from stem cell therapy arises through EVs released from transplanted cells created interest in using EVs for clinical applications. EVs from various cellular sources, including mesenchymal stem cells, neural stem cells, and glia, are efficacious in models of neurological disease. In these models, EVs attenuate reactive gliosis, neuronal death, pro-inflammatory signaling, as well as reduce cognitive, behavioral, and motor deficits. EVs are naturally permeable to the blood-brain barrier and can be modified to contain molecules of interest, thereby also serving as a vehicle to transport therapeutics into the brain. This review summarizes the current state of research using EVs as a treatment in models of neurological disorders and highlights considerations for future research.
Assuntos
Encéfalo/metabolismo , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/metabolismo , Doenças do Sistema Nervoso/terapia , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/terapia , Comunicação Celular , Morte Celular , Vesículas Extracelulares/metabolismo , Gliose , Humanos , Doenças do Sistema Nervoso/metabolismo , Doenças Neuroinflamatórias , Fagocitose , Transplante de Células-Tronco , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/terapiaRESUMO
Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Nanofios/ultraestrutura , Periplasma/fisiologia , Shewanella/metabolismo , Shewanella/ultraestrutura , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Biocombustíveis , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons/fisiologia , Regulação Bacteriana da Expressão Gênica , Microscopia de Força Atômica , Modelos Químicos , Oxirredução , Periplasma/genéticaRESUMO
OBJECTIVE: Evidence is mixed on how young adults' cannabis and alcohol use and co-use patterns have changed following recreational cannabis legalization (RCL). Incorporating measures of frequency and intensity of use we examined changes in college students' use and co-use patterns following RCL. METHOD: Four-year college students (n = 845,589) ages 18-24 years participated in the National College Health Assessment between 2008 and 2018, including students from 7 states that enacted RCL and 42 that did not. Latent profile analyses identified six patterns of use from four indicator variables tapping frequency of cannabis use and frequency and intensity of alcohol use: Abstainers, Light Alcohol Only, Heavy Alcohol Only, Predominantly Heavy Cannabis Use, Moderate Co-use, and Heavy Co-use. RESULTS: Regression models that adjusted for time and person- and institution-level covariates indicated that students' exposure to RCL was associated with lower odds of being in the two alcohol-only use classes, higher odds of being in the Predominantly Heavy Cannabis Use, Heavy Co-Use and Abstainers classes, and was not significantly related to Moderate Co-Use class membership. CONCLUSIONS: RCL was positively associated with patterns of frequent cannabis use and frequent and intense co-use but also with abstinence. Use of alcohol-only became less prevalent after RCL. Research on how RCL influences the prevalence of problematic patterns of substance use will inform and improve prevention efforts.
Assuntos
Cannabis , Transtornos Relacionados ao Uso de Substâncias , Adulto Jovem , Humanos , Consumo de Bebidas Alcoólicas/epidemiologia , Estudantes , UniversidadesRESUMO
BACKGROUND/AIM: Activin A is involved in the pathogenesis of human liver diseases, but its therapeutic targeting is not fully explored. Here, we tested the effect of novel, highly specific small-molecule-based activin A antagonists (NUCC-474/555) in improving liver regeneration following partial hepatectomy and halting fibrosis progression in models of chronic liver diseases (CLDs). METHODS: Cell toxicity of antagonists was determined in rat hepatocytes and Huh-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Hepatocytes and hepatic stellate cells (HSCs) were treated with activin A and NUCC-555 and analyzed by reverse transcription-polymerase chain reaction and immunohistochemistry. Partial hepatectomized Fisher (F)344 rats were treated with NUCC-555, and bromodeoxyuridine (BrdU) incorporation was determined at 18/24/36/120/240 h. NUCC-555 was administered into thioacetamide- or carbon tetrachloride-treated F344 rats or C57BL/6 mice, and the fibrosis progression was studied. RESULTS: NUCC-474 showed higher cytotoxicity in cultured hepatic cells; therefore, NUCC-555 was used in subsequent studies. Activin A-stimulated overexpression of cell cycle-/senescence-related genes (e.g., p15INK4b, DEC1, Glb1) was near-completely reversed by NUCC-555 in hepatocytes. Activin A-mediated HSC activation was blocked by NUCC-555. In partial hepatectomized rats, antagonizing activin A signaling resulted in a 1.9-fold and 2.3-fold increase in BrdU+ cells at 18 and 24 h, respectively. Administration of NUCC-555 in rats and mice with progressing fibrosis significantly reduced collagen accumulation (7.9-fold), HSC activation indicated by reduced alpha smooth muscle actin+ and vimentin+ cells, and serum aminotransferase activity. CONCLUSIONS: Our studies demonstrate that activin A antagonist NUCC-555 promotes liver regeneration and halts fibrosis progression in CLD models, suggesting that blocking activin A signaling may represent a new approach to treating people with CLD.
Assuntos
Ativinas , Hepatopatias , Transdução de Sinais , Animais , Humanos , Camundongos , Ratos , Bromodesoxiuridina , Fibrose , Hepatopatias/tratamento farmacológico , Camundongos Endogâmicos C57BL , Ratos Endogâmicos F344 , Transdução de Sinais/efeitos dos fármacosRESUMO
The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.
Assuntos
Amidoidrolases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genoma Bacteriano/fisiologia , NAD/genética , Amidoidrolases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , NAD/metabolismo , Homologia de Sequência de Aminoácidos , Shewanella/enzimologia , Shewanella/genéticaRESUMO
Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated the HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of the Cra regulator characteristic of Enterobacteriales. In this study, we combined a large scale comparative genomic reconstruction of HexR-controlled regulons in 87 species of Proteobacteria with the detailed experimental analysis of the HexR regulatory network in the Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1 to 2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa was confirmed as a HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6-phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatic analysis and experimentally verified by changed gene expression pattern in S. oneidensis ΔhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using [(13)C]lactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Shewanella/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Proteínas de Bactérias/genética , Carbono/metabolismo , Mutação , Fosfotransferases (Aceptores Pareados)/biossíntese , Fosfotransferases (Aceptores Pareados)/genética , Ácido Pirúvico/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição/genéticaRESUMO
The ability to use lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal-reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial d- or l-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. By using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO_1522-SO_1518) containing lactate permease and candidate genes for both d- and l-lactate dehydrogenase enzymes. The predicted d-LDH gene (dld-II, SO_1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted l-LDH is encoded by 3 genes with previously unknown functions (lldEGF, SO_1520-SO_1518). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dld-II and lldEFG encode fully functional d-and l-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is a previously uncharacterized example of a multisubunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld-II in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.
Assuntos
Genoma Bacteriano , Lactatos/metabolismo , Shewanella/genética , Bacillus subtilis/genética , Biocatálise , Escherichia coli/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Shewanella/metabolismo , EstereoisomerismoRESUMO
Many animals frequently transition between different media while navigating their heterogeneous environments. These media vary in compliance, moisture content, and other characteristics that affect their physical properties. As a result, animals may need to alter their kinematics to adapt to potential changes in media while maintaining performance during predator escape and foraging. Due to its fluid nature, water is highly compliant, and although usually associated with swimming, water running has evolved in a variety of animals ranging from insects to mammals. While the best studied large water runners are the bipedal basilisk lizards (Basiliscus spp.), other lizards have also been observed to run across the surface of water, namely, Hemidactylus platyurus, a house gecko, and in this study, Anolis sagrei, the brown anole. Unlike the basilisk lizard, the primarily arboreal Anolis sagrei is not adapted for water running. Moreover, water running in A. sagrei, similar to that of the house gecko, was primarily quadrupedal. Here, we tested for performance and kinematic differences between aquatic and terrestrial running and if the variance in performance and kinematic variables differed between the two media. We found no difference in average and maximum velocity between running on land and water. We also found that Anolis sagrei had higher hindlimb stride frequencies, decreased duty factor, and shorter stride lengths on water, as well as more erect postures. Finally, we found that most kinematics did not differ in variance between the two media, but of those that were different, almost all were more variable during terrestrial running. Our findings show that animals may be capable of specialized modes of locomotion, even if they are not obviously adapted for them, and that they may do this by modulating their kinematics to facilitate locomotion through novel environments.
Assuntos
Lagartos , Corrida , Animais , Fenômenos Biomecânicos , Lagartos/anatomia & histologia , Locomoção , Água , MamíferosRESUMO
Aberrations in the centrosome number and structure can readily be detected at all stages of tumor progression and are considered hallmarks of cancer. Centrosome anomalies are closely linked to chromosome instability and, therefore, are proposed to be one of the driving events of tumor formation and progression. This concept, first posited by Boveri over 100 years ago, has been an area of interest to cancer researchers. We have now begun to understand the processes by which these numerical and structural anomalies may lead to cancer, and vice-versa: how key events that occur during carcinogenesis could lead to amplification of centrosomes. Despite the proliferative advantages that having extra centrosomes may confer, their presence can also lead to loss of essential genetic material as a result of segregational errors and cancer cells must deal with these deadly consequences. Here, we review recent advances in the current literature describing the mechanisms by which cancer cells amplify their centrosomes and the methods they employ to tolerate the presence of these anomalies, focusing particularly on centrosomal clustering.
RESUMO
Shewanella oneidensis MR-1 is a metal reducer that uses a large number of electron acceptors including thiosulfate, polysulfide and sulfite. The enzyme required for thiosulfate and polysulfide respiration has been recently identified, but the mechanisms of sulfite reduction remained unexplored. Analysis of MR-1 cultures grown anaerobically with sulfite suggested that the dissimilatory sulfite reductase catalyses six-electron reduction of sulfite to sulfide. Reduction of sulfite required menaquinones but was independent of the intermediate electron carrier CymA. Furthermore, the terminal sulfite reductase, SirA, was identified as an octahaem c cytochrome with an atypical haem binding site. The sulfite reductase of S. oneidensis MR-1 does not appear to be a sirohaem enzyme, but represents a new class of sulfite reductases. The gene that encodes SirA is located within a 10-gene locus that is predicted to encode a component of a specialized haem lyase, a menaquinone oxidase and copper transport proteins. This locus was identified in the genomes of several Shewanella species and appears to be linked to the ability of these organisms to reduce sulfite under anaerobic conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Shewanella/enzimologia , Sulfitos/metabolismo , Proteínas de Bactérias/genética , Teste de Complementação Genética , Mutação , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Shewanella/genética , Vitamina K 2/metabolismoRESUMO
The dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1 produced γ-MnS (rambergite) nanoparticles during the concurrent reduction of MnO2 and thiosulfate coupled to H2 oxidation. To investigate effect of direct microbial reduction of MnO2 on MnS formation, two MR-1 mutants defective in outer membrane c-type cytochromes (ΔmtrC/ΔomcA and ΔmtrC/ΔomcA/ΔmtrF) were also used and it was determined that direct reduction of MnO2 was dominant relative to chemical reduction by biogenic sulfide generated from thiosulfate reduction. Although bicarbonate was excluded from the medium, incubations of strain MR-1 with lactate as the electron donor produced MnCO3 (rhodochrosite) as well as MnS in nearly equivalent amounts as estimated by micro X-ray diffraction (micro-XRD) analysis. It was concluded that carbonate released from lactate metabolism promoted MnCO3 formation and that Mn(II) mineralogy was strongly affected by carbonate ions even in the presence of abundant sulfide and weakly alkaline conditions expected to favour the precipitation of MnS. Formation of MnS, as determined by a combination of micro-XRD, transmission electron microscopy, energy dispersive X-ray spectroscopy, and selected area electron diffraction analyses was consistent with equilibrium speciation modelling predictions. Biogenic manganese sulfide may be a manganese sink in the Mn biogeochemical cycle in select environments such as deep anoxic marine basins within the Baltic Sea.
Assuntos
Compostos de Manganês/metabolismo , Óxidos/metabolismo , Shewanella/metabolismo , Sulfetos/metabolismo , Tiossulfatos/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Carbonatos/metabolismo , Grupo dos Citocromos c/genética , Lactatos/metabolismo , Manganês/metabolismo , Mutação , Oxirredução , Shewanella/genética , Difração de Raios XRESUMO
Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level.
Assuntos
Biologia Computacional/métodos , Modelos Biológicos , Shewanella/crescimento & desenvolvimento , Shewanella/metabolismo , Trifosfato de Adenosina/metabolismo , Biomassa , Ácido Láctico/metabolismo , Modelos Lineares , Redes e Vias Metabólicas , Oxigênio/metabolismo , Fenótipo , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Carbohydrates are a primary source of carbon and energy for many bacteria. Accurate projection of known carbohydrate catabolic pathways across diverse bacteria with complete genomes constitutes a substantial challenge due to frequent variations in components of these pathways. To address a practically and fundamentally important challenge of reconstruction of carbohydrate utilization machinery in any microorganism directly from its genomic sequence, we combined a subsystems-based comparative genomic approach with experimental validation of selected bioinformatic predictions by a combination of biochemical, genetic and physiological experiments. RESULTS: We applied this integrated approach to systematically map carbohydrate utilization pathways in 19 genomes from the Shewanella genus. The obtained genomic encyclopedia of sugar utilization includes ~170 protein families (mostly metabolic enzymes, transporters and transcriptional regulators) spanning 17 distinct pathways with a mosaic distribution across Shewanella species providing insights into their ecophysiology and adaptive evolution. Phenotypic assays revealed a remarkable consistency between predicted and observed phenotype, an ability to utilize an individual sugar as a sole source of carbon and energy, over the entire matrix of tested strains and sugars.Comparison of the reconstructed catabolic pathways with E. coli identified multiple differences that are manifested at various levels, from the presence or absence of certain sugar catabolic pathways, nonorthologous gene replacements and alternative biochemical routes to a different organization of transcription regulatory networks. CONCLUSIONS: The reconstructed sugar catabolome in Shewanella spp includes 62 novel isofunctional families of enzymes, transporters, and regulators. In addition to improving our knowledge of genomics and functional organization of carbohydrate utilization in Shewanella, this study led to a substantial expansion of our current version of the Genomic Encyclopedia of Carbohydrate Utilization. A systematic and iterative application of this approach to multiple taxonomic groups of bacteria will further enhance it, creating a knowledge base adequate for the efficient analysis of any newly sequenced genome as well as of the emerging metagenomic data.
Assuntos
Metabolismo dos Carboidratos/genética , Genoma Bacteriano/genética , Redes e Vias Metabólicas/genética , Shewanella/genética , Shewanella/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carbono/metabolismo , Enterobacteriaceae/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Dados de Sequência Molecular , Fenótipo , Regulon/genética , Reprodutibilidade dos Testes , Shewanella/enzimologia , Shewanella/isolamento & purificação , Transcrição GênicaRESUMO
Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracellular UO(2) nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2) nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO(2)-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2) nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2) nanoparticles. In the environment, such association of UO(2) nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2) or transport in soils and sediments.
Assuntos
Grupo dos Citocromos c/metabolismo , Shewanella/metabolismo , Compostos de Urânio/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Biodegradação Ambiental , Glicocálix/química , Ferro/metabolismo , Nanopartículas Metálicas/química , Oxirredução , Fósforo/metabolismo , Polissacarídeos Bacterianos/metabolismo , Distribuição Tecidual , Urânio/farmacocinética , Compostos de Urânio/farmacocinéticaRESUMO
Pertechnetate, (99)Tc(VII)O(4)(-), is a highly mobile radionuclide contaminant at US Department of Energy sites that can be enzymatically reduced by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble Tc(IV)O(2(s)). In other microorganisms, Tc(VII)O(4)(-) reduction is generally considered to be catalysed by hydrogenase. Here, we provide evidence that although the NiFe hydrogenase of MR-1 was involved in the H(2)-driven reduction of Tc(VII)O(4)(-)[presumably through a direct coupling of H(2) oxidation and Tc(VII) reduction], the deletion of both hydrogenase genes did not completely eliminate the ability of MR-1 to reduce Tc(VII). With lactate as the electron donor, mutants lacking the outer membrane c-type cytochromes MtrC and OmcA or the proteins required for the maturation of c-type cytochromes were defective in reducing Tc(VII) to nanoparticulate TcO(2) x nH(2)O((s)) relative to MR-1 or a NiFe hydrogenase mutant. In addition, reduced MtrC and OmcA were oxidized by Tc(VII)O(4)(-), confirming the capacity for direct electron transfer from these OMCs to TcO(4)(-). c-Type cytochrome-catalysed Tc(VII) reduction could be a potentially important mechanism in environments where organic electron donor concentrations are sufficient to allow this reaction to dominate.
Assuntos
Grupo dos Citocromos c/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Shewanella/metabolismo , Pertecnetato Tc 99m de Sódio/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Transporte de Elétrons , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Oxirredução , Óxidos/química , Óxidos/metabolismo , Shewanella/química , Shewanella/enzimologia , Shewanella/genética , Pertecnetato Tc 99m de Sódio/química , Vitamina K 2/químicaRESUMO
Biofilms possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments that may ultimately drive species diversity, determine biofilm structure and the spatial distribution of the community members. Using non-invasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated the kinetics and stratification of anaerobic metabolism within live biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1. Biofilms were pre-grown using a defined minimal medium in a constant-depth film bioreactor and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. Biofilms generated in this manner were subjected to changing substrate/electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate) and the metabolic responses measured. Localized NMR spectroscopy was used to non-invasively measure hydrogen-containing metabolites at high temporal resolution (4.5 min) under O(2)-limited conditions. Reduction of electron acceptor under anaerobic conditions was immediately observed upon switching feed solutions indicating that no gene induction (transcriptional response) was needed for MR-1 to switch metabolism from O(2) to fumarate, dimethyl sulfoxide or nitrate. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate changes in population response to the availability of electron acceptor and to probe metabolic competition under O(2)-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how these factors relate to environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described herein.
Assuntos
Biofilmes/crescimento & desenvolvimento , Shewanella/química , Shewanella/fisiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Transporte de Elétrons , Espectroscopia de Ressonância Magnética , Microscopia Confocal , Shewanella/citologia , Shewanella/genéticaRESUMO
Recent studies have strongly suggested a role for the synaptic scaffolding protein SHANK1 in normal synaptic structure and signaling. Global SHANK1 knockout (SHANK1-/-) mice demonstrate reduced dendritic spine density, an immature dendritic spine phenotype and impairments in various cognitive tasks. SHANK1 overexpression is associated with increased dendritic spine size and impairments in fear conditioning. These studies suggest proper regulation of SHANK1 is crucial for appropriate synaptic structure and cognition. However, little is known regarding SHANK1's developmental expression in brain regions critical for learning. The current study quantified cell specific developmental expression of SHANK1 in the hippocampus, a brain region critically involved in various learning paradigms shown to be disrupted by SHANK1 dysregulation. Consistent with prior studies, SHANK1 was found to be strongly co-expressed with dendritic markers, with significant increased co-expression at postnatal day (P) 15, an age associated with increased synaptogenesis in the hippocampus. Interestingly, SHANK1 was also found to be expressed in astrocytes and microglia. To our knowledge, this is the first demonstration of glial SHANK1 localization; therefore, these findings were further examined via a glial purified primary cell culture fraction using magnetic cell sorting. This additional analysis further demonstrated that SHANK1 was expressed in glial cells, supporting our immunofluorescence co-expression findings. Developmentally, astroglial SHANK1 co-expression was found to be significantly elevated at P5 with a reduction into adulthood, while SHANK1 microglial co-expression did not significantly change across development. These data collectively implicate a more global role for SHANK1 in mediating normal cellular signaling in the brain. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 363-373, 2018.
Assuntos
Astrócitos/metabolismo , Região CA1 Hipocampal/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/citologia , Região CA1 Hipocampal/citologia , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismo , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Sinapses/metabolismoRESUMO
Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.
Assuntos
Citocromos c/metabolismo , Metais/metabolismo , Shewanella/enzimologia , Shewanella/metabolismo , Aerobiose , Anaerobiose , Citocromos c/genética , Deleção de Genes , Metais/toxicidade , Oxirredução , Shewanella/genéticaRESUMO
In the genome of Shewanella oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA) have been identified. These two systems seem to be atypical because the genome lacks genes encoding cytoplasmic membrane electron transport proteins, NapC for NAP and NrfBCD/NrfH for NRF, respectively. Here, we present evidence that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Transcriptional and mutational analyses suggest that CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfH in S. oneidensis. Surprisingly, a strain devoid of napB encoding the small subunit of nitrate reductase exhibited the maximum cell density sooner than the wild type. Further characterization of this strain showed that nitrite was not detected as a free intermediate in its culture and NapB provides a fitness gain for S. oneidensis to compete for nitrate in the environments. On the basis results from mutational analyses of napA, napB, nrfA and napBnrfA in-frame deletion mutants, we propose that NapB is able to favor nitrate reduction by routing electrons to NapA exclusively.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Shewanella/metabolismo , Análise Mutacional de DNA , Transporte de Elétrons , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Oxirredução , Compostos de Amônio Quaternário/metabolismo , Shewanella/enzimologiaRESUMO
Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.