Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

The death rate ofMethanobacterium thermoautotrophicum strain Marburg upon exposure toN-methyl-N-nitro-N-nitrosoguanidine under anaerobic conditions was of the same order of magnitude as the death rates that have been reported forEscherichia coli. Cultures of the methanogenic bacterium, mutagenized by nitrosoguanidine-treatment and grown under non-selective conditions, yielded mutants resistant toDL-ethionine (30 mM) or to 2-bromoethane sulfonic acid (3.8 mM). No mutants were observed in untreated controls. Among 1500 clones obtained from nitrosoguanidine-treated cell suspensions there were 6 mutants requiring a single growth factor each, namelyl-leucine,l-phenylalanine, thiamine (2 mutants) or adenosine (2 mutants). Three mutant-strains were studied in more detail. They were genetically stable (no revertants among 10⁹ cells), and wild type growth rates were restored by 5 mml-leucine, 0.4 mM adenosine and 0.03 mM thiamine, respectively.Abbreviations 2-BES 2-bromoethanesulfonic acid - MIC minimum inhibitory concentration     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Simultaneous utilization of cysteine and thiosulfate for growth ofMethanobacterium thermoautotrophicum strain ΔH

Summary The sulfur-containing compounds cysteine, thiosulfate and dithionite, were investigated to substitute for sulfide as a sulfur source for cultivation ofMethanobacterium thermoautotrophicum. although none of the three compounds was suitable as the sole sulfur source, a combination of thiosulfate and cysteine supported growth and methanogenesis of the methanogen in batch and continuous culture as efficiently as sulfide.     

Non-enzymatic ammonia formation from glutamine under growth conditions for Methanobacterium thermoautotrophicum

Glutamine was found to be converted to NH₃ and pyroglutamic acid (5-oxo-2-pyrrolidine carboxylic acid; 5-oxoproline) under conditions of growth for Methanobacterium thermoautotrophicum at rates sufficient to satisfy the organism's nitrogen requirements.     

Molecular dynamics of the diatom thylakoid membrane under different light conditions

During the last years significant progress was achieved in unraveling molecular characteristics of the thylakoid membrane of different diatoms. With the present review it is intended to summarize the current knowledge about the structural and functional changes within the thylakoid membrane of diatoms acclimated to different light conditions. This aspect is addressed on the level of the organization and regulation of light-harvesting proteins, the dissipation of excessively absorbed light energy by the process of non-photochemical quenching, and the lipid composition of diatom thylakoid membranes. Finally, a working hypothesis of the domain formation of the diatom thylakoid membrane is presented to highlight the most prominent differences of heterokontic thylakoids in comparison to vascular plants and green algae during the acclimation to low and high light conditions.     

The light-dependent effect of external pH on the membrane potential of Nitella

In the light the membrane potential of Nitella flexilis andNitella axilliformis was hyperpolarized by raising the externalpH above pH 5.5, at the rate of 30–40 mV/pH below pH 8.This hyperpolarization was largely reduced in the dark. The membrane potential was sensitive to the external pH of mediawith a low potassium concentration, where it was relativelyinsensitive to potassium concentration. In media of a high concentrationwhere it was sensitive to the potassium concentration, the membranebecame insensitive to the external pH. The transition from apH-sensitive to a pH-insensitive state occurred rather abruptlyon increasing the external potassium concentration. (Received September 1, 1972; )     

Convenient fluorescence-based methods to measure membrane potential and intracellular pH in the Archaeon Methanobacterium thermoautotrophicum

New and improved methods to determine the membrane potential (Delta Psi) and the Delta pH in methanogenic archaea were developed and tested in Methanobacterium thermoautotrophicum strain Delta H. The Delta pH measurements took advantage of the pH-dependent fluorescence properties of coenzyme F(420), the major intracellular electron carrier in the organism. The protonophore p-nitrophenol did not show any interference with the F(420) fluorescence spectra and was therefore suitable to equalize internal and external pH. The method developed allowed the determination of the intracellular pH with an error of less than 0.05 pH units.Membrane potentials could easily be assessed using the fluorescent probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)) with an accuracy of approximately 10 mV.Both methods were tested with cell suspensions of M. thermoautrophicum incubated at medium pH values between 5.5 and 8. It was found that Delta Psi and Delta pH values remained constant under these conditions. Membrane potentials were about -160 mV and Delta pH was kept at 0.35 pH units (inside minus outside) resulting in a total proton motive force of about -180 mV (inside negative).     

Decay of membrane potential under phosphorylating conditions in chloroplasts with in vivo activated ATPase

(1) The relation between the membrane potential and phosphorylation was studied in chloroplasts rapidly prepared from illuminated spinach leaves (light chloroplasts) and from dark-adapted leaves (dark chloroplasts). Light chloroplasts had a higher ATP hydrolysis activity than dark chloroplasts. (2) In the presence of ADP or ATP, a rapidly decaying phase of the field-indicating 518 nm absorbance change with a half-time of 15 ms became apparent in addition to the slow phase with a half-time of more than 300 ms in either type of chloroplast. Under these conditions, light chloroplasts showed a larger rapid phase than dark chloroplasts. (3) The rapid phase was suppressed by dicyclohexylcarbodiimide and was assumed to reflect the dissipation of membrane potential due to proton movements inside the CF₁-CF₀ ATP synthetase. (4) A model for the proton movement in ATP synthetase is proposed.     

The development of someLemnaceae under different nutrient conditions

Plants ofSpirodela polyrrhiza, Lemna gibba, L. minor andL. minuscula were grown experimentally in nutrient media under various nutrient conditions involving different concentrations and forms of nitrogen and different concentrations of phosphorus. The treatments affected the multiplication rate of the plants, their root length and formation of anthocyanin and of turions.     

Gating current in the membrane of nodes of Ranvier under conditions of linear alteration of the membrane potential

Asymmetrical displacement currents (Id) in the frog Ranvier node (R. ridibunda) treated with tetrodotoxin and tetraethylammonium were studied by the use of ramp-voltage pulses. In some experiments both ramp- and step-voltage pulses were used. The net Id consists of two components, one of which Id (I) can be blocked by local anesthetic trimecaine, or inactivated with the 10 ms depolarizing prepulse sufficiently large to inactivate the sodium current in the same node (before Na+ removal and TTX application). Parameters of the steady state charge distribution are very close (though not identical) to that of the peak sodium permeability vs. potential relation. The charge carrying Id (I) is estimated as 0.3--0.5 of the net displaced charge. The results suggest that trimecaine- and inactivation-sensitive component of Id may be the true gating current of Na+ channels.     

The disappearance of leaf litter under different woodland conditions

Summary A method is described by which quantitative studies can be made on the course of disappearance of leaf litter under woodland conditions. The litter samples are placed in the field in nylon hairnets. Results are given for a variety of litters placed on different soils.Roudsea Wood Nature Reserve, Publication No. 1.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.     

Improved growth and methane production conditions for Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was grown on a defined mineral salts medium under strictly anaerobic conditions with H₂ and CO₂ as the sole energy and carbon sources, respectively. The cultivation medium was optimized with respect to non-organic components including Se(IV), W(VI), N, Ni(II), Fe(II), Co(II) and Mo(VI). Sulphide concentration in the medium was maintained constant using an on-line regulatory system by the addition of 0.5 M Na₂S. A maximum supply rate of 0.6 vvm of a mixture of 80% H₂ and 20% CO₂ was achieved for the gaseous substrates. Under these conditions a specific growth rate of 0.30 h^–1 and a cell concentration of 4.8 g cell dry weight (DW) l^–1, representing a 140% increase over previously published results, were obtained. The growth yield of 2.3 g DW mol^–1 CH₄ was similar to published values. However, the overall specific productivity was enhanced from 11 mmol CH₄ g^–1 DW h^–1 to 24 mmol CH₄ g^–1 DW h^–1, corresponding to an improvement of 120%. Correspondence to: U. von Stockar     

The growth of isolated barley embryos cultivated under different conditions

Isolated barley embryos were cultivated aseptically in three different complete media. The growth of the primary root of each embryo was measured during four days of cultivation. Embryos were cultivated on three different consistencies of media: on agar plates, on cellulose tissue moistened with the medium and in liquid shaken cultures. The last way of cultivation yielded the highest degree of the growth of roots. Optimum combination of conditions in shaken liquid medium was selected on the basis of systematic study. The growth of roots of isolated embryos under appropriate conditions approaches that of roots of seedlings cultived on moistened blotting paper.