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PubMed2026

High-Quality Genome Assembly, Metabolome, Pangenome, and Metabolic Models of Megasphaera hexanoica KCCM 43214T.

Megasphaera hexanoica KCCM 43214T, isolated from cow rumen, is capable of producing medium-chain carboxylic acids such as hexanoate and octanoate. In this study, we present a high-quality genome assembly, along with intracellular metabolomic profiling and pangenomic analysis. Illumina sequencing generated 2.3 Gbp from 15,293,634 reads with a GC content of 49.5%, while PacBio HiFi sequencing produced 331.5 Mbp across 45,266 reads, with an average read length of 7,323 bp and a HiFi read N50 of 8,214 bp. Hybrid assembly of short and long reads resulted in a single 2.88 Mbp contig, containing 2,835 protein-coding genes. Genome-scale metabolic models were constructed to evaluate its metabolic capabilities under specific growth conditions. Intracellular metabolomic analysis of cells grown in medium containing fructose and lactate revealed key metabolic activities associated with chain elongation. Pangenomic analysis across nine annotated genomes identified 6,721 orthologous genes using OrthoMCL, emphasizing the genetic and functional diversity within the Megasphaera genus. This dataset offers valuable insights into the metabolism and biotechnological potential of M. hexanoica KCCM 43214T.

Metabolome↗
PubMed1979

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii.

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.

Amino Acids↗
PubMed1979

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.

Computers↗
PubMed1979

Megasphaera elsdenii endocarditis.

A case of endocarditis caused by Megasphaera elsdenii is reported. This anaerobic grim-negative coccus has rarely been associated with human infections and has not previously been described as a cause of endocarditis.

Anaerobiosis↗
PubMed1978

Nutritional characteristics of Megasphaera elsdenii.

Megasphaera elsdenii B159 (formerly Peptostreptococcus elsdenii; Rogosa 1971) is able to grow in a chemically defined medium containing minerals, NH4+ as the sole nitrogen source, sulfate as the source of sulfur, acetate and glucose as the carbon and energy sources, and the vitamins biotin, pyridoxine, and calcium pantothenate. When lactate serves as the carbon source, with cysteine present as the reducing agent, acetate is not required. Valine and threonine appeared to be required although direct inoculation of unwashed cells into media lacking these amino acids permitted growth in their absence after an extended incubation period. Sulfur sources utilized included sulfate, sulfide, thiosulfate, cysteine, and glutathionine; methionine was utilized less readily. When glucose was autoclaved in the medium, the generation time of M. elsdenii was 170--200 min, but increased to more than 400 min when glucose was autoclaved separately and added to the cooled sterile medium. When fructose, which had been sterilized separately, was utilized as the carbon and energy source, the generation time was 200 min.

Acetates↗
PubMed1979

Characterization of endotoxin from the rumen bacterium Megasphaera elsdenii.

Phenol-water extraction of Megasphaera elsdenii, a predominant gram-negative coccus in rumens of cattle fed high-grain diets, yielded material that exhibited typical characteristics of endotoxin. The extract was lethal to mice and to chicken embryos, caused biphasic fever in rabbits, leukopenia in mice, and local and generalized Shwartzman reactions; and induced tolerance to the lethal effect of the endotoxin in mice. The material contained carbohydrate, protein, lipid, phosphorus, and 2-keto-3-deoxyoctonate, but no nucleic acid. The beta-hydroxymyristic acid was absent. Results imply that M elsdenii endotoxin has many biological and chemical characteristics common to enterobacterial endotoxin. However, the median lethal doses in mice and in chicken embryos, and minimal dose required to elicit a local Shwartzman reaction, indicate that M elsdenii endotoxin's potency is low, which may explain why the large gram-negative bacterial population in the rumen of cattle is generally innocuous.

Animals↗
PubMed1979

Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by alpha-hydroxybutynoate.

The acetylenic alpha-hydroxy acid 2-hydroxy-3-butynoate (alpha HB) is a substrate and an irreversible inactivator of the FAD-containing flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii. On the average, the enzyme undergoes five catalytic turnovers with alpha HB in air at pH 7.0 before being inactivated. Irreversible inactivation is due to the conversion of the flavin to a pink adduct with visible absorption peaks at 522, 382, and 330 nm and weak fluorescence with an emission maximum at 635 nm. The adduct is stable and can be released from the enzyme and purified. It retains a structure analogous to FAD since it binds to the FAD-specific apo-D-amino acid oxidase. It can be further converted to an FMN analogue with phosphodiesterase which binds to the FMN-specific apoflavodoxin. Experiments were conducted to test whether inactivation was initiated by an alpha HB allene carbanion or the dehydrogenation product of alpha HB. Kinetic studies proved inconclusive in that a rapid equilibrium between an oxidized enzyme--allene carbanion pair and reduced enzyme--keto acid pair would make these two species kinetically equivalent. The olefinic substrate 2-hydroxy-3-butenoate, however, produced no flavin adduct. Since the keto acid derived from the oxidation of this alpha-hydroxy acid is expected to be as reactive as 2-keto-3-butynoate, it is concluded that an allene carbanion produced by abstraction of the alpha-hydrogen of alpha HB is the reactive species which covalently adds to the flavin.

Alkynes↗
PubMed1979

Structure of the flavin adduct formed in the suicide reaction of alpha-hydroxybutynoate with D-lactate dehydrogenase.

The Zn-dependent flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii is irreversibly inactivated by the D form of the suicide substrate 2-hydroxy-3-butynoic acid. The process of inactivation involves formation of a new pink chromophore, which can be released in intact form from the protein and which was purified to homogeneity by affinity chromatography. Inactivation involves covalent addition of the suicide substrate to the flavin coenzyme. The optical spectra indicate an elongation of the flavin chromophore, and the chemical reactivity suggests a derivative of reduced flavin. The structure of this adduct was deduced from Fourier transform NMR, from the chemical properties, and from comparison with appropriate models, which were synthesized chemically. This structure involves the covalent linkage of the acetylenic inhibitor to positions N(5) and C(6) of the flavin coenzyme via carbon atoms 2 and 4 of the inhibitor to form an additional fused aromatic ring. The pink adduct can be reconverted to an isoalloxazine chromophore by reduction with borohydride and subsequent reoxidation with oxygen. This new isoalloxazine has the spectral properties of an isoflavin, and it is proposed to carry the moiety of the inactivator molecule as substituent at position C(6). The structure of the pink chromophore representing a cyclic adduct to the flavin positions N(5) and C(6) is compared to that of the adduct obtained from L-lactate oxidase from Mycobacterium smegmatis and the L form of the same inhibitor [C(4a)--N(5) cyclic adduct; Schonbrunn, A., Abeles, R. H., Walsh, C. T., Ghisla, S., Ogata, H., and Massey, V. (1976) Biochemistry 15, 1978]. This comparison allows deductions about the relative orientation of substrate, coenzyme, and active center functional groups in the two enzymes.

Alkynes↗
PubMed1978

A reappraisal of the reaction of butyryl-coenzyme A dehydrogenase with phenylmercuric acetate. Evidence that de-greening involves a reaction of the tightly bound thioester.

Phenylmercuric acetate reversibly de-greens butyryl-CoA dehydrogenase from Megasphaera elsdenii, abolishing the absorption band at 710nm. The view that this is a result of modification of a protein thiol group is re-examined in the light of the following new observations. (i) After treatment with phenylmercuric acetate, the enzyme's ability to be re-greened by addition of thiols was not decreased by gel filtration or precipitation with (NH(4))(2)SO(4). (ii) Phenylmercuric acetate caused the same extent of de-greening whether added in a few large amounts or many small ones. The overall time taken for de-greening was, however, greatly extended when many small additions were made. (iii) In Tris/acetate buffer, pH7.5, 3.5mol of phenylmercuric acetate/mol of enzyme subunit was required for complete de-greening, compared with only 2.5mol/mol in phosphate buffer, pH7. (iv) None of the groups that react with phenylmercuric acetate is accessible to iodoacetate or iodoacetamide. (v) On a molar basis dithiothreitol, mercaptoethanol and CoA are equally effective in re-greening the enzyme. (vi) Provided that phenylmercuric acetate is not present in excess, the de-greened enzyme forms normal and stable complexes with crotonyl-CoA and acetoacetyl-CoA. (vii) When a small excess of phenylmercuric acetate is present, full stable development of the enzyme-acetoacetyl-CoA complex requires addition of several mol of acetoacetyl-CoA/mol of enzyme subunit. (viii) The ability of de-greened enzyme to be immediately re-greened by an excess of thiol declines with time, more rapidly at pH6 than at pH7 or 8, but at all three pH values the instantaneous re-greening was followed by a slow phase of further increase in A(710). This further recovery was most extensive and most rapid at pH8. These findings are reminiscent of the previously described reversible decline in the re-greening capacity of a protein-free acid extract of green butyryl-CoA dehydrogenase. It is concluded that the likely cause of de-greening is chemical modification of the tightly bound thioester rather than a protein thiol group. The reversibility would be explained if the thioester exists on the surface of the enzyme in equilibrium with free CoA and a lactone, or if the acyl group is readily and reversibly transferred from the thiol of CoA to a protein side chain.

Acyl Coenzyme A↗
PubMed1978

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.

Anaerobiosis↗
PubMed1979

Transformation of mercuric chloride and methylmercury by the rumen microflora.

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.

Aerobiosis↗
PubMed1975

Rapid screening of Veillonella by ultraviolet fluorescence.

Among 51 strains of anaerobic gram-negative cocci belonging to the family Veillonellaceae, all strains of Veillonella (V. parvula and V. alcalescens) displayed red fluorescence under long-wave (366 nm) ultraviolet light, whereas no Acidaminococcus or Megasphaera demonstrated fluorescence. In contrast to Bacteroides melaninogenicus, growth of Veillonella does not require hemin and menadione, and flourescence is rapidly lost upon exposure to air. The fluorescent component of a strain of V. parvula examined could not be extracted in solution with water, ether, methanol, or chloroform, but was readily extracted with 0.4 N NaOH. Spectrophotofluorometrically, the fluorescence maximum of this extract was 660 nm with an excitation maximum of 300 nm, when measured at pH 7.2 and 25 C. Coupled with the Gram stain, ultraviolet fluorescence may be a useful tool for rapid screening of Veillonella and is particularly helpful for detection and, isolation of this organism from mixed culture.

Air↗
PubMed1977

Studies on some characteristics of hydrogen production by cell-free extracts of rumen anaerobic bacteria.

Hydrogen production was studied in the following rumen anaerobes: Bacteroides clostridiiformis, Butyrivibrio fibrisolvens, Enbacterium limosum, Fusobacterium necrophorum, Megasphaera elsdenii, Ruminococcus albus, and Ruminococcus flavefaciens. Clostridium pasteurianum and Escherichia coli were included for comparative purposes. Hydrogen production from dithionite, dithionite-reduced methyl viologen, pyruvate, and formate was determined. All species tested produced hydrogen from dithionite-reduce methyl viologen, but only C. pasteurianum, B. clostridiiformis, E. limosum, and M. elsdenii produced hydrogen from dithionite. All species except E. coli produced hydrogen from pyruvate, but activity was low or absent in extracts of E. limosum, F. necrophorum, R. albus, and R. flavefaciens unless methyl viologen was added. Hydrogen was produced from formate only by E. coli, B. clostridiiformis, E. limosum, F. necrophorum, and R. flavefaciens. Extracts were subjected to ultracentrifugation in an effort to determine the solubility of hydrogenase. The hydrogenase of all species except E. coli appeared to be soluble, although variable amounts of hydrogenase activity were detected in the pellet. Treatment of extracts of the rumen microbial species with DEAE-cellulose resulted in loss ofhydrogen production from pyruvate. Activity was restored by the addition of methyl viologen. It is concluded that hydrogen production in these rumen microorganisms is similar to that in the saccharolytic clostridia.

Anaerobiosis↗
PubMed1978

Some effects of arsenic on the rumen microflora; an in vitro study.

The rate of fermentation of the rumen microflora was inhibited almost 30% by 5 micrograms/ml of arsenic added in the form of arsenite, although 304 micrograms/ml was required to cause 50% inhibition. Arsenate was less inhibitory. The rate of fermentation of a separated bacterial fraction was inhibited 37% by 1 microgram of arsenite per millilitre, whereas 100 micrograms/ml had little effect on the fermentation of a separated protozoal fraction. Similar results were obtained for arsenate. Both fractions had the capacity to take up arsenate, but the protozoa took it up more readily to a higher intracellular concentration. Both arsenate and arsenite inhibited the growth of a number of rumen bacteria in pure culture at concentrations as low as 5 micrograms of arsenic per millilitre. The greater resistance of Megasphaera elsdenii to arsenate as compared with that of Bacteroides succinogenes was not related to the inability to take up the element. In conclusion, the concentrations of arsenic causing a significant inhibitory effect on the fermentative activity and growth of some rumen bacteria are less than that reported to be toxic to ruminant animals.

Anaerobiosis↗
PubMed1977

[Fecal flora of man. V. communication: The fluctuation of the fecal flora of the healthy adult (author's transl)].

Seven fecal specimens from each of healthy persons, aged 25 to 42 years, during the period of 2 months were analyzed to obtain the knowledge on the day-to-day variation in the same person and the person-to-person variation. Irrespective of individuals, bacteroidaceae, catenabacteria (eubacteria and strict anaerobic lactobacilli), peptostreptococci (anaerobic gram positive cocci) and bifidobacteria constituted the predominant flora. Spirillaceae, megasphaerae and clostridia (except Cl. perfringens) were also found as predominant flora in specific individuals. In all cases, enterobacteria and streptococci constitute the accompanying flora. In addition, veillonallae and lactobacilli were found as predominant flora in most cases. The remaining flora, consisting of Cl. perfringens, staphylococci and yeast, occurred occasionally in low numbers. With regard to the composition of the fecal flora of each person, a great individual variation was recognized and confirmed statistically. Each person had a characteristic fecal flora. In the same person, the numbers of bacteria of the predominating flora were over a 2 months period relatively constant. On the other hand, a great day-to-day variation in the numbers of bacteria of the accompanying flora as well as the remaining flora was demonstrated.

Adult↗
PubMed1979

Comparison of three methods of susceptibility testing of bacteroidaceae, peptococcaceae and other anaerobes to doxycycline.

The in vitro activity of doxycycline against 147 strains of gram-positive and gram-negative anaerobes was determined by broth dilution, agar dilution and agar diffusion tests. The strains were isolated from clinical specimens in 1977. Saccharolytic Bacteroides strains (39 B. fragilis, 6 B. thetaiotaomicron, 4 B. vulgatus) showed broth dilution MICs of less than or equal to 0.0625-4.0 microgram/ml after 5 h incubation and MICs of less than or equal to 16 microgram/ml after 15 h incubation at 37 degrees C. With strains of B. oralis, B. melaninogenicus ss. intermedius, B. corrodens, Veillonella sp. and Megasphaera elsdenii the broth dilution MICs were less than or equal to 0.0625-32 microgram/ml after 15 h incubation. Peptostreptococcus spp., Propionibacterium acnes, Eubacterium sp., Bifidobacterium sp. and Clostridium sp. had broth dilution MICs of less than or equal to 2 microgram/ml, whereas Peptococcus spp. were inhibited by less than or equal to 0.0625 to greater than 32 microgram/ml. With the great majority of strains tested, MICs were 2 to 256 times higher in agar than in broth dilution tests. Clinical and experimental studies seem to be needed to determine which of the in vitro data are correlated to the outcome of doxycycline therapy. Several groups and species were separately considered for statistical analysis of the relationship between zone size and MIC. With gram-positive anaerobes, correlation was poor between broth dilution MIC and zone size (correlation coefficients r = - 0.168 for Peptococcaceae, and r = - 0.108 for P. acnes). When calculating the regression lines for agar dilution MICs and zone diameters, a higher correlation was found (r = - 0.9 for Peptococcaceae; r = - 0.397 for P. acnes). With B. fragilis and other Bacteroides species correlation coefficients were r = - 0.807 to r = - 0.891 for broth and agar dilution MICs and zone size but stochastic linearity was lacking.

Anaerobiosis↗
PubMed1976

[The fecal flora of man. IV. Communication: Comparison of the newly developed method with the old conventional method for the analysis of intestinal flora (author's transl)].

The results of 42 fecal flora analysis of healthy adult men were compared by using a newly developed method and an old conventional method. Statistical estimate indicate, that with the new method the total counts and the counts of bifidobacteria, catenabacteria (eubacteria and anaerobic lactobacilli), peptostreptococci, and clostridia (except Cl. perfringens) as well as the occurences of peptostreptococci and spirillaceae were significant higher than with the use of the old method. On the other hand the counts and occurences of bifidobacteria, veillonellae, Cl. perfringens, Megasphaerae and aerobes were similar with both method. With the new method the total counts of feces of healthy adult men nubmered 1-2 X 10(11) per g wet weight, and fastidious anaerobes, such as bacteroides, catenabacteria and peptostreptococci, were always occurred as predominant flora. The results suggest that the conventional anaerobic jar method is inadequate for the isolation of fastidiuos anaerobes and indicate that the use of modified medium 10 in combination with strict anaerobic technique is necessary for the analysis of fecal flora.

Adult↗