RESUMEN
Sequencing of two cDNAs from the anaerobic fungi Piromyces equi and Piromyces sp. strain E2 revealed that they both encode a glycoside hydrolase (GH) family 48 cellulase, containing two C-terminal fungal dockerin domains. N-terminal sequencing of the major component of the Piromyces multi-enzyme cellulase/hemicellulase complex, termed the cellulosome, showed that these 80 kDa proteins corresponded to the GH family 48 enzyme. These data show for the first time that GH family 48 cellulases are not confined to bacteria, and that bacterial and fungal cellulosomes share the same pivotal component.
Asunto(s)
Glicósido Hidrolasas/genética , Piromyces/genética , Dominio Catalítico , Glicósido Hidrolasas/metabolismo , Filogenia , Piromyces/metabolismo , Análisis de Secuencia de ADNRESUMEN
Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.
Asunto(s)
Dimetilsulfóxido/metabolismo , Agua Dulce/microbiología , Sedimentos Geológicos/microbiología , Methanosarcinaceae/aislamiento & purificación , Methanosarcinaceae/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Bases , Biodegradación Ambiental , Recuento de Colonia Microbiana , ADN de Archaea/análisis , Desoxirribonucleasa HindIII/metabolismo , Sedimentos Geológicos/química , Methanosarcinaceae/clasificación , Methanosarcinaceae/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Methanosarcina semesiae MD1T (T = type strain), a novel obligately methylotrophic methanogenic archaeon is described. Strain MD1T was isolated from an enrichment on dimethylsulfide inoculated with mangrove sediment. The cells were irregularly coccoid, non-motile, 1.4+/-0.2 microm in diameter and stained Gram-positive. The catabolic substrates used included dimethylsulfide, methanethiol, methanol and methylated amines, but not acetate, formate, H2/CO2 or a combination of these substrates. When cells grown on dimethylsulfide were transferred to trimethylamine or methanol and vice versa, a lag phase was observed. The same lag phase occurred when cells grown on trimethylamine were transferred to methanol and vice versa, indicating that for each substrate different enzymes were induced. Fastest growth occurred within a temperature range of 30-35 degrees C and a pH of 6.5-7.5. Both Na+ and Mg2+ were required for growth, with maximum growth rates at 200-600 mM Na+ and 20-100 mM Mg2+. The cells exhibited specific growth rates (h-1) of 0.07+/-0.02, 0.15+/-0.04 and 0.18-/+0.05 on dimethylsulfide, methanol and trimethylamine, respectively. Analysis of the 16S rRNA gene sequence showed that strain MD1T was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (94-97% sequence similarity).
Asunto(s)
Sedimentos Geológicos/microbiología , Metano/metabolismo , Methanosarcina/clasificación , Sulfuros/metabolismo , Árboles , Medios de Cultivo , ADN de Archaea/genética , ADN Ribosómico/genética , Genes de ARNr , Metanol/metabolismo , Methanosarcina/citología , Methanosarcina/aislamiento & purificación , Methanosarcina/fisiología , Metilaminas/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.
Asunto(s)
Cilióforos/fisiología , Euryarchaeota/fisiología , Filogenia , ARN Ribosómico 16S/genética , Simbiosis , Anaerobiosis , Animales , Cilióforos/genética , Cucarachas , ADN de Archaea/genética , ADN Ribosómico/genética , Euryarchaeota/genética , ARN de Archaea/genética , Rana ridibundaRESUMEN
During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent Km(H2) decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H2-dependent methenyl-tetrahydromethanopterin dehydrogenase (H2-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H2-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H2-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.
Asunto(s)
Adaptación Fisiológica , Enzimas/metabolismo , Metano/metabolismo , Methanobacterium/metabolismo , Biomasa , Fermentación , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Methanobacterium/enzimología , Methanobacterium/crecimiento & desarrollo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Temperatura , Tiosulfatos/químicaRESUMEN
Three strains of Agaricus bisporus (B430, 116, and 155.8), which share the ability to form hyphal aggregates on solid media under axenic conditions, were investigated with respect to carbohydrate levels and activities of enzymes involved in their carbon metabolism. The size and macroscopic appearance of the aggregates, when grown on diluted medium, suggest that substrate limitation plays a role in the process of fruiting body development in A. bisporus. The enzymes trehalose phosphorylase (TP), mannitol dehydrogenase (MD), and glucose-6-phosphate dehydrogenase (G6PD) seem to be developmentally regulated, in contrast to hexokinase (HK). Activities of TP (measured in the direction of trehalose degradation), MD, and G6PD were higher in the hyphal aggregates compared with the mycelium, whereas HK activity varied little. In the period preceding the axenic formation of hyphal aggregates, synthesis of trehalose by TP approximately doubled in the mycelium. The carbohydrate levels, which were measured by HPLC, varied in a way similar to their corresponding enzymes. The results indicate synthesis of trehalose in the mycelium of A. bisporus before the hyphal aggregates arise. Subsequently, translocation of the trehalose takes place from the mycelium to the emerging aggregates. In these small aggregates the trehalose is rapidly broken down to yield glucose and glucose-1-phosphate, serving as carbon and energy sources for further growth of the aggregates and for the synthesis of the osmolyte mannitol.
Asunto(s)
Agaricus/enzimología , Agaricus/crecimiento & desarrollo , Metabolismo de los Hidratos de Carbono , Glucosiltransferasas/metabolismo , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Glucosa/metabolismo , Manitol/metabolismo , Trehalosa/metabolismoRESUMEN
Aerobic and anaerobic ciliates swim towards the cathode when they are exposed to a constant DC field. Nyctotherus ovalis from the intestinal tract of cockroaches exhibits a different galvanotactic response: at low strength of the DC field the ciliates orient towards the anode whereas DC fields above 2-4 V/cm cause cathodic swimming. This reversal of the galvanotactic response is not due to backward swimming. Rather the ciliates turn around and orient to the cathode with their anterior pole. Exposure to various cations, chelators, and Ca(2+)-channel inhibitors suggests that Ca(2+)-channels similar to the "long lasting" Ca(2+)-channels of vertebrates are involved in the voltage-dependent anodic galvanotaxis. Evidence is presented that host-dependent epigenetic factors can influence the voltage-threshold for the switch from anodic to cathodic swimming.
Asunto(s)
Canales de Calcio/fisiología , Cilióforos/genética , Cilióforos/fisiología , Cucarachas/parasitología , Animales , Calcio/metabolismo , ADN Protozoario/genética , ADN Ribosómico/genética , Electrodos , Campos Electromagnéticos , Electrofisiología , Magnesio/metabolismo , Datos de Secuencia Molecular , Movimiento , Filogenia , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
A newly isolated methanogen, strain DMS1(T), is the first obligately anaerobic archaeon which was directly enriched and isolated from a freshwater sediment in defined minimal medium containing dimethyl sulfide (DMS) as the sole carbon and energy source. The use of a chemostat with a continuous DMS-containing gas stream as a method of enrichment, followed by cultivation in deep agar tubes, resulted in a pure culture. Since the only substrates utilized by strain DMS1(T) are methanol, methylamines, methanethiol (MT), and DMS, this organism is considered an obligately methylotrophic methanogen like most other DMS-degrading methanogens. Strain DMS1(T) differs from all other DMS-degrading methanogens, since it was isolated from a freshwater pond and requires NaCl concentrations (0 to 0.04 M) typical of the NaCl concentrations required by freshwater microorganisms for growth. DMS was degraded effectively only in a chemostat culture in the presence of low hydrogen sulfide and MT concentrations. Addition of MT or sulfide to the chemostat significantly decreased degradation of DMS. Transient accumulation of DMS in MT-amended cultures indicated that transfer of the first methyl group during DMS degradation is a reversible process. On the basis of its low level of homology with the most closely related methanogen, Methanococcoides burtonii (94.5%), its position on the phylogenetic tree, its morphology (which is different from that of members of the genera Methanolobus, Methanococcoides, and Methanohalophilus), and its salt tolerance and optimum (which are characteristic of freshwater bacteria), we propose that strain DMS1(T) is a representative of a novel genus. This isolate was named Methanomethylovorans hollandica. Analysis of DMS-amended sediment slurries with a fluorescence microscope revealed the presence of methanogens which were morphologically identical to M. hollandica, as described in this study. Considering its physiological properties, M. hollandica DMS1(T) is probably responsible for degradation of MT and DMS in freshwater sediments in situ. Due to the reversibility of the DMS conversion, methanogens like strain DMS1(T) can also be involved in the formation of DMS through methylation of MT. This phenomenon, which previously has been shown to occur in sediment slurries of freshwater origin, might affect the steady-state concentrations and, consequently, the total flux of DMS and MT in these systems.
Asunto(s)
Sedimentos Geológicos/microbiología , Methanosarcinaceae/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Ecosistema , Agua Dulce/microbiología , Methanosarcinaceae/genética , Methanosarcinaceae/crecimiento & desarrollo , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Filogenia , Compuestos de Sulfhidrilo/metabolismo , Sulfuros/metabolismoRESUMEN
The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.
Asunto(s)
Acetiltransferasas/metabolismo , Hidrógeno/metabolismo , Orgánulos/enzimología , Piromyces/enzimología , Ácido Pirúvico/metabolismo , Acetiltransferasas/química , Acetiltransferasas/genética , Secuencia de Aminoácidos , Anaerobiosis , Secuencia de Bases , Northern Blotting , Western Blotting , ADN Complementario , ADN de Hongos , Endopeptidasa K/metabolismo , Biblioteca de Genes , Genes Fúngicos , Mitocondrias/enzimología , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Piromyces/genética , Alineación de SecuenciaRESUMEN
The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 micromol of DMS was stoichiometrically converted into 112 micromol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.
Asunto(s)
Bacterias Anaerobias/metabolismo , Dimetilsulfóxido/metabolismo , Euryarchaeota/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Anaerobiosis , Bacterias Anaerobias/efectos de los fármacos , Biodegradación Ambiental/efectos de los fármacos , Euryarchaeota/efectos de los fármacos , Agua Dulce/microbiología , Sedimentos Geológicos/microbiología , Hidrógeno/farmacología , Cinética , Sulfatos/farmacologíaRESUMEN
Degradation of dimethyl sulfide and methanethiol in slurries prepared from sediments of minerotrophic peatland ditches were studied under various conditions. Maximal aerobic dimethyl sulfide-degrading capacities (4.95 nmol per ml of sediment slurry. h-1), measured in bottles shaken under an air atmosphere, were 10-fold higher than the maximal anaerobic degrading capacities determined from bottles shaken under N2 or H2 atmosphere (0.37 and 0. 32 nmol per ml of sediment slurry. h-1, respectively). Incubations under experimental conditions which mimic the in situ conditions (i. e., not shaken and with an air headspace), however, revealed that aerobic degradation of dimethyl sulfide and methanethiol in freshwater sediments is low due to oxygen limitation. Inhibition studies with bromoethanesulfonic acid and sodium tungstate demonstrated that the degradation of dimethyl sulfide and methanethiol in these incubations originated mainly from methanogenic activity. Prolonged incubation under a H2 atmosphere resulted in lower dimethyl sulfide degradation rates. Kinetic analysis of the data resulted in apparent Km values (6 to 8 microM) for aerobic dimethyl sulfide degradation which are comparable to those reported for Thiobacillus spp., Hyphomicrobium spp., and other methylotrophs. Apparent Km values determined for anaerobic degradation of dimethyl sulfide (3 to 8 microM) were of the same order of magnitude. The low apparent Km values obtained explain the low dimethyl sulfide and methanethiol concentrations in freshwater sediments that we reported previously. Our observations point to methanogenesis as the major mechanism of dimethyl sulfide and methanethiol consumption in freshwater sediments.
Asunto(s)
Bacterias/metabolismo , Agua Dulce/microbiología , Sedimentos Geológicos/microbiología , Compuestos de Sulfhidrilo/metabolismo , Sulfuros/metabolismo , Aerobiosis , Anaerobiosis , Biodegradación AmbientalRESUMEN
Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30 degrees C. The optimum pH ranges for trehalose degradation and synthesis were 6.0-7.5 and 6.0-7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P(i) (K(i)=2.0 mM). The enzyme was highly specific towards trehalose, P(i), glucose and alpha-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P(i), glucose and alpha-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K(m) values were 61, 4.7, 24 and 6.3 mM for trehalose, P(i), glucose and alpha-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.
Asunto(s)
Agaricus/enzimología , Glucosiltransferasas/aislamiento & purificación , Adenosina Trifosfato/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Glucosa/metabolismo , Glucofosfatos/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Metales/farmacología , Peso Molecular , Conformación Proteica , Especificidad por Sustrato , Temperatura , Trehalosa/análogos & derivados , Trehalosa/metabolismo , Trehalosa/farmacologíaRESUMEN
The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.
Asunto(s)
Cilióforos/genética , Sistema Digestivo/parasitología , Evolución Molecular , Filogenia , Animales , Secuencia de Bases , Cilióforos/ultraestructura , Cucarachas , ADN Ribosómico/genética , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.
Asunto(s)
Cobamidas/aislamiento & purificación , Cobamidas/metabolismo , Methanosarcina barkeri/enzimología , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Cinética , Methanosarcina barkeri/crecimiento & desarrollo , Metilaminas/metabolismo , Modelos Químicos , Peso MolecularRESUMEN
By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F390, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Methanobacterium/aislamiento & purificación , ARN Mensajero/metabolismo , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/análisis , Recuento de Colonia Microbiana , Flavinas , Hidrógeno/fisiología , Methanobacterium/enzimología , Methanobacterium/crecimiento & desarrollo , Mutagénesis , Riboflavina/análogos & derivados , Riboflavina/análisisRESUMEN
The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences.
Asunto(s)
Aconitato Hidratasa/clasificación , Oxidorreductasas de Alcohol/clasificación , Malato Deshidrogenasa/clasificación , Piromyces/enzimología , Regiones no Traducidas 5' , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Anaerobiosis , Secuencia de Bases , Citosol/enzimología , ADN Complementario , ADN de Hongos , Dosificación de Gen , Expresión Génica , Cetoácido Reductoisomerasa , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Piromyces/genética , ARN de Hongos , ARN MensajeroRESUMEN
Methanobacterium thermoautotrophicum deltaH was grown in a fed-batch fermentor and in a chemostat under a variety of 80% hydrogen-20% CO2 gassing regimes. During growth or after the establishment of steady-state conditions, the cells were analyzed for the content of adenylylated coenzyme F420 (factor F390-A) and other methanogenic cofactors. In addition, cells collected from the chemostat were measured for methyl coenzyme M reductase isoenzyme (MCR I and MCR II) content as well as for specific activities of coenzyme F420-dependent and H2-dependent methylenetetrahydromethanopterin dehydrogenase (F420-MDH and H2-MDH, respectively), total (viologen-reducing) and coenzyme F420-reducing hydrogenase (FRH), factor F390 synthetase, and factor F390 hydrolase. The experiments were performed to investigate how the intracellular F390 concentrations changed with the growth conditions used and how the variations were related to changes in levels of enzymes that are known to be differentially expressed. The levels of factor F390 varied in a way that is consistently understood from the biochemical mechanisms underlying its synthesis and degradation. Moreover, a remarkable correlation was observed between expression levels of MCR I and II, F420-MDH, and H2-MDH and the cellular contents of the factor. These results suggest that factor F390 is a reporter compound for hydrogen limitation and may act as a response regulator of methanogenic metabolism.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Metano/metabolismo , Methanobacterium/enzimología , Methanobacterium/crecimiento & desarrollo , Riboflavina/análogos & derivados , Adenosina Monofosfato/análisis , Técnicas Bacteriológicas , Dióxido de Carbono/metabolismo , Fermentación , Flavinas , Isoenzimas/análisis , Nucleotidiltransferasas/análisis , Oxidación-Reducción , Oxidorreductasas/análisis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/análisis , Hidrolasas Diéster Fosfóricas/análisis , Riboflavina/análisisRESUMEN
The small subunit ribosomal DNA sequence (SSU rDNA) of the microaerophilic free-living amoeboflagellate Psalteriomonas lanterna has been sequenced and analyzed. The gene is 1,945 bp long and has a G + C content of 33.4%. Based upon ultrastructural studies, P. lanterna has been placed in the class Lyromonadea within the phylum Percolozoa Cavalier-Smith, 1991. However, based upon cytological characteristics, this microaerophilic free-living amoeboflagellate appears to be very primitive. It shares certain characteristics in common with some archezoans, i.e. it lacks mitochondria and dictyosomes but contains hydrogenosomes. Despite sharing these characteristics with the amitochondriate taxa, P. lanterna is not related to any of these taxa but instead to the Vahlkampfiidae. Therefore, we used primary sequence data and the secondary structure of the SSU rDNA gene to determine the placement P. lanterna in the phylogenetic tree. Our analyses showed that P. lanterna groups as a sister taxon to the Vahlkampfiidae but probably diverged from them quite early.
Asunto(s)
Eucariontes/genética , Filogenia , ARN Protozoario/genética , ARN Ribosómico/genética , Animales , Composición de Base , Clonación Molecular , ADN Protozoario/análisis , ADN Protozoario/química , ADN Ribosómico/análisis , ADN Ribosómico/química , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Several aspects of the endosymbiosis of methanogenic archaea with anaerobic protozoa are reviewed. Special attention is played to the role of hydrogenosomes and plastid-like organelles that seem to provide the substrates for the methanogenic endosymbionts. Evidence is presented that hydrogenosomes evolved several times in the various protoctistan taxa. Hydrogenosomes are seemingly different, and their common denominator is the production of hydrogen. The absence of nucleic acids and a protein-synthesizing machinery hampers the analysis of their divergent evolutionary history, and molecular genetic data argue not only for different but even a chimeric origin of the hydrogenosomes.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Eucariontes/microbiología , Euryarchaeota/fisiología , Hidrógeno/metabolismo , Orgánulos/metabolismo , Simbiosis , Anaerobiosis , Animales , Eucariontes/metabolismoRESUMEN
The anaerobic fungus Piromyces sp. strain E2 produces extracellular cellulolytic enzymes present both in a high molecular mass (HMM) complex or as individual proteins. Although the HMM complex was present in the culture fluid during all growth stages, the highest amounts of complex were obtained when cultures were harvested at the end of fungal growth. The complex obtained after gel-filtration chromatography on Sephacryl S-300 HR was found to be the major factor in hydrolysis of cellulose to glucose (sole product, up to 250 mM). The complex was very stable as demonstrated by identical hydrolysis patterns with fresh preparations or preparations stored at 4 degrees C for 2 months. From inhibition experiments with gluconic acid lactone and glucose, it was concluded that the HMM complex must contain at least one glucohydrolase. SDS-PAGE analysis revealed that a partially purified HMM complex was composed of at least ten polypeptides and contained numerous endoglucanases and one beta-glucosidase.