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1.
mBio ; 10(2)2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992355

RESUMO

Microbially produced electrically conductive protein filaments are of interest because they can function as conduits for long-range biological electron transfer. They also show promise as sustainably produced electronic materials. Until now, microbially produced conductive protein filaments have been reported only for bacteria. We report here that the archaellum of Methanospirillum hungatei is electrically conductive. This is the first demonstration that electrically conductive protein filaments have evolved in Archaea Furthermore, the structure of the M. hungatei archaellum was previously determined (N. Poweleit, P. Ge, H. N. Nguyen, R. R. O. Loo, et al., Nat Microbiol 2:16222, 2016, https://doi.org/10.1038/nmicrobiol.2016.222). Thus, the archaellum of M. hungatei is the first microbially produced electrically conductive protein filament for which a structure is known. We analyzed the previously published structure and identified a core of tightly packed phenylalanines that is one likely route for electron conductance. The availability of the M. hungatei archaellum structure is expected to substantially advance mechanistic evaluation of long-range electron transport in microbially produced electrically conductive filaments and to aid in the design of "green" electronic materials that can be microbially produced with renewable feedstocks.IMPORTANCE Microbially produced electrically conductive protein filaments are a revolutionary, sustainably produced, electronic material with broad potential applications. The design of new protein nanowires based on the known M. hungatei archaellum structure could be a major advance over the current empirical design of synthetic protein nanowires from electrically conductive bacterial pili. An understanding of the diversity of outer-surface protein structures capable of electron transfer is important for developing models for microbial electrical communication with other cells and minerals in natural anaerobic environments. Extracellular electron exchange is also essential in engineered environments such as bioelectrochemical devices and anaerobic digesters converting wastes to methane. The finding that the archaellum of M. hungatei is electrically conductive suggests that some archaea might be able to make long-range electrical connections with their external environment.


Assuntos
Condutividade Elétrica , Flagelos/fisiologia , Methanospirillum/fisiologia , Eletricidade , Transporte de Elétrons , Fenilalanina/química
2.
Environ Microbiol ; 21(5): 1833-1846, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30895699

RESUMO

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.


Assuntos
Ácidos/metabolismo , Proteínas de Bactérias/metabolismo , Deltaproteobacteria/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Ácidos/química , Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Benzoatos/metabolismo , Ácidos Cicloexanocarboxílicos/metabolismo , Deltaproteobacteria/enzimologia , Deltaproteobacteria/genética , Transporte de Elétrons , Metano/metabolismo , Methanospirillum/metabolismo , Proteômica
3.
Proc Natl Acad Sci U S A ; 116(14): 6897-6902, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30886102

RESUMO

The exergonic reaction of FeS with H2S to form FeS2 (pyrite) and H2 was postulated to have operated as an early form of energy metabolism on primordial Earth. Since the Archean, sedimentary pyrite formation has played a major role in the global iron and sulfur cycles, with direct impact on the redox chemistry of the atmosphere. However, the mechanism of sedimentary pyrite formation is still being debated. We present microbial enrichment cultures which grew with FeS, H2S, and CO2 as their sole substrates to produce FeS2 and CH4 Cultures grew over periods of 3 to 8 mo to cell densities of up to 2 to 9 × 106 cells per mL-1 Transformation of FeS with H2S to FeS2 was followed by 57Fe Mössbauer spectroscopy and showed a clear biological temperature profile with maximum activity at 28 °C and decreasing activities toward 4 °C and 60 °C. CH4 was formed concomitantly with FeS2 and exhibited the same temperature dependence. Addition of either penicillin or 2-bromoethanesulfonate inhibited both FeS2 and CH4 production, indicating a coupling of overall pyrite formation to methanogenesis. This hypothesis was supported by a 16S rRNA gene-based phylogenetic analysis, which identified at least one archaeal and five bacterial species. The archaeon was closely related to the hydrogenotrophic methanogen Methanospirillum stamsii, while the bacteria were most closely related to sulfate-reducing Deltaproteobacteria, as well as uncultured Firmicutes and Actinobacteria. Our results show that pyrite formation can be mediated at ambient temperature through a microbially catalyzed redox process, which may serve as a model for a postulated primordial iron-sulfur world.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Ferro/metabolismo , Methanospirillum , Filogenia , RNA Arqueal , RNA Ribossômico 16S , Sulfetos/metabolismo , Methanospirillum/genética , Methanospirillum/metabolismo , Oxirredução , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo
4.
Environ Microbiol ; 20(12): 4503-4511, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30126076

RESUMO

Under methanogenic conditions, short-chain fatty acids are common byproducts from degradation of organic compounds and conversion of these acids is an important component of the global carbon cycle. Due to the thermodynamic difficulty of propionate degradation, this process requires syntrophic interaction between a bacterium and partner methanogen; however, the metabolic strategies and behaviour involved are not fully understood. In this study, the first genome analysis of obligately syntrophic propionate degraders (Pelotomaculum schinkii HH and P. propionicicum MGP) and comparison with other syntrophic propionate degrader genomes elucidated novel components of energy metabolism behind Pelotomaculum propionate oxidation. Combined with transcriptomic examination of P. schinkii behaviour in co-culture with Methanospirillum hungatei, we found that formate may be the preferred electron carrier for P. schinkii syntrophy. Propionate-derived menaquinol may be primarily re-oxidized to formate, and energy was conserved during formate generation through newly proposed proton-pumping formate extrusion. P. schinkii did not overexpress conventional energy metabolism associated with a model syntrophic propionate degrader Syntrophobacter fumaroxidans MPOB (i.e., CoA transferase, Fix and Rnf). We also found that P. schinkii and the partner methanogen may also interact through flagellar contact and amino acid and fructose exchange. These findings provide new understanding of syntrophic energy acquisition and interactions.


Assuntos
Peptococcaceae/metabolismo , Propionatos/metabolismo , Deltaproteobacteria/metabolismo , Metabolismo Energético , Formiatos/metabolismo , Methanospirillum/metabolismo , Oxirredução
5.
Environ Microbiol ; 20(5): 1842-1856, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29611893

RESUMO

Syntrophobacter fumaroxidans is a sulfate-reducing bacterium able to grow on propionate axenically or in syntrophic interaction with methanogens or other sulfate-reducing bacteria. We performed a proteome analysis of S. fumaroxidans growing with propionate axenically with sulfate or fumarate, and in syntrophy with Methanospirillum hungatei, Methanobacterium formicicum or Desulfovibrio desulfuricans. Special attention was put on the role of hydrogen and formate in interspecies electron transfer (IET) and energy conservation. Formate dehydrogenase Fdh1 and hydrogenase Hox were the main confurcating enzymes used for energy conservation. In the periplasm, Fdh2 and hydrogenase Hyn play an important role in reverse electron transport associated with succinate oxidation. Periplasmic Fdh3 and Fdh5 were involved in IET. The sulfate reduction pathway was poorly regulated and many enzymes associated with sulfate reduction (Sat, HppA, AprAB, DsrAB and DsrC) were abundant even at conditions where sulfate was not present. Proteins similar to heterodisulfide reductases (Hdr) were abundant. Hdr/Flox was detected in all conditions while HdrABC/HdrL was exclusively detected when sulfate was available; these complexes most likely confurcate electrons. Our results suggest that S. fumaroxidans mainly used formate for electron release and that different confurcating mechanisms were used in its sulfidogenic metabolism.


Assuntos
Técnicas de Cocultura , Deltaproteobacteria/metabolismo , Desulfovibrio/metabolismo , Methanobacterium/metabolismo , Methanospirillum/metabolismo , Propionatos/metabolismo , Transporte de Elétrons , Formiato Desidrogenases/metabolismo , Formiatos , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Oxirredução , Proteoma/metabolismo , Sulfatos/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-30596343

RESUMO

In this study, a continuous feeding experiment was conducted with synthetic iso-plophyl alcohol (2-propanol)-containing wastewater using a lab-scale psychrophilic UASB reactor to evaluate process performance and retained sludge properties. For smooth acclimation, methanogenic granular sludge was seeded and a proportion of 2-propanol in the synthetic wastewater containing sucrose and volatile fatty acids was increased stepwise from 0% to 30%, 60% and then 90% of COD (chemical oxygen demand). As a result, after a 4-week period for acclimation to 2-propanol degradation, a COD removal rate of 95% was achieved at an organic loading rate (OLR) of 8.4 kg COD/m3/day. Additionally, the physical properties of the retained granular sludge were maintained even when the reactor was supplied with 2-propanol-rich wastewater for more than 200 days. From the batch assays using serum bottles, methanogenic degradation of 2-propanol was observed with acetone accumulation. By comparison, 2-propanol degradation was clearly inhibited in the presence of chloroform as a specific inhibitor of methanogen. A domain archaeal community structure analysis targeting 16S rRNA genes showed the relative abundance of the genus Methanospillium was increased in the 2-propanol acclimated sludge. These results suggested Methanospillium related species in the granular sludge appreciably contributed to the direct degradation of 2-proapanol into acetone under an anaerobic condition.


Assuntos
2-Propanol/análise , Archaea/isolamento & purificação , Reatores Biológicos/microbiologia , Esgotos/química , Archaea/metabolismo , Metano/metabolismo , Methanospirillum/isolamento & purificação , Methanospirillum/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Eliminação de Resíduos Líquidos/métodos , Qualidade da Água
7.
Environ Microbiol ; 19(7): 2727-2739, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28447396

RESUMO

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid-degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L-1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (∼1.5 times) in the presence of CNT (5 g·L-1 ), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re-frame discussions and re-interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.


Assuntos
Bactérias Anaeróbias/metabolismo , Metano/biossíntese , Methanobacterium/metabolismo , Methanospirillum/metabolismo , Nanotubos de Carbono/química , Butiratos/química , Técnicas de Cocultura , Transporte de Elétrons/fisiologia
8.
Nat Microbiol ; 2: 16222, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27922015

RESUMO

Archaea use flagella known as archaella-distinct both in protein composition and structure from bacterial flagella-to drive cell motility, but the structural basis of this function is unknown. Here, we report an atomic model of the archaella, based on the cryo electron microscopy (cryoEM) structure of the Methanospirillum hungatei archaellum at 3.4 Šresolution. Each archaellum contains ∼61,500 archaellin subunits organized into a curved helix with a diameter of 10 nm and average length of 10,000 nm. The tadpole-shaped archaellin monomer has two domains, a ß-barrel domain and a long, mildly kinked α-helix tail. Our structure reveals multiple post-translational modifications to the archaella, including six O-linked glycans and an unusual N-linked modification. The extensive interactions among neighbouring archaellins explain how the long but thin archaellum maintains the structural integrity required for motility-driving rotation. These extensive inter-subunit interactions and the absence of a central pore in the archaellum distinguish it from both the bacterial flagellum and type IV pili.


Assuntos
Microscopia Crioeletrônica , Flagelos/ultraestrutura , Methanospirillum/ultraestrutura
9.
Appl Microbiol Biotechnol ; 100(10): 4685-98, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26810199

RESUMO

The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae, Methanospirillaceae, and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae, and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus- and Nitrosotalea-like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of ß-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities.


Assuntos
Archaea/crescimento & desenvolvimento , Biomassa , Sedimentos Geológicos/microbiologia , Archaea/classificação , China , DNA Arqueal/isolamento & purificação , Euryarchaeota/classificação , Euryarchaeota/crescimento & desenvolvimento , Methanobacteriaceae/classificação , Methanobacteriaceae/crescimento & desenvolvimento , Methanobrevibacter/classificação , Methanobrevibacter/crescimento & desenvolvimento , Methanosarcinales/classificação , Methanosarcinales/crescimento & desenvolvimento , Methanospirillum/classificação , Methanospirillum/crescimento & desenvolvimento , Nitrogênio/análise , Fósforo/análise , Filogenia , RNA Ribossômico 16S/isolamento & purificação , Rios/microbiologia , Análise de Sequência de DNA , Microbiologia da Água
10.
Biotechnol Bioeng ; 113(3): 560-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26301789

RESUMO

The syntrophic cooperation between hydrogen-producing acetogens and hydrogenotrophic methanogens relies on a critical balance between both partners. A recent study, provided several indications for the dependence of the biomass-specific growth rate of a methanogenic coculture on the acetogen. Nevertheless, final experimental proof was lacking since biomass-specific rates were obtained from a descriptive model, and not from direct measurement of individual biomass concentrations. In this study, a recently developed quantitative PCR approach was used to measure the individual biomass concentrations in the coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1 on lactate, formate or both. The model-derived growth yields and biomass-specific rates were successfully validated. Experimental findings identified the acetogen as the growth-limiting partner in the coculture on lactate. While the acetogen was operating at its maximum biomass-specific lactate consumption rate, the hydrogenotrophic methanogen showed a significant overcapacity. Furthermore, this study provides experimental evidence for different growth strategies followed by the syntrophic partners in order to maintain a common biomass-specific growth rate. During syntrophic lactate conversion, the biomass-specific electron transfer rate of Methanospirillum hungatei JF1 was three-fold higher compared to Desulfovibrio sp. G11. This is to compensate for the lower methanogenic biomass yield per electron-mole of substrate, which is dictated by the thermodynamics of the underlying reaction.


Assuntos
Meios de Cultura/química , Desulfovibrio/crescimento & desenvolvimento , Desulfovibrio/metabolismo , Formiatos/metabolismo , Ácido Láctico/metabolismo , Methanospirillum/crescimento & desenvolvimento , Methanospirillum/metabolismo , Biomassa , Técnicas de Cocultura , Desulfovibrio/genética , Transporte de Elétrons , Methanospirillum/genética , Reação em Cadeia da Polimerase em Tempo Real
11.
Appl Environ Microbiol ; 81(24): 8434-44, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26431966

RESUMO

The anaerobic metabolism of crotonate, benzoate, and cyclohexane carboxylate by Syntrophus aciditrophicus grown syntrophically with Methanospirillum hungatei provides a model to study syntrophic cooperation. Recent studies revealed that S. aciditrophicus contains Re-citrate synthase but lacks the common Si-citrate synthase. To establish whether the Re-citrate synthase is involved in glutamate synthesis via the oxidative branch of the Krebs cycle, we have used [1-(13)C]acetate and [1-(14)C]acetate as well as [(13)C]bicarbonate as additional carbon sources during axenic growth of S. aciditrophicus on crotonate. Our analyses showed that labeled carbons were detected in at least 14 amino acids, indicating the global utilization of acetate and bicarbonate. The labeling patterns of alanine and aspartate verified that pyruvate and oxaloacetate were synthesized by consecutive carboxylations of acetyl coenzyme A (acetyl-CoA). The isotopomer profile and (13)C nuclear magnetic resonance (NMR) spectroscopy of the obtained [(13)C]glutamate, as well as decarboxylation of [(14)C]glutamate, revealed that this amino acid was synthesized by two pathways. Unexpectedly, only the minor route used Re-citrate synthase (30 to 40%), whereas the majority of glutamate was synthesized via the reductive carboxylation of succinate. This symmetrical intermediate could have been formed from two acetates via hydration of crotonyl-CoA to 4-hydroxybutyryl-CoA. 4-Hydroxybutyrate was detected in the medium of S. aciditrophicus when grown on crotonate, but an active hydratase could not be measured in cell extracts, and the annotated 4-hydroxybutyryl-CoA dehydratase (SYN_02445) lacks key amino acids needed to catalyze the hydration of crotonyl-CoA. Besides Clostridium kluyveri, this study reveals the second example of a microbial species to employ two pathways for glutamate synthesis.


Assuntos
Deltaproteobacteria/metabolismo , Ácido Glutâmico/biossíntese , Hidroliases/metabolismo , Redes e Vias Metabólicas/genética , Interações Microbianas/fisiologia , Acetatos/metabolismo , Acetilcoenzima A/química , Acil Coenzima A/metabolismo , Citrato (si)-Sintase/genética , Hidroxibutiratos/metabolismo , Espectroscopia de Ressonância Magnética , Methanospirillum/metabolismo , Oxirredução , Ácido Succínico/química
12.
Mar Pollut Bull ; 99(1-2): 104-11, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26231062

RESUMO

Little knowledge is available about the potential impact of commercial silver nanoparticles (Ag-NPs) on estuarine microbial communities. The Hugli river estuary, India, is susceptible to heavy metals pollution through boat traffic, and there is the potential for Ag-NP exposure via effluent discharged from ongoing municipal and industrial activities located in close proximity. This study investigated the effects of commercial Ag-NPs on native microbial communities in estuarine sediments collected from five stations, using terminal restriction fragment length polymorphism (T-RFLP) technique. An increase in the number of bacteria in consortium in sediments was observed following exposure to Ag-NPs. In general microbial communities may be resistant in estuarine systems to the antimicrobial effects of commercial Ag-NPs, but key microorganisms, such as Pelobacter propionicus, disappeared following exposure to Ag-NPs. In conclusion, the T-RFLP analysis indicated that Ag-NPs have the potential to shape estuarine sediment bacterial community structure.


Assuntos
Sedimentos Geológicos/microbiologia , Consórcios Microbianos/efeitos dos fármacos , Nanopartículas/toxicidade , Prata/toxicidade , Poluentes Químicos da Água/toxicidade , Bactérias/efeitos dos fármacos , Meio Ambiente , Estuários , Índia , Metais Pesados/toxicidade , Methanospirillum/efeitos dos fármacos , Consórcios Microbianos/genética , Polimorfismo de Fragmento de Restrição , Células Procarióticas/efeitos dos fármacos , Rios , Prata/química , Clima Tropical , Poluentes Químicos da Água/análise
13.
PLoS Comput Biol ; 11(7): e1004364, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26147299

RESUMO

Microorganisms in nature do not exist in isolation but rather interact with other species in their environment. Some microbes interact via syntrophic associations, in which the metabolic by-products of one species serve as nutrients for another. These associations sustain a variety of natural communities, including those involved in methanogenesis. In anaerobic syntrophic communities, energy is transferred from one species to another, either through direct contact and exchange of electrons, or through small molecule diffusion. Thermodynamics plays an important role in governing these interactions, as the oxidation reactions carried out by the first community member are only possible because degradation products are consumed by the second community member. This work presents the development and analysis of genome-scale network reconstructions of the bacterium Syntrophobacter fumaroxidans and the methanogenic archaeon Methanospirillum hungatei. The models were used to verify proposed mechanisms of ATP production within each species. We then identified additional constraints and the cellular objective function required to match experimental observations. The thermodynamic S. fumaroxidans model could not explain why S. fumaroxidans does not produce H2 in monoculture, indicating that current methods might not adequately estimate the thermodynamics, or that other cellular processes (e.g., regulation) play a role. We also developed a thermodynamic coculture model of the association between the organisms. The coculture model correctly predicted the exchange of both H2 and formate between the two species and suggested conditions under which H2 and formate produced by S. fumaroxidans would be fully consumed by M. hungatei.


Assuntos
Deltaproteobacteria/metabolismo , Hidrogênio/metabolismo , Metano/metabolismo , Methanospirillum/metabolismo , Modelos Biológicos , Simbiose/fisiologia , Simulação por Computador , Transferência de Energia/fisiologia , Consórcios Microbianos/fisiologia , Termodinâmica
14.
Appl Microbiol Biotechnol ; 99(5): 2313-27, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25467924

RESUMO

To better understand the quantitative relationships between messenger RNA (mRNA) and protein biomarkers relevant to bioremediation, we quantified and compared respiration-associated gene products in an anaerobic syntrophic community. Respiration biomarkers for Dehalococcoides, an organohalide reducer, and Methanospirillum, a hydrogenotrophic methanogen, were quantified via qRT-PCR for mRNA and multiple reaction monitoring (MRM) of proteotypic peptides for protein. mRNA transcripts of the Dehalococcoides reductive dehalogenases PceA, TceA, and DMC1545, and hydrogenase HupL, as well as the Methanospirillum oxidoreductases MvrD and FrcA were shown to be similarly regulated with respect to their temporal responses to substrate addition. However, MvrD was two orders of magnitude lower in mRNA abundance. Per cell, Dehalococcoides protein biomarkers quantified were more abundant than Methanospirillum proteins. Comparing mRNA with protein abundance, poor correlations were observed between mRNA transcript levels and the net protein produced. For example, Dehalococcoides HupL and TceA transcripts were similarly abundant though TceA was far more abundant at the protein level (167 ± 121 vs. 1095 ± 337 proteins per cell, respectively). In Methanospirillum, MvrD maintained comparable per-cell protein abundance to FrcA (42 ± 14 vs. 60 ± 1 proteins per cell, respectively) despite the significantly lower transcript levels. Though no variability in protein decay rates was observed, the mRNA translation rate quantified for TceA was greater than the other Dehalococcoides targets monitored. These data suggest that there is considerable variation in the relationship between mRNA abundance and protein production both across transcripts within an organism and across organisms. This highlights the importance of empirically based studies for interpreting biomarker levels in environmentally relevant organisms.


Assuntos
Proteínas de Bactérias/análise , Biomarcadores/análise , Biotransformação , Chloroflexi/metabolismo , Perfilação da Expressão Gênica , Methanospirillum/metabolismo , RNA Mensageiro/análise , Anaerobiose , Proteínas de Bactérias/genética , Chloroflexi/genética , Methanospirillum/genética , Consórcios Microbianos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
15.
PLoS One ; 9(12): e115902, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25536080

RESUMO

Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost "Methanobacillus omelianskii" coculture. During growth on ethanol, both Pelobacter species exhibited NAD+-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD+-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be EH2 = -358±12 mV and EHCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was -35 to -28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.


Assuntos
Acetaldeído/metabolismo , Deltaproteobacteria/metabolismo , Acetilcoenzima A/metabolismo , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Deltaproteobacteria/enzimologia , Deltaproteobacteria/crescimento & desenvolvimento , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Hidrogênio/metabolismo , Methanospirillum/enzimologia , Methanospirillum/crescimento & desenvolvimento , Methanospirillum/metabolismo , Oxirredução
16.
Int J Syst Evol Microbiol ; 64(Pt 1): 180-6, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24048867

RESUMO

A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3-8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4-0.5×7.5-25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5-37 °C (optimum at 20-30 °C), at pH 6.0-10 (optimum 7.0-7.5) and with 0-0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1(T) and 94 % sequence similarity to Methanospirillum lacunae Ki8-1(T). DNA-DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1(T) revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum, for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1(T) ( = DSM 26304(T) = VKM B-2808(T)).


Assuntos
Reatores Biológicos/microbiologia , Methanospirillum/classificação , Filogenia , Esgotos/microbiologia , Composição de Bases , Temperatura Baixa , DNA Arqueal/genética , Methanospirillum/genética , Methanospirillum/crescimento & desenvolvimento , Methanospirillum/isolamento & purificação , Dados de Sequência Molecular , Países Baixos , Hibridização de Ácido Nucleico , Oxirredutases/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Temperatura
17.
Int J Syst Evol Microbiol ; 64(Pt 2): 638-41, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24158951

RESUMO

A psychrotolerant methanogenic strain, X-18(T), was isolated from the soil of the Madoi wetland at Qinghai, Tibetan plateau, China. Cells were wavy rods (11-62 µm long) with blunt tapered ends and Gram-stain-negative. Strain X-18(T) grew strictly anaerobically and produced methane exclusively from H2/CO2. Growth occurred in the temperature range of 4-32 °C and optimally at 25 °C. Growth pH ranged from 6.5 to 8.0 and the optimum was 7.0. The G+C content of the genomic DNA of strain X-18(T) was 44.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences and the alpha subunit of methyl-coenzyme M reductase indicated that strain X-18(T) was affiliated to the genus Methanospirillum and was most closely related to Methanospirillum lacunae Ki8-1(T), with 96.3% 16S rRNA gene sequence similarity. However, strain X-18(T) could be distinguished from the existing species of the genus Methanospirillum by its lower growth temperature and obligate hydrogenotrophic methanogenesis. On the basis of phenotypic characteristics and phylogenetic analysis, strain X-18(T) represents a novel species of the genus Methanospirillum, for which the name Methanospirillum psychrodurum sp. nov. is proposed and strain X-18(T) is assigned as the type strain ( = CGMCC 1.5186(T) = JCM 19216(T)).


Assuntos
Methanospirillum/classificação , Filogenia , Microbiologia do Solo , Áreas Alagadas , Composição de Bases , China , DNA Arqueal/genética , Metano/biossíntese , Methanospirillum/genética , Methanospirillum/isolamento & purificação , Dados de Sequência Molecular , Oxirredutases/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
18.
World J Microbiol Biotechnol ; 30(2): 539-45, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23990068

RESUMO

In a study during the 1970s co-variation of nitrogenase activity and methane formation associated with Sphagnum riparium was observed. This was suggested as evidence for a possible mechanism of hydrogen transfer from cyanobacteria to methanogens. We show experimentally that such a pathway is feasible. In a series of laboratory experiments, using a hydrogenase deficient strain of the heterocystous cyanobacterium Nostoc punctiforme and the hydrogenotrophic methanogen Methanospirillum hungateii in co-cultures, increasing light intensities resulted in elevated nitrogenase activity and methane production. The increase in methane production can be directly deduced from the nitrogenase activity of the N. punctiforme based on hydrogen balance calculations. These experimental results clearly suggest the possible existence of a novel photosynthetically regulated pathway for methane formation.


Assuntos
Hidrogênio/metabolismo , Metano/metabolismo , Methanospirillum/metabolismo , Nostoc/metabolismo , Luz , Nitrogenase/metabolismo , Fotossíntese
19.
Appl Environ Microbiol ; 79(20): 6325-30, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23934484

RESUMO

Methylmercury (MeHg), a neurotoxic substance that accumulates in aquatic food chains and poses a risk to human health, is synthesized by anaerobic microorganisms in the environment. To date, mercury (Hg) methylation has been attributed to sulfate- and iron-reducing bacteria (SRB and IRB, respectively). Here we report that a methanogen, Methanospirillum hungatei JF-1, methylated Hg in a sulfide-free medium at comparable rates, but with higher yields, than those observed for some SRB and IRB. Phylogenetic analyses showed that the concatenated orthologs of the Hg methylation proteins HgcA and HgcB from M. hungatei are closely related to those from known SRB and IRB methylators and that they cluster together with proteins from eight other methanogens, suggesting that these methanogens may also methylate Hg. Because all nine methanogens with HgcA and HgcB orthologs belong to the class Methanomicrobia, constituting the late-evolving methanogenic lineage, methanogenic Hg methylation could not be considered an ancient metabolic trait. Our results identify methanogens as a new guild of Hg-methylating microbes with a potentially important role in mineral-poor (sulfate- and iron-limited) anoxic freshwater environments.


Assuntos
Mercúrio/metabolismo , Methanospirillum/metabolismo , Compostos de Metilmercúrio/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Análise por Conglomerados , Meios de Cultura/química , Methanospirillum/genética , Metilação , Filogenia , Análise de Sequência de DNA , Sulfetos/metabolismo
20.
J Bacteriol ; 195(21): 4900-14, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23974031

RESUMO

The mineralization of organic matter in anoxic environments relies on the cooperative activities of hydrogen producers and consumers obligately linked by interspecies metabolite exchange in syntrophic consortia that may include sulfate reducing species such as Desulfovibrio. To evaluate the metabolic flexibility of syntrophic Desulfovibrio to adapt to naturally fluctuating methanogenic environments, we studied Desulfovibrio alaskensis strain G20 grown in chemostats under respiratory and syntrophic conditions with alternative methanogenic partners, Methanococcus maripaludis and Methanospirillum hungatei, at different growth rates. Comparative whole-genome transcriptional analyses, complemented by G20 mutant strain growth experiments and physiological data, revealed a significant influence of both energy source availability (as controlled by dilution rate) and methanogen on the electron transfer systems, ratios of interspecies electron carriers, energy generating systems, and interspecies physical associations. A total of 68 genes were commonly differentially expressed under syntrophic versus respiratory lifestyle. Under low-energy (low-growth-rate) conditions, strain G20 further had the capacity to adapt to the metabolism of its methanogenic partners, as shown by its differing gene expression of enzymes involved in the direct metabolic interactions (e.g., periplasmic hydrogenases) and the ratio shift in electron carriers used for interspecies metabolite exchange (hydrogen/formate). A putative monomeric [Fe-Fe] hydrogenase and Hmc (high-molecular-weight-cytochrome c3) complex-linked reverse menaquinone (MQ) redox loop become increasingly important for the reoxidation of the lactate-/pyruvate oxidation-derived redox pair, DsrC(red) and Fd(red), relative to the Qmo-MQ-Qrc (quinone-interacting membrane-bound oxidoreductase; quinone-reducing complex) loop. Together, these data underscore the high enzymatic and metabolic adaptive flexibility that likely sustains Desulfovibrio in naturally fluctuating methanogenic environments.


Assuntos
Adaptação Fisiológica/fisiologia , Desulfovibrio/enzimologia , Desulfovibrio/genética , Transporte de Elétrons/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Ácido Láctico/metabolismo , Metano/metabolismo , Mathanococcus , Methanospirillum , Mutação , Oxirredução
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