Microbial cycling of volatile organic sulfur compounds.

Microbial cycling of volatile organic sulfur compounds (VOSCs), especially dimethyl sulfide (DMS) and methanethiol (MT), is intensively studied because these compounds play an important role in the processes of global warming, acid precipitation, and the global sulfur cycle. VOSC concentrations in freshwater sediments are low due to the balance between the formation and degradation of these compounds. These reactions occur for the greater part at the oxic/anoxic interphase of sediment and water column. In contrast to marine ecosystems, where dimethylsulfoniopropionate is the main precursor of MT and DMS, in freshwater ecosystems, VOSCs are formed mainly by methylation of sulfide and to a lesser extent from the degradation of S-containing amino acids. One of the major routes for DMS and MT formation through sulfide methylation is anaerobic O-demethylation of methoxylated aromatic compounds. Inhibition studies have revealed that the major part of the endogenously produced MT and DMS is degraded anaerobically by methanogens. The major bacterial groups involved in formation and consumption of VOSCs are described.

Purine degradation in the edible mushroom Agaricus bisporus.

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts from fruit bodies.

Obligate sulfide-dependent degradation of methoxylated aromatic compounds and formation of methanethiol and dimethyl sulfide by a freshwater sediment isolate, Parasporobacterium paucivorans gen. nov., sp. nov.

Methanethiol (MT) and dimethyl sulfide (DMS) have been shown to be the dominant volatile organic sulfur compounds in freshwater sediments. Previous research demonstrated that in these habitats MT and DMS are derived mainly from the methylation of sulfide. In order to identify the microorganisms that are responsible for this type of MT and DMS formation, several sulfide-rich freshwater sediments were amended with two potential methyl group-donating compounds, syringate and 3,4,5-trimethoxybenzoate (0.5 mM). The addition of these methoxylated aromatic compounds resulted in excess accumulation of MT and DMS in all sediment slurries even though methanogenic consumption of MT and DMS occurred. From one of the sediment slurries tested, a novel anaerobic bacterium was isolated with syringate as the sole carbon source. The strain, designated Parasporobacterium paucivorans, produced MT and DMS from the methoxy groups of syringate. The hydroxylated aromatic residue (gallate) was converted to acetate and butyrate. Like Sporobacterium olearium, another methoxylated aromatic compound-degrading bacterium, the isolate is a member of the XIVa cluster of the low-GC-content Clostridiales group. However, the new isolate differs from all other known methoxylated aromatic compound-degrading bacteria because it was able to degrade syringate in significant amounts only in the presence of sulfide.

Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments.

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.

HPLC detection of soluble carbohydrates involved in mannitol and trehalose metabolism in the edible mushroom Agaricus bisporus.

A convenient and sensitive method was developed to separate and detect various types of carbohydrates (polyols, mono- and disaccharides, and phosphorylated sugars) simultaneously using high-performance liquid chromatography (HPLC). The method consists of a chromatographic separation on a CarboPac PA1 anion-exchange analytical column followed by pulsed amperometric detection. In a single run (43 min) 13 carbohydrates were readily resolved. Calibration plots were linear over the ranges of 5-25 microM to 1. 0-1.5 mM. The reliable and fast analysis technique, avoiding derivatization steps and long run times, was used to determine the levels of carbohydrates involved in mannitol and trehalose metabolism in the edible mushroom Agaricus bisporus. Moreover, the method was used to study the trehalose phosphorylase reaction.

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus.

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.

Trehalose phosphorylase activity and carbohydrate levels during axenic fruiting in three Agaricus bisporus strains.

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.

Transport of amino acids and ammonium in mycelium of Agaricus bisporus.

Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed.

Isolation and characterization of Methanomethylovorans hollandica gen. nov., sp. nov., isolated from freshwater sediment, a methylotrophic methanogen able to grow on dimethyl sulfide and methanethiol.

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.

NAD+-dependent glutamate dehydrogenase of the edible mushroom Agaricus bisporus: biochemical and molecular characterization.

The NAD+-dependent glutamate dehydrogenase (NAD-GDH) of Agaricus bisporus, a key enzyme in nitrogen metabolism, was purified to homogeneity. The apparent molecular mass of the native enzyme is 474 kDa comprising four subunits of 116 kDa. The isoelectric point of the enzyme is about 7.0. Km values for ammonium, 2-oxoglutarate, NADH, glutamate and NAD+ were 6.5, 3.5, 0.06, 37.1 and 0.046 mM, respectively. The enzyme is specific for NAD(H). The gene encoding this enzyme (gdhB) was isolated from an A. bisporus H39 recombinant lambda phage library. The deduced amino acid sequence specifies a 1029-amino acid protein with a deduced molecular mass of 115,463 Da, which displays a significant degree of similarity with NAD-GDH of Saccharomyces cerevisiae and Neurospora crassa. The ORF is interrupted by fifteen introns. Northern analysis combined with enzyme activity measurements suggest that NAD-GDH from A. bisporus is regulated by the nitrogen source. NAD-GDH levels in mycelium grown on glutamate were higher than NAD-GDH levels in mycelium grown on ammonium as a nitrogen source. Combined with the kinetic parameters, these results suggest a catabolic role for NAD-GDH. However, upon addition of ammonium to the culture transcription of the gene is not repressed as strongly as that of the gene encoding NADP-GDH (gdhA). To date, tetrameric NAD-GDHs with large subunits, and their corresponding genes, have only been isolated from a few species. This enzyme represents the first NAD-GDH of basidiomycete origin to be purified and is the first such enzyme from basidiomycetes whose sequence has been determined.

Role of methanogens and other bacteria in degradation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.

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.

Regulation of glutamine synthetase from the white button mushroom Agaricus bisporus.

The regulation of glutamine synthetase (GS) from Agaricus bisporus was studied at the posttranscriptional level using a specific antibody fraction directed against purified GS. The cross-reactivity of the antiserum against various Agaricus species and other fungi was tested and low reactivity with the Ascomycetes was found. GS protein and activity levels were measured in cell-free extracts of mycelium grown on different N sources. In mycelium grown on glutamine or ammonium as N source, the biosynthetic GS activity is higher than the transferase activity. Moreover, the results show a correlation between GS biosynthetic activity, GS protein, and previously reported mRNA levels. Also, after addition of ammonium or glutamine to glutamate-utilizing cultures, transferase activity decreased more rapidly than biosynthetic activity and GS protein level. This suggests a conformational modification which only affects transferase activity.

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus.

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.

Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.

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.

Activation and reaction kinetics of the dimethylamine/coenzyme M methyltransfer in Methanosarcina barkeri strain Fusaro.

Dimethylamine/5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) from Methanosarcina barkeri Fusaro catalyzes (Vmax = 4700 nmol x min(-1) x mg(-1) protein; k(cat) = 7.8 s(-1)) the transfer of a methyl group from dimethylamine (apparent Km = 0.45 mM) to its corrinoid prosthetic group to yield monomethylamine (MMA) and the methylated enzyme. The product, MMA, is a competitive inhibitor of the reaction (apparent Ki = 5.5 mM). The methyl group bound to the corrinoid prosthetic group of DMA-MT is subsequently transferred to coenzyme M in a reaction mediated by methylcobalamin/coenzyme M methyltransferase isoenzyme II [MT2(II)], which binds with high affinity to DMA-MT (apparent Km = 0.22 microM). As isolated, DMA-MT is inactive, but it can enzymically be reactivated by methyltransferase activating protein (MAP), ATP, and hydrogenase. Apart from the established role in corrinoid activation, ATP was found to act as a powerful allosteric effector on the methyltransferase reaction. The results of kinetic studies, supported by the resolution of as-yet partially purified auxiliary protein fractions, demonstrate that DMA-MT, MT2(II), MAP, and hydrogenase are the only enzymic components involved in the dimethylamine/coenzyme M methyltransfer in M. barkeri Fusaro.

Medium-reductant directed expression of methyl coenzyme M reductase isoenzymes in Methanobacterium thermoautotrophicum (strain deltaH).

Methanobacterium thermoautotrophicum was grown in a chemostat under various controlled conditions in the presence of either sodium sulfide or sodium thiosulfate. After establishment of the steady state, cells were taken and examined for expression of the mRNA transcripts coding for the different forms of methyl coenzyme M reductase (MCR) and methylene tetrahydomethanopterin dehydrogenase (MDH). MCR isoenzyme II expression varied most markedly. Expression was found not only to depend on known parameters temperature, pH and gassing rate, but also on the medium composition, especially the reductant present.

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growthand Production of (Hemi)Cellulolytic Enzymes on a Range of CarbohydrateSubstrates

The anaerobic fungus Neocallimastix sp. strain L2,isolated from the feces of a llama, was tested for growth on a range ofsoluble and insoluble carbohydrate substrates. The fungus was able to fermentglucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch,inulin, filter paper cellulose, and Avicel. No growth was observed onarabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan,glycerol, citrate, soya, and wheat bran. The fermentation products aftergrowth were hydrogen, formate, acetate, ethanol, and lactate. Thefermentation pattern was dependent on the carbon source. In general, higherhydrogen production resulted in decreased formation of lactate and ethanol.Recovery of the fermented carbon in products at the end of growth ranged from50% to 80%. (Hemi)cellulolytic enzyme activities were affectedby the carbon source. Highest activities were found in filtrates fromcultures grown on cellulose. Growing the fungus on inulin and lactose yieldedthe lowest cellulolytic activities. Highest specific activities foravicelase, endoglucanase, beta-glucosidase, and xylanase were obtained withAvicel as the substrate for growth (0.29, 5.9, 0.57, and 13IU · mg-1 protein, respectively). Endoglucanase activitybanding patterns after SDS-PAGE were very similar for all substrates. Minordifferences indicated that enzyme activities may in part be the result ofsecretion of different sets of isoenzymes.

The role of the cellulolytic high molecular mass (HMM) complex of the anaerobic fungus Piromyces sp. strain E2 in the hydrolysis of microcrystalline cellulose.

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.

Formation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.

Concentrations of volatile organic sulfur compounds (VOSC) were measured in water and sediment columns of ditches in a minerotrophic peatland in The Netherlands. VOSC, with methanethiol (4 to 40 nM) as the major compound, appeared to be mainly of sediment origin. Both VOSC and hydrogen sulfide concentrations decreased dramatically towards the water surface. High methanethiol and high dimethyl sulfide concentrations in the sediment and just above the sediment surface coincided with high concentrations of hydrogen sulfide (correlation factors, r = 0.91 and r = 0.81, respectively). Production and degradation of VOSC were studied in 32 sediment slurries collected from various freshwater systems in The Netherlands. Maximal endogenous methanethiol production rates of the sediments tested (up to 1.44 (mu)mol per liter of sediment slurry (middot) day(sup-1)) were determined after inhibition of methanogenic and sulfate-reducing populations in order to stop VOSC degradation. These experiments showed that the production and degradation of VOSC in sediments are well balanced. Statistical analysis revealed multiple relationships of methanethiol production rates with the combination of methane production rates (indicative of total anaerobic mineralization) and hydrogen sulfide concentrations (r = 0.90) or with the combination of methane production rates and the sulfate/iron ratios in the sediment (r = 0.82). These findings and the observed stimulation of methanethiol formation in sediment slurry incubations in which the hydrogen sulfide concentrations were artificially increased provided strong evidence that the anaerobic methylation of hydrogen sulfide is the main mechanism for VOSC formation in most freshwater systems. Methoxylated aromatic compounds are likely a major source of methyl groups for this methylation of hydrogen sulfide, since they are important degradation products of the abundant biopolymer lignin. Increased sulfate concentrations in several freshwater ecosystems caused by the inflow of water from the river Rhine into these systems result in higher hydrogen sulfide concentrations. As a consequence, higher fluxes of VOSC towards the atmosphere are conceivable.