Unusual stability of the Methanospirillum hungatei sheath.

The proteinaceous sheath of Methanospirillum hungatei was isolated by lysing cells in 50 mM dithiothreitol, separating the sheath from other cellular material by discontinuous sucrose density centrifugation, and removing the "cell spacers" with dilute NaOH. The isolated sheath material consisted of hollow tubes which had a highly ordered surface array. The stability of the sheath to treatment with denaturants and to enzymatic digestion was examined by a turbidimetric assay in conjunction with electron microscopy and optical or electron diffraction. The sheath was resistant to a range of proteases and also was not digested by peptidoglycan-degrading enzymes, a lipase, a cellulase, a glucosidase, or Rhozyme (a mixture of galactosidases, acetylglucosaminidase, acetylgalactosaminidase, fucosidase, and mannosidases). In addition to being unaffected by common salts, thiol-reducing agents, and EDTA, the layer was resistant to powerful denaturants such as 6 M urea, 6 M guanidinium hydrochloride, 10 M LiSCN, cyanogen bromide, sodium periodate, and 1% sodium dodecyl sulfate. Strong bases, boiling 3 N HCl, and performic acid did attack the sheath; in these cases, the array was systematically disassembled in a progressive manner, which was followed by electron microscopy. The layer was slightly modified by N-bromosuccinimide in urea, but the array remained intact. The stability of the sheath was remarkable, not only as compared to other bacterial surface arrays, but also as compared to proteins generally, and possibly indicated the presence of covalent cross-links between protein subunits.     

Three-dimensional architecture of the cell sheath and septa of Methanospirillum hungatei.

The methanogenic bacterium Methanospirillum hungatei exists as filaments which have a very unusual cell wall architecture, comprising a long cylindrical sheath within which there may be many individual cells arranged in a line. The sheath has a two-dimensional crystalline structure, and the cells are separated within the tube by septa which also have a crystalline structure. We have used computer image processing of tilted-view electron micrographs to analyze the structure in negative stains of both of these components in three dimensions. The repeating unit of the sheath consists of four approximately spherical domains ca. 2.5 nm in diameter arranged in a row. Based on observations of the type of lattice imperfections that occur, we suggest that each of the domains represents a separate polypeptide subunit and that the subunits are incorporated into the wall one by one. The septa are circular plates of remarkably constant size. They are normally found as double layers. They are hexagonally symmetrical and consist of trimerically associated subunits which interact about dimer axes to form an open network containing large pores ca. 15 nm in diameter.     

Dissolution and immunochemical analysis of the sheath of the archaeobacterium Methanospirillum hungatei GP1.

The sheath of Methanospirillum hungatei GP1 was degraded by three dissolution techniques, which produced a range of soluble products. By using 0.05 M L-arginine buffer (pH 12.6) at 90 degrees C for 10 min, 74% (dry weight) of the sheath was dissolved; however, the solubilized polypeptides were extensively degraded. Treatment with 2% beta-mercaptoethanol and 2% sodium dodecyl sulfate at 90 degrees C in 0.05 M 2(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 9.0) solubilized 42% (dry weight) of the sheath as a group of polypeptides of 30 to 40 kDa. At 100 degrees C for 2 h, 5% beta-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 20 mM EDTA released 74% of the sheath's mass as a group of polypeptides of 10 to 40 kDa. All solubilized products were examined by SDS-polyacrylamide gel electrophoresis, and a range of high- and low-molecular-weight polypeptides was identified. None were glycoproteins. Hoops, which comprise the sheath's structure, were seen by electron microscopy after all of the attempted dissolutions. Monoclonal antibodies were produced against the 10- to 40-kDa range of solubilized products and against the approximately 40-kDa polypeptides, and polyclonal antiserum was produced against an 18-kDa polypeptide. These immunological markers were used in Western immunoblotting and protein A-colloidal gold-antibody probing by electron microscopy to identify the structural location of the various polypeptides. Native sheath, which possesses 2.8-nm particles on its outer surface (M. Stewart, T.J. Beveridge, and G.D. Sprott, J. Mol. Biol. 183:509-515, 1985; P.J. Shaw, G.J. Hills, J.A. Henwood, J.E. Harris, and D.B. Archer, J. Bacteriol. 161:750-757, 1985), presented a gentle wave-form surface in platinum-shadowed specimens. In contrast, the inner face of the sheath was highlighted by ridges lying perpendicular to the longitudinal axis of the sheath and likely corresponded to hoop boundaries. Both the polyclonal and monoclonal antibodies were specific for different faces; polyclonal antibodies labeled the inner face, whereas monoclonal antibodies labeled the outer face. Accordingly, the apparent asymmetry of structure between the two faces of the sheath can be correlated by our immunochemical probing with a distinct asymmetry in the distribution of exposed polypeptides between the faces. The possible implications of this asymmetry for growth and maturation of the sheath are explained.     

High-resolution topography of the S-layer sheath of the archaebacterium Methanospirillum hungatei provided by scanning tunneling microscopy.

The inner and outer surfaces of the sheath of Methanospirillum hungatei GP1 have been imaged for the first time by using a bimorph scanning tunneling microscope (STM) on platinum-coated or uncoated specimens to a nominal resolution in height of ca. 0.4. nm. Unlike more usual types of microscopy (e.g., transmission electron microscopy), STM provided high-resolution topography of the surfaces, giving good depth detail which confirmed the sheath to be a paracrystalline structure possessing minute pores and therefore impervious to solutes possessing a hydrated radius of greater than 0.3 nm. STM also confirmed that the sheath consisted of a series of stacked hoops approximately 2.5 nm wide which were the remnants of the sheath after treatment with 2% (wt/vol) sodium dodecyl sulfate-2% (vol/vol) beta-mercaptoethanol (pH 9.0). No topographical infrastructure could be seen on the sides of the hoops. This research required the development of a new long-range STM capable of detecting small particles such as bacteria on graphite surfaces as well as a new "hopping" STM mode which did not deform the poorly conducting bacterial surface during high-resolution topographical analysis.     

Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1.

Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol-insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material).     

Physical characterization of the flagella and flagellins from Methanospirillum hungatei.

Flagellar filaments from Methanospirillum hungatei GP1 and JF1 were isolated and subjected to a variety of physical and chemical treatments. The filaments were stable to temperatures up to 80 degrees C and over the pH range of 4 to 10. The flagellar filaments were dissociated in the detergents (final concentration of 0.5%) Triton X-100, Tween 20, Tween 80, Brij 58, N-octylglucoside, cetyltrimethylammonium bromide, and Zwittergent 3-14, remaining intact in only two of the detergents tested, sodium deoxycholate and 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS). Spheroplasting techniques were used to separate the internal cells from the complex sheath, S-layer (cell wall), and end plugs of M. hungatei. The flagellar basal structure was visualized after solubilization of membranes by CHAPS or deoxycholate. The basal structure appeared to be a simple knob with no apparent ring or hook structures. The multiple, glycosylated flagellins constituting the flagellar filaments were cleaved by proteases and cyanogen bromide. The cyanogen bromide-generated fragments of M. hungatei GP1 flagellins were partially sequenced to provide internal sequence information. In addition, the amino acid composition of each flagellin was determined and indicated that the flagellins are distinct gene products, rather than differentially glycosylated forms of the same gene product.     

Crystalline order to high resolution in the sheath of Methanospirillum hungatei: a cross-beta structure

Electron microscopy and electron diffraction indicate that the outer sheath of the cell wall of the archaebacterium Methanospirillum hungatei contains a two-dimensional crystalline lattice having, at least to low resolution, p2 symmetry in projection with a = 5.66 nm, b = 2.81 nm and gamma = 85.6 degrees. At a resolution of 2 nm, the unit cell contains two lobes, whereas high-angle electron diffraction shows the presence of a substantial quantity of beta structure, with the 0.47 nm spacing (between polypeptide chains within a sheet) oriented circumferentially. The sheath is unusual when compared to other regular surface arrays found on bacteria in that it is a compact structure with small subunits. It may have a structural role analogous to barrel hoops since it tends to fragment perpendicular to its axis to give rings or hoops.     

Monolayer properties of archaeol and caldarchaeol polar lipids of a methanogenic archaebacterium, Methanospirillum hungatei, at the air/water interface.

Monolayer studies at the air/water interface were carried out on the major tetraether (caldarchaeol-) derived phosphoglycolipid, Glcp-alpha(1-2)-Galf-beta(1-1)-caldarchaeol-phosphoglycerol (PGC-I), the major diether (archaeol-) derived glycolipid, Glcp-alpha(1-2)-Galf-beta(1-1)-archaeol (DGA-I), the major archaeol-derived phospholipids, phosphatidyl-N,N dimethylaminopentanetetrol (PPDAA) and phosphatidyl-N,N,N-trimethylaminopentanetetrol (PPTAA) and the minor caldarchaeol-derived glycolipid, Glcp-alpha(1-2)-Galf-beta(1-1)-caldarchaeol (DGC-I) isolated from the methanogenic archaebacterium, Methanospirillum hungatei. The compression isotherms obtained showed that the two tetraether lipids had molecular surface areas about twice those of the diether lipids at all surface pressures, suggesting that both polar headgroups of the tetraether lipids are anchored into the aqueous subphase, even at the collapse pressure pi c. A U-shaped hydrocarbon chain conformation thus appears to be preferred for the tetraether lipids at the air/water interface, rather than an extended chain arrangement. The compression isotherms of the two tetraether lipids PGC-I and DGC-I were very similar at pH 0, both molecules being uncharged, but at pH 5.6 or 8, PGC-I films were much more expanded than the neutral DGC-I, due to ionization of the phosphate group in PGC-I and the resulting charge-charge repulsion. Monolayers of the zwitterionic diether phospholipids PPDAA and PPTAA were much less compressible than the glycosylated lipids, PGC-I, DGC-I and DGA-I, because the latter lipids contain the more compressible diglycosyl headgroup, oriented in horizontal conformation at low surface pressures, compared to the lower compressibility of the zwitterionic headgroup in the vertical conformation, particularly at pH 0 and 5.6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the cell wall of the sheathed methanogen Methanospirillum hungatei GP1 as an S layer.

The cell wall of Methanospirillum hungatei GP1 is a labile structure that has been difficult to isolate and characterize because the cells which it encases are contained within a sheath. Cell-sized fragments, 560 nm wide by several micrometers long, of cell wall were extracted by a novel method involving the gradual drying of the filaments in 2% (wt/vol) sodium dodecyl sulfate and 10% (wt/vol) sucrose in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer containing 10 mM EDTA. The surface was a hexagonal array (a = b = 15.1 nm) possessing a helical superstructure with a ca. 2.5 degrees pitch angle. In shadowed relief, the smooth outer face was punctuated with deep pits, whereas the inner face was relatively featureless. Computer-based two-dimensional reconstructed views of the negatively stained layer demonstrated 4.0- and 2.0-nm-wide electron-dense regions on opposite sides of the layer likely corresponding to the openings of funnel-shaped channels. The face featuring the larger openings best corresponds to the outer face of the layer. The smaller opening was encircled by a stalk-like mass from which 2.2-nm-wide protrusions were resolved. The cell wall in situ was degraded at pH 9.6 at 56 degrees C but was unaffected at pH 7.4 at the same temperature. The cell wall was composed of two nonglycosylated polypeptides (114 and 110 kDa). The cell wall resembled an archaeal S layer and may function in regulating the passage of small (< 10-kDa) sheath precursor proteins.     

Isolation and chemical composition of the cytoplasmic membrane of the archaebacterium Methanospirillum hungatei

The cytoplasmic membrane of Methanospirillum hungatei was isolated from osmotic lysates of spheroplasts, with yields of 7-8% of the cell dry weight. Cytoplasmic contamination was negligible, as judged by the removal of soluble enzymes. The cytoplasmic membrane consists of lipid (35-37%), primarily as a biphytanyldiglycerol tetraether glycolipid; protein (45-50%); and carbohydrate (10-12%). Ultra-thin sections showed that the trilaminar membrane formed vesicles with a maximum diameter of 0.4 microns. Protrusions of membrane projecting from the vesicles were seen often in negatively stained preparations. Fractionation of M. hungatei cells grown in the presence of [14C]mevalonic acid revealed that 90% of the phytanyl lipids were present in the cytoplasmic membrane band, with two minor bands accounting for the remainder of the label. Approximately 50% of the galactose, glucose, and mannose present in the cytoplasmic membrane was found in lipid extracts, while the remainder of these sugars and 98% of the rhamnose were present as nonlipid sugars. The cell sheath, isolated with a yield of 13% of the cell dry weight, contained the same sugars as the cytoplasmic membrane, but in very different proportions. Amino acid analysis of the membrane proteins showed that hydrophobic amino acid residues made up 37% of the total, neutral amino acids, 39%, basic, 8%, acidic, 16%, and that half-cysteine was present. Sodium dodecyl sulfate-polyacrylamide gel patterns of solubilized cytoplasmic membrane proteins revealed major bands at 195, 74.5, 44, 32, and 30 KDa. Significant amounts of nickel co-isolated with the cytoplasmic membrane, accounting for 0.16% of the membrane dry weight.     

Detection of growth sites in and protomer pools for the sheath of Methanospirillum hungatei GP1 by use of constituent organosulfur and immunogold labeling.

Methanospirillum hungatei GP1 integrated approximately 9% of cellular [35S]cysteine into its sheath. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels revealed that [35S]cysteine was confined to the proteins released by the sodium dodecyl sulfate-beta-mercaptoethanol-EDTA solubilization method (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) and was not present in the proteins released by treatment with phenol (G. Southam and T. J. Beveridge, J. Bacteriol. 174:935-946, 1992). Limited labeling of exposed sulfhydryl groups on hoops produced from sheath material suggested that most organosulfur groups occur within hoops and therefore help contribute to resilience. Electron microscopic autoradiography demonstrated that sheath growth, which is most active at the sites of cell division (spacer region), occurs through the de novo development of hoops. For growth to occur in the spacer region, sheath precursors must transverse several periodic envelope layers, including the cell wall (a single layer) and the various lamellae of the spacer plug (T. J. Beveridge, G. D. Sprott, and P. Whippey, J. Bacteriol. 173:130-140, 1991).     

Isolation and Ultrastructure of the Flagella of Methanococcus thermolithotrophicus and Methanospirillum hungatei

The flagella of the archaebacteria Methanococcus thermolithotrophicus and Methanospirillum hungatei enter the cells in regions with ultrastructure resembling that of the polar organelles found in a variety of eubacteria. Flagella of both organisms consist of a filament, a hook, and a basal body with two rings similar to those of gram-positive eubacteria. The integrity of the flagella of M. thermolithotrophicus is lost in the absence of high salt concentrations, and those of both organisms are unstable at high pH. The flagellar filaments of M. hungatei are composed of two flagellins of 24 and 26 kilodaltons.     

Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei.

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.     

Physiological and 15N-NMR analysis of molecular nitrogen fixation by Methanococcus thermolithotrophicus, Methanobacterium bryantii and Methanospirillum hungatei

Two mesophilic methanogenic bacteria, Methanobacterium bryantii strain MOH and Methanospirillum hungatei strain GP1 were demonstrated, using several different experimental approaches, to fix dinitrogen. Evidence includes (1) growth with N2 as the sole nitrogen source; (2) incorporation of 15N2 into cellular material (both soluble amino acid pools and insoluble cell protein and other macromolecules) detected by 15N-NMR spectroscopy; (3) acetylene reduction to ethylene by the cells, and inhibition of this reaction by bromoethanesulfonic acid (BES), a methanogen inhibitor. High-resolution 15N-NMR analysis of ethanol extracts of these organisms and cross-polarization magic-angle sample spinning analysis of the solid debris from these extracts are compared to labeled material from Methanococcus thermolithotrophicus, a methanogen previously determined to fix dinitrogen.     

Ultrastructure, inferred porosity, and gram-staining character of Methanospirillum hungatei filament termini describe a unique cell permeability for this archaeobacterium.

By light microscopy, Methanospirillum hungatei GP1 stains gram positive at the terminal ends of each multicellular filament and gram negative at all regions in between. This phenomenon was studied further by electron microscopy and energy-dispersive X-ray spectroscopy of Gram-stained cells, using a platinum compound to replace Gram's iodine (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Crystal violet-platinum precipitates could be found only in the terminal cells of each filament, which suggested that the multilamellar plugs at the filament ends were involved with stain penetration. When sheaths were isolated by sodium dodecyl sulfate-dithiothreitol treatment, the end plugs could be ejected and their layers could be separated from one another by 0.1 M NaOH treatment. Each plug consisted of at least three individual layers; two were particulate and possessed 14.0-nm particles hexagonally arranged on their surfaces with a spacing of a = b = 18.0 nm, whereas the other was a netting of 12.5-nm holes with spacings and symmetry identical to those of the particulate layers. Optical diffraction and computer image reconstruction were used to clarify the structures of each layer in an intact plug and to provide a high-resolution image of their interdigitated structures. The holes through this composite were three to six times larger than those through the sheath. Accordingly, we propose that the terminal plugs of M. hungatei allow the access of larger solutes than does the sheath and that this is the reason why the end cells of each filament stain gram positive whereas more internal cells are gram negative. Intuitively, since the cell spacers which partition the cells from one another along the filament contain plugs identical in structure to terminal plugs, the diffusion of large solutes for these cells would be unidirectional along the filament-cell axis.     

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1.

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.     

Effect of changes in mineral composition and growth temperature on filament length and flagellation in the Archaeon Methanospirillum hungatei

Methanospirillum hungatei strains GP1 and JF1 when cultivated at 37°C in JMA medium grew as motile single cells or short chains of cells (typically 10–30 m long). When M. hungatei was grown in low Ca²⁺ concentrations or with the divalent cation chelator EDTA, the organism grew as long non-flagellated filaments (up to 900 m long). The two strains had different thresholds of calcium concentrations for long filament formation (<0.25 mM for GP1 and <0.15 mM for JF1) as well as different minimal Ca²⁺ requirements for growth. Both strains produced long, almost straight, filaments at Ca²⁺ concentrations near the minimum required for growth. At suboptimal growth temperatures the organisms still grew as short filaments but no longer possessed flagella. Western blot analysis indicated that flagellin monomer was present in cultures of long non-flagellated filaments and short non-flagellated cultures grown at suboptimal temperatures. The amount of flagellin present appeared to be equal in both non-flagellated and flagellated cultures. When cells were grown as long non-flagellated filaments and switched to growth conditions inducing short, flagellated forms, flagella were first observed at 2.5 h after this switch.Portions of this work were previously presented at the 91st General Meeting of the American Society for Microbiology, May 5–9 1991, Dallas, Texas (abstract I-81) and at the 41st Annual Meeting of the Canadian Society of Microbiologists, June 3–6 1991, London, Ontario (Abstract MP-1)     

Methanogenesis and the K+ transport system are activated by divalent cations in ammonia-treated cells of Methanospirillum hungatei

We describe a K+ transport system in Methanospirillum hungatei cells depleted of cytoplasmic K+ via an ammonia/K+ exchange reaction (Sprott, G. D., Shaw, K. M., and Jarrell, K. F. (1984) J. Biol. Chem. 259, 12602-12608). Ammonia-treated cells contained low concentrations of ATP and were unable to make CH4 or to transport 86Rb+. All of these properties were restored by CaCl2, MgCl2, or MnCl2, and not by CoCl2 or NiCl2. The Rb+ transport system had a Km of 0.42 and Vmax of 29 nmol/min X mg; K+ inhibited competitively. Both H2 and CO2 were required for appreciable transport, whereas air, valinomycin, or nigericin were potent inhibitors. The influx of Rb+ was electrogenic and associated with proton efflux, producing a delta pH (alkaline inside) in acidic media. In the absence of K+ (or Rb+), the activation of CH4 synthesis by Mg2+ produced little change in the cytoplasmic pH, showing that methanogenesis did not elicit a net efflux of protons. The pH optimum for transport was in the range 6.0-7.3 where the transmembrane pH gradient would contribute minimally to the proton motive force. Protonophores at pH 6.3 caused a partial decline in CH4 synthesis and the ATP content and dramatically collapsed Rb+ transport. These and other inhibitor experiments, coupled with the fact that the Rb+ gradient was too large to be in equilibrium with the proton motive force alone, suggest a role for both ATP and the proton motive force in Rb+ transport. Also, a role for K+ in osmoregulation is indicated.     

Glycerol and propanediols degradation by Desulfovibrio alcoholovorans in pure culture in the presence of sulfate, or in syntrophic association with Methanospirillum hungatei

Abstract In a mineral medium containing sulfate as terminal electron acceptor, the sulfate-reducing bacterium Desulfovibrio alcoholovorans oxidized stoichiometrically 1 mol glycerol to 1 mol acetate and 1 mol 1,3-propanediol to 1 mol acetate with the concomitant reduction of 0.75 and 1 mol sulfate, respectively; 1 mol 1,2-propanediol was degraded to 0.8 mol acetate and 0.1 mol proprionate, with the reduction of approximately 1 mol sulfate. The maximum specific growth rates (μ_max in h⁻¹) were 0.22, 0.086 and 0.09 with glycerol, 1,3-propanediol and 1,2-propanediol, respectively. The growth yields were 12.7 g, 11.1 g and 7.2 g dry weight/mol 1,3-propanediol, glycerol and 1,2-propanediol degraded, respectively. The growth yields and maximum specific growth rates of the H₂-transferring associations were also calculated. In the absense of sulfate, all these reduced substrates were degraded to acids and methane when D. alcoholovorans was cocultured with Methanospirillum hungatei . Changes in the metabolic pathway were observed in the degradation of 1,2- and 1,3-propanediol. The metabolic efficiency of D. alcoholovorans to degrade glycerol, 1.2- and 1,3-propanediol is discussed.