RESUMO
The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Oxigênio , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Oxigênio/metabolismo , Methanosarcina/genética , Methanosarcina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredutases/metabolismo , Metano/metabolismo , Citocromos/metabolismo , Acetatos , Lactatos/metabolismoRESUMO
To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.
Assuntos
Proteínas Arqueais/metabolismo , Frutose-Bifosfatase/metabolismo , Methanosarcina/metabolismo , Fosfofrutoquinase-1/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Galinhas , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/isolamento & purificação , Frutosefosfatos/metabolismo , Genes Arqueais , Cinética , Methanosarcina/genética , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/isolamento & purificação , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent Km values for Na+ and Li+ were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent Km for K+ Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea IMPORTANCE: The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria A mechanism is proposed in which an MrpAD subcomplex catalyzes Na+/H+ antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways.
Assuntos
Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea/fisiologia , Methanosarcina/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Arqueais/genética , Transporte Biológico , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Lítio/metabolismo , Methanosarcina/genética , Modelos Moleculares , Filogenia , Conformação Proteica , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Methanosarcina acetivorans, considered a strict anaerobic archaeon, was cultured in the presence of 0.4-1% O2 (atmospheric) for at least 6 months to generate air-adapted cells; further, the biochemical mechanisms developed to deal with O2 were characterized. Methane production and protein content, as indicators of cell growth, did not change in air-adapted cells respect to cells cultured under anoxia (control cells). In contrast, growth and methane production significantly decreased in control cells exposed for the first time to O2. Production of reactive oxygen species was 50 times lower in air-adapted cells versus control cells, suggesting enhanced anti-oxidant mechanisms that attenuated the O2 toxicity. In this regard, (i) the transcripts and activities of superoxide dismutase, catalase and peroxidase significantly increased; and (ii) the thiol-molecules (cysteine + coenzyme M-SH + sulfide) and polyphosphate contents were respectively 2 and 5 times higher in air-adapted cells versus anaerobic-control cells. Long-term cultures (18 days) of air-adapted cells exposed to 2% O2 exhibited the ability to form biofilms. These data indicate that M. acetivorans develops multiple mechanisms to contend with O2 and the associated oxidative stress, as also suggested by genome analyses for some methanogens.
Assuntos
Metano/biossíntese , Methanosarcina/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Ar , Genoma Microbiano , Metano/metabolismo , Methanosarcina/genética , Methanosarcina/crescimento & desenvolvimento , Peroxidase/genética , Polifosfatos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
To assess what defence mechanisms are triggered by Cd(2+) stress in Methanosarcina acetivorans, cells were cultured at different cadmium concentrations. In the presence of 100 µM CdCl2, the intracellular contents of cysteine, sulfide and coenzyme M increased, respectively, 8, 27 and 7 times versus control. Cells incubated for 24 h in medium with less cysteine and sulfide removed up to 80% of Cd(2+) added, whereas their cysteine and coenzyme M contents increased 160 and 84 times respectively. Cadmium accumulation (5.2 µmol/10-15 mg protein) resulted in an increase in methane synthesis of 4.5 times in cells grown on acetate. Total phosphate also increased under high (0.5 mM) Cd(2+) stress. On the other hand, cells preadapted to 54 µM CdCl2 and further exposed to > 0.63 mM CdCl2 developed the formation of a biofilm with an extracellular matrix constituted by carbohydrates, DNA and proteins. Biofilm cells were able to synthesize methane. The data suggested that increased intracellular contents of thiol molecules and total phosphate, and biofilm formation, are all involved in the cadmium resistance mechanisms in this marine archaeon.
Assuntos
Biofilmes/crescimento & desenvolvimento , Cádmio/farmacologia , Farmacorresistência Bacteriana/fisiologia , Mesna/metabolismo , Methanosarcina/efeitos dos fármacos , Citratos/metabolismo , Cisteína/metabolismo , DNA Bacteriano/metabolismo , Matriz Extracelular/metabolismo , Malatos/metabolismo , Metano/biossíntese , Methanosarcina/genética , Methanosarcina/metabolismo , Fosfatos/metabolismo , Sulfetos/metabolismoRESUMO
Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos-São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30 degrees C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH(4)/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.
Assuntos
Metano/biossíntese , Methanobacteriaceae/metabolismo , Methanosarcina/metabolismo , Microbiologia da Água , Poluentes da Água , Brasil , Methanobacteriaceae/genética , Methanobacteriaceae/crescimento & desenvolvimento , Methanosarcina/genética , Methanosarcina/crescimento & desenvolvimentoRESUMO
This paper discusses the results of pentachlorophenol (PCP) anaerobic biodegradation in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor operated under methanogenic and halophylic conditions. The system was inoculated with autochthonous microorganisms taken from a site in the Santos-São Vicente Estuary (state of São Paulo, Brazil) severely contaminated with PCP, phenolic compounds, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and heavy metals. The inoculum was previously enriched for methanogenesis activity by changing glucose concentrations and under halophylic condition. PCP was added to the HAIB reactor as sodium salt (NaPCP) at an initial concentration of 5 mg l(-1) and increased to 13, 15, and 21 mg l(-1). Organic matter removal efficiency ranged from 77 to 100%. PCP removal efficiency was 100%. Denaturing gradient gel electrophoresis profile showed changes in the structure of Bacteria domain, which was associated with NaPCP and glucose amendments. The diversity of Archaea remained unaltered during the different phases. Scanning electron microscope examinations showed that cells morphologically resembling Methanosarcina and Methanosaeta predominated in the biofilm. These cells were detected by fluorescence in situ hybridization with the Methanosarcinales (MSMX860) specific probe. The results are of great importance in planning the estuary's restoration by using anaerobic technology and autochthonous microorganisms for bioremediation.
Assuntos
Reatores Biológicos/microbiologia , Sedimentos Geológicos/microbiologia , Pentaclorofenol/metabolismo , Microbiologia da Água , Anaerobiose , Bactérias/genética , Bactérias/metabolismo , Bactérias/ultraestrutura , Biodegradação Ambiental , Biofilmes/crescimento & desenvolvimento , Biomassa , Brasil , Eletroforese/métodos , Hibridização in Situ Fluorescente , Methanosarcina/genética , Methanosarcina/metabolismo , Methanosarcina/ultraestrutura , Microscopia Eletrônica de Varredura , Pentaclorofenol/química , RNA Ribossômico 16S/genéticaRESUMO
Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (gammaCA), a Zn containing enzyme from Methanosarcina thermophila (CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of gammaCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from alpha and gamma proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain gammaCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.