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PubMed1992

Phylogenetic and chemotaxonomic characterization of Acidaminococcus fermentans.

The phylogenetic position of Acidaminococcus fermentans was determined by comparative sequence analysis of the 16S rRNA. This Gram-negative bacterium is a member of the Sporomusa cluster that is defined by other Gram-negative bacteria, i.e. Sporomusa, Megasphaera, Selenomonas, Butyrivibrio, Pectinatus, and Zymophilus. The branching point of this group within the radiation of Gram-positive bacteria of the Clostridium/Bacillus subphylum and adjacent to Peptococcus niger could be confirmed. Chemotaxonomic data were provided for a more detailed characterization of A. fermentans.

Amino Acids, Diamino↗

PubMed2005

Characterization of vaginal microflora of healthy, nonpregnant women by chaperonin-60 sequence-based methods.

OBJECTIVE: The purpose of this study was to use a novel method that was based on the application of chaperonin-60 sequencing to describe the vaginal microflora of 16 healthy women. STUDY DESIGN: Asymptomatic women consented for vaginal swabs to be collected at the time of a clinical pelvic examination. Total genomic DNA was isolated from the vaginal swabs. Degenerate, universal polymerase chain reaction primers were used to amplify an approximately 555 base pair region of the universal chaperonin-60 gene, which is found in all eubacteria and eukaryotes, from the total genomic DNA and libraries of cloned polymerase chain reaction products were constructed. Library clones were sequenced, and the resulting sequences were assigned to taxonomic groups on the basis of similarity to reference sequence data. Presence of Chlamydophila psittaci sequences in the samples was confirmed by species-specific polymerase chain reaction. RESULTS: Sixteen of the 23 women who were enrolled had normal flora by Nugent's score of <4 and had adequate polymerase chain reaction product for assessment. Vaginal flora libraries were dominated by a variety of sequences with similarity to Lactobacillus spp L. crispatus, L. iners, L. gasseri, L. jensenii, and L. buchneri. Other sequences that were identified included representatives of Gardnerella spp, sequences with similarity to Porphyromonas spp and Megasphaera spp and sequences identical to C psittaci. CONCLUSION: Culture-independent, chaperonin-60 sequence-based molecular methods can lead to the identification of greater diversity within defined taxa compared with those that are identified by standard culture-based methods and to the identification of novel organisms that were not previously associated with vaginal flora.

Adult↗

PubMed2007

Bacterial flora of the female genital tract: function and immune regulation.

The use of non-culture gene amplification techniques has improved our understanding of the composition of the vaginal bacterial ecosystem. In most healthy women in the reproductive period the predominant vaginal bacteria are one or more of the following species of Lactobacillus: L. crispatus, L. iners and L. gasseri. However, in other apparently healthy women lactobacilli may be deficient or absent, being replaced by other lactic-acid-producing bacteria: Atopobium, Megasphaera and/or Leptotrichia species. Infection and/or proliferation of pathogenic bacteria in the vagina is suppressed by lactic acid production, bacteria-generated antimicrobial products, and the local activities of the innate and cell-mediated immune systems. Vaginal epithelial cells produce a range of compounds with antimicrobial activities. These cells also possess membrane-bound Toll-like receptors that recognize pathogen-associated molecular patterns. Recognition leads to pro-inflammatory cytokine production and antigen-specific immunity. Local production of IgG and IgA antibodies can also be initiated in the endocervix and vagina in response to infection.

Female↗

PubMed2006

Composition of the bacterial flora in tonsilloliths.

Tonsilloliths are a potential cause of oral malodor. In this study, microbial profiles and composition of tonsilloliths were determined using culture-independent molecular methods and scanning electron microscopy. 16S ribosomal RNA bacterial genes (16S rDNAs) isolated from tonsilloliths of 6 individuals were amplified by PCR and cloned into Escherichia coli. Partial 16S rDNA sequences of approximately 600 bases of cloned inserts were used to determine species identity by comparison with sequences of known species. Characteristics of bacteria on the surface and inside the tonsillolith were analyzed using scanning electron microscopy. Anaerobic bacteria detected in tonsilloliths belonged to the genera Eubacterium, Fusobacterium, Megasphaera, Porphyromonas, Prevotella, Selenomonas and Tannerella, all of which appear to be associated with production of volatile sulfur compounds. Electron microscopy revealed cocci and rods on the surface and rods predominating inside the tonsilloliths. These results support the tonsillolith as an origin of oral malodor.

Adult↗

PubMed2003

Beer spoilage bacteria and hop resistance.

For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as Pectinatus cerevisiiphilus, Pectinatus frisingensis and Megasphaera cerevisiae. They can spoil beer by turbidity, acidity and the production of unfavorable smell such as diacetyl or hydrogen sulfide. For the microbiological control, many advanced biotechnological techniques such as immunoassay and polymerase chain reaction (PCR) have been applied in place of the conventional and time-consuming method of incubation on culture media. Subsequently, a method is needed to determine whether the detected bacterium is capable of growing in beer or not. In lactic acid bacteria, hop resistance is crucial for their ability to grow in beer. Hop compounds, mainly iso-alpha-acids in beer, have antibacterial activity against Gram-positive bacteria. They act as ionophores which dissipate the pH gradient across the cytoplasmic membrane and reduce the proton motive force (pmf). Consequently, the pmf-dependent nutrient uptake is hampered, resulting in cell death. The hop-resistance mechanisms in lactic acid bacteria have been investigated. HorA was found to excrete hop compounds in an ATP-dependent manner from the cell membrane to outer medium. Additionally, increased proton pumping by the membrane bound H(+)-ATPase contributes to hop resistance. To energize such ATP-dependent transporters hop-resistant cells contain larger ATP pools than hop-sensitive cells. Furthermore, a pmf-dependent hop transporter was recently presented. Understanding the hop-resistance mechanisms has enabled the development of rapid methods to discriminate beer spoilage strains from nonspoilers. The horA-PCR method has been applied for bacterial control in breweries. Also, a discrimination method was developed based on ATP pool measurement in lactobacillus cells. However, some potential hop-resistant strains cannot grow in beer unless they have first been exposed to subinhibitory concentration of hop compounds. The beer spoilage ability of Pectinatus spp. and M. cerevisiae has been poorly studied. Since all the strains have been reported to be capable of beer spoiling, species identification is sufficient for the breweries. However, with the current trend of beer flavor (lower alcohol and bitterness), there is the potential risk that not yet reported bacteria will contribute to beer spoilage. Investigation of the beer spoilage ability of especially Gram-negative bacteria may be useful to reduce this risk.

ATP-Binding Cassette Transporters↗

PubMed1986

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases.

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, Mössbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.

Anaerobiosis↗

PubMed1982

Mechanism of action of butyryl-CoA dehydrogenase: reactions with acetylenic, olefinic, and fluorinated substrate analogues.

The acetylenic thio ester (3-pentynoyl)pantetheine irreversibly inactivates butyryl-CoA dehydrogenase from Megasphaera elsdenii. The inactivator becomes covalently attached to the protein (0.61 +/- 0.1 mol of 14C-labeled inactivator/mol of enzyme flavin). No modification of the flavin cofactor is seen. The covalent enzyme-inactivator adduct is labile toward base and neutral hydroxylamine. These treatments release 85 +/- 5% of the incorporated 14C label from the protein. Base-catalyzed hydrolysis of the adduct releases 3-oxopentanoic acid (0.6 mol/mol of incorporated inactivator). Treatment with hydroxylamine leads to formation of a hydroxamic acid on the protein (0.64 +/- 0.09 mol/mol of incorporated inactivator). The covalent adduct can be reduced with sodium borohydride with release of 1,3-pentanediol. Hydrolysis of the protein with 6 N HCl after sodium borohydride reduction yields 2-amino-5-hydroxyvaleric acid and proline. We conclude that the inactivator has reacted with the gamma-carboxyl group of a glutamate residue at the enzyme active site. The inactivation proceeds through enzyme-catalyzed rearrangement of the acetylene to an allene, followed by nucleophilic addition of the carboxyl group to the allene. (3-Chloro-3-butenoyl)pantetheine irreversibly inactivates the enzyme in a fashion similar to the acetylenic thio ester and also modifies a glutamate residue. Butyryl-CoA dehydrogenase catalyzes the isomerization of (3-butenoyl)pantetheine to (2-butenoyl)pantetheine. The enzyme catalyzes the elimination of HF from 3-fluoropropionyl-CoA and (3,3-difluorobutyryl)pantetheine. We suggest, that these results together support an oxidation mechanism for butyryl-CoA dehydrogenase which is initiated by alpha-proton abstraction.

Acetylation↗

PubMed1986

Oxidation-reduction potentials of butyryl-CoA dehydrogenase.

In order to obtain butyryl-CoA dehydrogenase from Megasphaera elsdenii in pure enough form to perform redox studies, the existing purification procedures first had to be modified and clarified [Engel, P. (1981) Methods Enzymol. 71, 359-366]. These modifications are described, and the previously unpublished spectral properties of the electrophoretically pure CoA-free butyryl-CoA dehydrogenase are presented. In our spectral reductive titration of pure enzyme, we show that although blue neutral flavin radical is stabilized in nonquantitative amounts in dithionite titrations (19%) or in electrochemical reductions mediated by methylviologen (5%), it is not thermodynamically stabilized; therefore, only a midpoint potential for butyryl-CoA dehydrogenase is obtained. The electron-transfer behavior from pH 5.5 to pH 7.0 indicates reversible two-electron transfer accompanied by one proton: EFlox + 2e- + H+ = EFlredH- Em7 = -0.079 V vs. SHE where EFlox is oxidized butyryl-CoA dehydrogenase, EFlredH- is two electron reduced enzyme, and Em7 is the midpoint potential at pH 7.0 at 25 degrees C. Redox data and activity data both indicate that the enzyme loses activity rapidly at pH values above 7.0. The Em7 of the butyryl-CoA dehydrogenase is 40 mV positive of the Em7 of the butyryl-CoA/crotonyl-CoA couple [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. Binding of substrate analogue acetoacetyl-CoA caused the potential of butyryl-CoA dehydrogenase to shift 100 mV negative of the free enzyme. The negative shift in potential makes electron transfer from enzyme to substrate more probable, which is consistent with the direction of electron transfer in the bacterial system.(ABSTRACT TRUNCATED AT 250 WORDS)

Butyryl-CoA Dehydrogenase↗

PubMed1990

Spectral and electrochemical properties of glutaryl-CoA dehydrogenase from Paracoccus denitrificans.

Studies of the spectral (UV/vis and resonance Raman) and electrochemical properties of the FAD-containing enzyme glutaryl-CoA dehydrogenase (GCD) from Paracoccus denitrificans reveal that the properties of the oxidized enzyme (GCDox) appear to be invariant from those properties known for other acyl-CoA dehydrogenases such as mammalian general acyl-CoA dehydrogenase (GACD) and butyryl-CoA dehydrogenase (BCD) from Megasphaera elsdenii. However, when either free or complexed GCD is reduced, its spectral and electrochemical behavior differs from that of both GACD and BCD. Free GCD does not stabilize any form of one-electron-reduced GCD, but when GCD is complexed to its inhibitor, aceto-acetyl-CoA, the enzyme stabilizes 20% of the blue neutral radical form of FAD (FADH.) upon reduction. Like GACD, when crotonyl-CoA- (CCoA) bound GCD is reduced, the red anionic form of FAD radical (FAD.-) is stabilized, and excess reduction equivalents are necessary to effect full reduction of the complex. A comproportionation reaction is proposed between fully reduced crotonyl-CoA-bound GCD (GCD2e-CCoA) and GCDox-CCoA to partially explain the stabilization of GCD-bound FAD.- by CCoA. When GCD is reduced by its optimal substrate, glutaryl-CoA, a two-electron reduction is observed with concomitant formation of a long-wavelength charge-transfer band. It is proposed that the ETF specific for GCD abstracts one electron from this charge-transfer species and this is followed by the decarboxylation of the oxidized substrate. At pH 6.4, potential values measured for free GCD and GCD bound to acetoacetyl-CoA are -0.085 and -0.129 V, respectively. Experimental evidence is given for a positive shift in the reduction potential of GCD when the enzyme is bound to a 1:1 mixture of butyryl-CoA and CCoA. However, significant GCD hydratase activity is observed, preventing quantitation of the potential shift.

Chemical Phenomena↗

PubMed1990

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins.

Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins. The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M. elsdenii and C. beijerinckii (a.k.a. Clostridium MP) with association constants (Ka) of 1.0 x 10(7) M-1 and 3.1 x 10(6) M-1, values about 10(2) less than the corresponding Ka values for FMN. X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C. beijerinckii at 2.5-A resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 A. The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation. The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M. elsdenii have been determined in the pH range 4.5-9.2. The oxidized/semiquinone equilibrium (E'0 = -160 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit; the semiquinone/reduced equilibrium (E'0 = -400 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4. Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 [Spencer, R., Fisher, J., & Walsh, C. (1977) Biochemistry 16, 3586-3593]. The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal.(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium↗

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↗

PubMed1998

Oxidase activity of the acyl-CoA dehydrogenases.

The medium chain acyl-CoA dehydrogenase catalyzes the flavin-dependent oxidation of a variety of acyl-CoA thioesters with the transfer of reducing equivalents to electron-transferring flavoprotein. The binding of normal substrates profoundly suppresses the reactivity of the reduced enzyme toward molecular oxygen, whereas the oxidase reaction becomes significant using thioesters such as indolepropionyl-CoA (IP-CoA) and 4-(dimethylamino)-3-phenylpropionyl-CoA (DP-CoA). Steady-state and stopped-flow studies with IP-CoA led to a kinetic model of the oxidase reaction in which only the free reduced enzyme reacts with oxygen (Johnson, J. K., Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 4738-4744). We have tested their proposal with IP-CoA and DP-CoA. The dependence of the oxidase reaction on oxygen concentration is biphasic with a major low affinity phase incompatible with a model predicting a simple Km for oxygen of 3 microM. If only free reduced enzyme reacts with oxygen, increasing IP-CoA would show strong substrate inhibition because it binds tightly to the reduced enzyme. Experimentally, IP-CoA shows simple saturation kinetics. The Glu376-Gln mutant of the medium chain dehydrogenase allows the oxygen reactivity of complexes of the reduced enzyme with IP-CoA and the corresponding product indoleacryloyl-CoA (IA-CoA) to be characterized without the subsequent redox equilibration that complicates analysis of the oxidase kinetics of the native enzyme. In sum, these data suggest that when bulky, nonphysiological substrates are employed, multiple reduced enzyme species react with molecular oxygen. The relatively high oxidase activity of the short chain acyl-CoA dehydrogenase from the obligate anaerobe Megasphaera elsdenii was studied by rapid reaction kinetics of wild-type and the Glu367-Gln mutant using butyryl-, crotonyl-, and 2-aza-butyryl-CoA thioesters. In marked contrast to those of the mammalian dehydrogenase, complexes of the reduced bacterial enzyme with these ligands react with molecular oxygen at rates similar to those of the free protein. Evolutionary and mechanistic aspects of the suppression of oxygen reactivity in the acyl-CoA dehydrogenases are discussed.

Acyl-CoA Dehydrogenase↗

PubMed1999

pH-dependent spectroscopic changes associated with the hydroquinone of FMN in flavodoxins.

Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.

Apoproteins↗

PubMed2004

Microbial monitoring by molecular tools of a two-phase anaerobic bioreactor treating fruit and vegetable wastes.

Microbial consortia in a two-phase, anaerobic bioreactor using a mixture of fruit and vegetable wastes were established. Bacterial and archaeal communities obtained by a culture-independent approach based on single strand conformation polymorphism analysis of total 16S rDNA showed the adaptation of the microflora to the process parameters. Throughout the 90 d of the study, the species composition of the bacterial community changed significantly. Bacterial 16S rDNA showed at least 7 different major species with a very prominent one corresponding to a Megasphaera elsdenii whereas bacterial 16S rDNA of a methanization bioreactor showed 10 different major species. After two weeks, Prevotella ruminicola became major and its dominance increased continuously until day 50. After an acid shock at pH 5, the 16S rDNA archaeal patterns in the acidogenic reactor showed two major prominent species corresponding to Methanosphaera stadtmanii and Methanobrevibacter wolinii, a hydrogenotrophic bacterium.

Archaea↗

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↗

PubMed1980

Efficiency of ferredoxins and flavodoxins as mediators in systems for hydrogen evolution.

1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.

Bacteria↗

PubMed1985

The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins.

The nucleotide sequence of a 629 base-pair segment of DNA spanning the nifF gene of Klebsiella pneumoniae is presented. The structural gene comprises 531 base-pairs (175 codons, excluding the translational initiator and terminator) encoding an acidic polypeptide of 18950 Da. The nifF product thus belongs to the long-chain class of flavodoxins. It shows some sequence homology to the short-chain flavodoxins from Desulfovibrio vulgaris, Clostridium MP and Megasphaera elsdenii, and much stronger homology to long-chain flavodoxins from Azotobacter vinelandii and Anacystis nidulans. The long chain flavodoxins thus seem to constitute a well-conserved sub-group. The homology with the A. vinelandii flavodoxin is particularly strong, which may reflect their common function in nitrogen fixation.

Amino Acid Sequence↗