Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport of Methanosarcina mazei Gö1

A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F₄₂₀H₂ dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F₄₂₀H₂ was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The K_m values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F₄₂₀-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.     

Energy Conservation by the H2:Heterodisulfide Oxidoreductase from Methanosarcina mazei Gö1: Identification of Two Proton-Translocating Segments

The membrane-bound H2:heterodisulfide oxidoreductase system of the methanogenic archaeon Methanosarcina mazei G?1 catalyzed the H2-dependent reduction of 2-hydroxyphenazine and the dihydro-2-hydroxyphenazine-dependent reduction of the heterodisulfide of HS-CoM and HS-CoB (CoM-S-S-CoB). Washed inverted vesicles of this organism were found to couple both processes with the transfer of protons across the cytoplasmic membrane. The maximal H+/2e- ratio was 0.9 for each reaction. The electrochemical proton gradient (DeltamicroH+) thereby generated was shown to drive ATP synthesis from ADP plus Pi, exhibiting stoichiometries of 0.25 ATP synthesized per two electrons transported for both partial reactions. ATP synthesis and the generation of DeltamicroH+ were abolished by the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847). The ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide did not affect H+ translocation but led to an almost complete inhibition of ATP synthesis and decreased the electron transport rates. The latter effect was relieved by the addition of SF 6847. Thus, the energy-conserving systems showed a stringent coupling which resembles the phenomenon of respiratory control. The results indicate that two different proton-translocating segments are present in the H2:heterodisulfide oxidoreductase system; the first involves the 2-hydroxyphenazine-dependent hydrogenase, and the second involves the heterodisulfide reductase.     

ATP-dependent H+ -pump activity in inverted vesicles of Methanosarcina mazei Gö1 and characterization of membrane ATPase.

ATP-dependent H+ -pump activity was found in inverted vesicles of Methanosarcina mazei Gö1 by using acridine orange as a fluorescent probe. The H+ -pump activity specifically required both Mg and sulfite ions, but azide, an inhibitor of F0F1-ATPase, did not inhibit the activity. The membranes prepared from M. mazei also had an Mg-ATPase activity, and at least the presence of vacuolar-type ATPase was detected.     

Electron paramagnetic resonance spectroscopic and electrochemical characterization of the partially purified N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanosarcina mazei Gö1.

The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase is a membrane-bound cobalamin-containing protein of Methanosarcina mazei Gö1 that couples the methylation of coenzyme M by methyltetra-hydrosarcinopterin to the translocation of Na+ across the cell membrane (B. Becher, V. Müller, and G. Gottschalk, J. Bacteriol. 174:7656-7660, 1992). We have partially purified this enzyme and shown that, in addition to the cobamide, at least one iron-sulfur cluster is essential for the transmethylation reaction. The membrane fraction or the partly purified protein contains a "base-on" cobamide with a standard reduction potential (Eo') for the Co2+/1+ couple of -426 mV. The iron-sulfur cluster appears to be a [4Fe-4S]2+/1+ type with an Eo' value of -215 mV. We have determined the methyltransferase activity at various controlled redox potentials and demonstrated that the enzyme activity is activated by a one-electron reduction with half-maximum activity occurring at -235 mV in the presence of ATP and -450 mV in its absence. No activation was observed when ATP was replaced by other nucleoside triphosphates or nonhydrolyzable ATP analogs.     

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain G?1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain G?1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.     

N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina strain Gö1 is an Na(+)-translocating membrane protein.

To determine the cellular localization of components of the methyltransferase system, we separated cell extracts of Methanosarcina strain G?1 into cytoplasmic and inverted-vesicle fractions. Measurements demonstrated that 83% of the methylene-tetrahydromethanopterin reductase activity resided in the cytoplasm whereas 88% of the methyl-tetrahydromethanopterin:coenzyme M methyltransferase (methyltransferase) was associated with the vesicles. The activity of the methyltransferase was stimulated 4.6-fold by ATP and 10-fold by ATP plus a reducing agent [e.g., Ti(III)]. In addition, methyltransferase activity depended on the presence of Na+ (apparent Km = 0.7 mM) and Na+ was pumped into the lumen of the vesicles in the course of methyl transfer from methyl-tetrahydromethanopterin not only to coenzyme M but also to hydroxycobalamin. Both methyl transfer reactions were inhibited by 1-iodopropane and reconstituted by illumination. A model for the methyl transfer reactions is presented.     

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.     

Overproduction of a functional A1 ATPase from the archaeon Methanosarcina mazei G?1 in Escherichia coli.

Single subunits of the A1 ATPase from the archaeon Methanosarcina mazei G?1 were produced in E. coli as MalE fusions and purified, and polyclonal antibodies were raised against the fusion proteins. A DNA fragment containing the genes ahaE, ahaC, ahaF, ahaA, ahaB, ahaD, and ahaG, encoding the hydrophilic A1 domain and part of the stalk of the A1AO ATPase of M. mazei G?1, was constructed, cloned into an expression vector and transformed into different strains of Escherichia coli. In any case, a functional, ATP-hydrolysing A1 ATPase was produced. Western blots demonstrated the production of subunits A, B, C, and F in E. coli, and minicell analyses suggested that subunits D, E, and G were produced as well. This is the first demonstration of a heterologous production of a functional ATPase from an archaeon. The A1 ATPase was sensitive to freezing but lost only about 50% of its activity within 18 days on ice. Inhibitor studies revealed that the heterologously produced A1 ATPase is insensitive to azide, dicyclohexylcarbodiimide and bafilomycin A1, but sensitive to diethylstilbestrol and its analogues dienestrol and hexestrol. The expression system described here will open new avenues towards the functional and structural analyses of this unique class of enzymes.     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

Escherichia coli iron enterobactin uptake monitored by Mössbauer spectroscopy.

Iron uptake by Escherichia coli under aerobic conditions of iron deficiency is mediated by a highly stable ferric enterobactin [Fe(ent)3-] siderophore complex. M?ssbauer spectroscopy has been used to monitor the fate of the iron as 57Fe(ent) was taken up by the cells. Osmotic shock experiments were used to distinguish between the iron present in the periplasmic space and that in the cytoplasm of the cell. Iron delivery by a synthetic analog of enterobactin, 1,3,5-N,N',N'- tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was also studied. Although Fe-MECAM was transported at the same rate as was Fe(ent) across the outer membrane and was apparently accumulated in the periplasmic space, the subsequent behaviors of Fe(ent) and Fe-MECAM were very different. After more than 30 min, a major fraction of the iron originally absorbed as ferric enterobactin appeared as Fe(II), apparently in the cytoplasm of the cell. However, little iron was delivered to the cytoplasm by the MECAM complex. The differences in specificity of these two stages of iron uptake by E. coli are discussed.     

Complementation of an Escherichia coli Polypeptide Deformylase Mutant with a Gene from Clostridium acetobutylicum ATCC 824

The Clostridium acetobutylicum ATCC 824 DNA containing the 3′ end of a PriA homolog, deformylase (def), and the 5′ end of formyltransferase (fmt) has been cloned, sequenced, and used to complement an Escherichia coli mutant. While def and fmt have been found sharing an operon in other organisms, the presence of a third gene within a putative operon has not previously been found. Received: 29 August 1997 / Accepted: 6 October 1997     

Discovery and Characterization of the First Archaeal Dihydromethanopterin Reductase,an Iron-Sulfur Flavoprotein from Methanosarcina mazei

The microbial production of methane by methanogenic archaea is dependent on the synthesis of the pterin-containing cofactor tetrahydromethanopterin (H₄MPT). The enzyme catalyzing the last step of H₄MPT biosynthesis (dihydromethanopterin reductase) has not previously been identified in methane-producing microorganisms. Previous complementation studies with the methylotrophic bacterium Methylobacterium extorquens have indicated that an uncharacterized archaeal-flavoprotein-like flavoprotein (AfpA) from Methylobacillusflagellatus or Burkholderiaxenovorans can replace the activity of a phylogenetically unrelated bacterial dihydromethanopterin reductase (DmrA). We propose that MM1854, a homolog of AfpA from Methanosarcina mazei, catalyzes the last step of H₄MPT biosynthesis in methane-producing microorganisms. To test this hypothesis, a six-histidine (His₆)-tagged version of MM1854 was produced. Bioinformatic analysis revealed the presence of one flavin mononucleotide (FMN)-binding site and two iron-sulfur cluster sites, consistent with an oxidoreductase enzyme. Purified His₆-MM1854 occurred as a homodimer of 29-kDa subunits, and the UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NAD(P)H was incapable of directly reducing the flavin cofactor, but dithionite eliminated the FMN peaks, indicating successful electron transfer to MM1854. An electron transfer system of NADPH, spinach NADPH-ferredoxin oxidoreductase, and ferredoxin could also reduce the FMN peaks. A newly developed assay indicated that dithiothreitol-reduced MM1854 could transfer electrons to dihydromethanopterin. This assay was also effective with a heat-stable DmrX analog from Methanocaldococcus jannaschii (MJ0208). These results provide the first biochemical evidence that MM1854 and MJ0208 function as archaeal dihydromethanopterin reductases (DmrX) and that ferredoxin may serve as an electron donor.     

Characterization of an Escherichia coli Mutant Deficient in Dihydrolipoyl Dehydrogenase Activity

A mutant of Escherichia coli deficient in dihydrolipoyl dehydrogenase (DHL) activity has been isolated and its characteristics have been studied. The activities of the pyruvic dehydrogenase (PDC) and alpha-ketoglutaric dehydrogenase complexes (KDC) are not present in extracts of the mutant unless purified dihydrolipoyl dehydrogenase is added. Experiments with antiserum to DHL have shown that cross-reacting material exists in mutant extracts. This suggests that the dihydrolipoyl dehydrogenase mutation (dhl(-)) is a missense structural mutation. The mutation maps very close to, if not adjacent to, the ace loci, and is not linked to the suc loci. This means the dhl locus is grouped with the genes for the other components of the PDC and not with the genes for KDC. The mutation is also transducible into prototrophic strains, demonstrating that no prior mutation is necessary for the DHL activity deficiency to exist. This evidence is consistent with the idea that there is only one gene for DHL and is supported by previous biochemical studies which have shown that DHL preparations from either enzyme complex are electrophoretically and immunochemically indistinguishable. Possible mechanisms for the genetic and metabolic control of DHL, PDC, and KDC are discussed.     

Deoxycytidine Triphosphate Deaminase: Characterization of an Escherichia coli Mutant Deficient in the Enzyme

A mutant of Escherichia coli, previously shown to contain abnormal nucleoside triphosphate pools, was found to be defective in its ability to synthesize thymidine nucleotides. The defect is not in the enzyme thymidylate synthetase but in deoxycytidine triphosphate deaminase, an enzyme that supplies deoxyuridine monophosphate, the substrate for thymidylate synthetase.     

Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA^Pyl pairs and cross recognition between nonsense codons and various tRNA^Pyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N^ε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.     

Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.

Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.     

Complementation of an Escherichia coli pyrF mutant with DNA from Desulfovibrio vulgaris.

A PyrF- mutant of Escherichia coli (SK1108, pyrF::Tn5 Kanr) was complemented with the Desulfovibrio vulgaris (Hildenborough) structural gene for orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). Either orientation of a 1.6-kilobase-pair D. vulgaris DNA fragment (pLP3B or pLP3A) complemented the PyrF- strain suggesting that the D. vulgaris pyrF promoter was functional. The apparent product of the D. vulgaris pyrF gene was a single 26-kilodalton polypeptide. These results demonstrate the utility of E. coli cloning systems in studying metabolic and energetic pathways in sulfate-reducing bacteria.     

Isolation and Characterization of an Escherichia coli K-12 Mutant with a Temperature-Sensitive RecA− Phenotype

A mutation that causes a temperature-sensitive RecA(-) phenotype was identified in a derivative of a PolA(-) strain that failed to grow at high temperature. The mutant allele (recA200) was shown to be linked to cysC, conferred a sharply temperature-sensitive, ultraviolet-sensitive Rec(-) phenotype in the range 35 to 42 C, and in crosses failed to show complementation at 42 C with Hfr's that transferred recA(-). Double mutants that carried both recA200 and polA were examined for ability to grow and synthesize DNA at restrictive temperatures.     

Complementation of an Escherichia coli DnaK defect by Hsc70-DnaK chimeric proteins

Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and lambda phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for lambda phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.