Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum . increased the extent of microcrystalline cellulose degradation by 10·12% and 7·96%, respectively. Biomass yield in co-cultures was increased by 89·9% and 59·4% for M. elsdenii and E. limosum . Y_cellulose for fungus alone was 52·29 g dry matter mol^-1 glucose. These values were 64·93 and 55·92 g mol^-1 glucose unit in co-culture with M. elsdenii and E. limosum , respectively.     

Catabolism of Amino Acids by Megasphaera elsdenii LC1

The amino acids in an acid hydrolysate of casein were catabolized more extensively by Megasphaera elsdenii than those in an enzymic hydrolysate. Threonine and serine were most actively degraded, but no resultant increase in growth yield occurred. Branched-chain volatile fatty acid production, which increased as the dilution rate of a glucose-limited chemostat decreased, seemed to be associated with maintenance rather than with growth.     

Disappearance of plasmalogen-containing phospholipids in Megasphaera elsdenii.

The plasmalogen content of phospholipids isolated from Megasphaera elsdenii ATCC 17752 decreased markedly in cultures passed serially at intervals of 3 to 6 weeks. From the wild-type ratio of vinyl ether to lipid phosphorus of 0.8, clones were isolated with ratios less than 0.05. Clonal analysis, as well as the reproducibility of the phenomenon and the long time course, suggest that the loss of plasmalogens is an adaptive process. Although small variations in cell morphology and ratios of end products of fermentation were detected, plasmalogen-rich and -deficient cells were virtually indistinguishable with respect to growth rates, range of fermentable carbohydrates, activities of selected enzymes, and electrophoretic patterns in both membrane and soluble proteins. Large decreases in saturated fatty acid production accompanied the decline of plasmalogens.     

Genome sequence of the ruminal bacterium Megasphaera elsdenii

Megasphaera elsdenii is a Gram-negative ruminal bacterium. It is being investigated as a probiotic supplement for ruminants as it may provide benefits for energy balance and animal productivity. Furthermore, it is of biotechnological interest due to its capability of producing various volatile fatty acids. Here we report the complete genome sequence of M. elsdenii DSM 20460, the type strain for the species.     

Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii catalyzes electron transfer from NADH or D-lactate dehydrogenase to butyryl-CoA dehydrogenase. As a basis for understanding the interactions of ETF with its substrates, we report here on the redox properties of ETF alone. ETF exhibited reversible, two-electron transfer during electrochemical reduction in the presence of mediator dyes. The midpoint redox potentials of the FAD cofactor were -0.185 V at pH 5.5, -0.259 V at pH 7.1 and -0.269 +/- 0.013 V at pH 8.4, all versus the standard hydrogen electrode In the presence of the indicator dye 1-deazariboflavin, the Nernst slopes were 0.029 V and 0.026 V at pH 5.5 and pH 7.1, respectively, compared with an expected value of 0.028 V at 10 degrees C. At pH 8.4, in the presence of 2-hydroxy-1,4-naphthoquinone or phenosafranine, the Nernst slope varied from 0.021 V to 0.041 V. In the experiments at pH 8.4, equilibration was very slow in the reductive direction and a difference of as much as 30 mV was observed between reductive and oxidative midpoints. ETF exhibited no thermodynamic stabilization of the radical form of the FAD cofactor during electrochemical reduction at pH 5.5, 7.1 or 8.4. However, up to 93% of kinetically stable, anionic radical was produced by dithionite titration at pH 8.5. Molar absorptivities of ETF radical were 17,000 M-1 X cm-1 at 365 nm and 5100 M-1 X cm-1 at 450 nm. The four ETF preparations used here contained less than 7% 6-OH-FAD. However, two of the preparations contained significant amounts (up to 30%) of flavin which stabilized radical and reduced at potentials 0.2 V more positive than those required for reduction of the major form of ETF. This is referred to as the B form of ETF. The proportion of ETF-FAD in the B form was increased by incubation with free FAD or by a cycle of reduction and reoxidation. These treatments caused marked changes in the absorption spectrum of oxidized ETF and decreases of 20-25% in ETF units/A450.     

Purification and properties of hydrogenase from Megasphaera elsdenii

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.     

Characterization of a reserve glucan from Megasphaera elsdenii.

A reserve glucan of Megasphaera elsdenii was studied by methylation analysis before and after treatment with isoamylase. The results of this study indicate that the glucan is of the amylopectin-glycogen type.     

The hydrogen-tritium exchange activity of Megasphaera elsdenii hydrogenase.

The hydrogenase of Megasphaera elsdenii was purified to a specific activity of 350 units/mg. The hydrogen-tritium exchange assay of Hallahan et al. [Hallahan, D.L., Fernandez, V. M., Hatchikian, E. C. and Hall, D. O. (1986) Biochimie (Paris) 68, 49-54] was adapted to allow its use in the study of the M. elsdenii hydrogenase preparation. Under the assay conditions routinely employed, the enzyme's exchange activity was inhibited by Tris/HCl and MgCl2; it was stimulated by ethylene glycol. Maximal activity in this standard assay occurred at pH 7.1. The effect of the concentration of molecular hydrogen (1H2 plus 3H1H) on the exchange activity was studied. The resulting double-reciprocal plot was linear; its slope and its intercepts on the ordinate and abscissa were pH-dependent. The rate equations for a number of models of the exchange activity were derived. Each model gave rise to a linear double-reciprocal plot at constant pH, but none could explain fully the observed effects of varying pH. The experimental data corresponded most closely to the predictions of models in which protons were treated both as substrates and as regulators of the enzyme's activity.     

Lactate and Acrylate Metabolism by Megasphaera elsdenii under Batch and Steady-State Conditions

The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied under batch and steady-state conditions. Under batch conditions, lactate was converted to acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl propenoate and 3-butenoate containing a terminal double bond were similarly converted into their respective saturated acids (isobutyrate and butyrate), while crotonate and lactate analogues 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. As the acrylate concentration in the feed was increased, butyrate and hydrogen formation decreased and propionate was increasingly generated, while the calculated ATP yield was unchanged. M. elsdenii metabolism differs substantially under batch and steady-state conditions. The results support the conclusion that propionate is not formed during lactate-limited steady-state growth because of the absence of this substrate to drive the formation of lactyl coenzyme A (CoA) via propionyl-CoA transferase. Acrylate and acrylate analogues are reduced under both batch and steady-state growth conditions after first being converted to thioesters via propionyl-CoA transferase. Our findings demonstrate the central role that CoA transferase activity plays in the utilization of acids by M. elsdenii and allows us to propose a modified acrylate pathway for M. elsdenii.     

Metabolism and some characteristics of ruminal strains of Megasphaera elsdenii

Megasphaera elsdenii belongs to the group comprising the ruminal and intestinal lactate- and sugar-fermenting species. In the present study the fermentation characteristics, metabolism of glucose and lactate, and susceptibility to antimicrobial agents of four ruminal strains were investigated. Particular attention was given to the mixed-substrate fermentation pattern and resultant fermentation acid profile. Lactate was utilized more rapidly than glucose in media with both carbon sources. Interaction of the two substrates changed the composition of fermentation end products toward more valerate and less propionate in cultures with glucose and lactate. Contrary to the indications in Bergey's Manual of Systematic Bacteriology, butyrate, not caproate, was the main end product of glucose metabolism. The strains examined were rather insensitive to many antimicrobial compounds, especially to ionophores and other antimicrobial feed additives.     

Factors Affecting Glucose and Maltose Phosphorylation by the Ruminal Bacterium Megasphaera elsdenii

The objectives of this study were to examine the effects of growth substrate and extracellular pH on phosphoenolpyruvate-dependent glucose phosphorylation as well as to examine how maltose is phosphorylated by the ruminal bacterium Megasphaera elsdenii B159. Phosphoenolpyruvate-dependent glucose phosphorylation by toluene-treated cells was constitutive, and glucose phosphorylation was reduced by 69% at pH 5.0. When toluene-treated cells were incubated in histidine buffer, little maltose phosphorylation occurred in the absence of inorganic phosphate. However, the addition of increasing concentrations of either potassium or sodium phosphate increased maltose phosphorylation. Maximal phosphorylation activity was observed at between 25 and 50 mM of either inorganic phosphate source. Compared with the control incubations, maltose phosphorylation was increased over threefold with 25 mM of either potassium or sodium phosphate. Phosphoglucomutase activity was detected in cell extracts of M. elsdenii B159, and this enzyme had a K _m of 3.2 mM for glucose-1-P and a V _max of 1836 nmol of NADP⁺ reduced/mg of protein per min. Maltose was also hydrolyzed by an inducible maltase (K _m , 1.19 mM). To our knowledge, this is the first report of a maltose phosphorylase and a maltase in M. elsdenii. Received: 3 November 1999 / Accepted: 5 January 2000     

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.     

Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction.

The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.     

Metabolism and some characteristics of ruminal strains of Megasphaera elsdenii.

Megasphaera elsdenii belongs to the group comprising the ruminal and intestinal lactate- and sugar-fermenting species. In the present study the fermentation characteristics, metabolism of glucose and lactate, and susceptibility to antimicrobial agents of four ruminal strains were investigated. Particular attention was given to the mixed-substrate fermentation pattern and resultant fermentation acid profile. Lactate was utilized more rapidly than glucose in media with both carbon sources. Interaction of the two substrates changed the composition of fermentation end products toward more valerate and less propionate in cultures with glucose and lactate. Contrary to the indications in Bergey's Manual of Systematic Bacteriology, butyrate, not caproate, was the main end product of glucose metabolism. The strains examined were rather insensitive to many antimicrobial compounds, especially to ionophores and other antimicrobial feed additives.     

Three-dimensional correlated NMR study of Megasphaera elsdenii flavodoxin in the oxidized state

The value of a three-dimensional (3D) non-selective total correlation/nuclear Overhauser enhancement spectroscopy (TOCSY-NOESY) spectrum for making sequential resonance assignments in proteins is demonstrated using the relatively large Megasphaera elsdenii flavodoxin (molecular mass 15 kDa) in the oxidized state. An easy and concise method for the analysis of 3D-NMR spectra and a strategy for the resonance assignment of 3D-NMR protein spectra is introduced. In this context, non-selective TOCSY-NOESY is compared with selective TOCSY-NOESY and non-selective NOESY-TOCSY. Sequential assignments in various secondary structure elements of flavodoxin are made using the method of analysis introduced. NOEs not previously identified in 2D-NMR spectra due to resonance overlap are found in the 3D Clean-TOCSY-NOESY spectrum. Also additional side-chain assignments could be made.     

Isolation of Acidaminococcus fermentans and Megasphaera elsdenii from Normal Human Feces

Fecal bacterial cultures from 40 normal humans yielded Megasphaera elsdenii from four individuals and Acidaminococcus fermentans from 10 individuals, with two individuals having both organisms.     

Rumen Bacterial Competition in Continuous Culture: Streptococcus bovis Versus Megasphaera elsdenii

Streptococcus bovis and Megasphaera elsdenii were grown in continuous culture with maltose as the limiting substrate at dilution rates of 0.36, 0.22, and 0.12 h. After each steady-state turnover, the pH was decreased by adding concentrated HCl to the medium reservoir. Relative counts of the two species at each dilution rate indicated that when the pH was high (6.6 to 6.0), higher dilution rates selected for a higher ratio of S. bovis to M. elsdenii. At intermediate pH (6.0 to 5.4), higher dilution rates once again selected for greater numbers of S. bovis in relation to M. elsdenii, but the increase in S. bovis numbers was much less at the 0.36-h dilution rate. Decreasing the pH below 5.4 caused the ratio of S. bovis to M. elsdenii to increase greatly, and no M. elsdenii cells were seen below pH 5.1. The ratio of the two species could be explained by their relative affinities for maltose if pH was greater than 6.0, but the lower relative numbers of S. bovis in the 0.36-h, intermediate-pH (6.0 to 5.4) incubations could not. Analysis of lactate production by S. bovis in pure culture showed that l-lactate was produced only if the pH was less than 5.2 at dilution rates of 0.22 and 0.12 h and less than 6.0 at a rate of 0.36 h. The lower numbers of S. bovis relative to M. elsdenii in the incubations with a dilution rate of 0.36 h and intermediate pH thus could be explained by utilization of l-lactate by M. elsdenii. The very high numbers of S. bovis at pH less than 5.4 were consistent with the greater tolerance of this organism to low pH.