Classification of methylotrophic bacteria according to 5S ribosomal RNA sequencing

5S ribosomal RNA sequences of 33 strains of methylotrophic bacteria were determined. Tentative phylogenetic tree was constructed using the maximum topological similarity principle. Strains under study can be divided into 7 separate branches consistently with the current classification of methylotrophic bacteria. More extensive tree was also built to show the position of methylotrophic bacteria with respect to non-methylotrophic ones. One can conclude that the in contrast to obligate methane-oxidizing bacteria, facultative methylotrophic bacteria do not comprise phylogenetically separate domain on the tree.     

One-carbon compounds transport studies in the obligate methylotroph

Cells of obligate methylotrophic Gram-negative bacterium Methylobacillus flagellatum KT which can only grow on methanol and methylamine media possess three different carriers mediating uptake of methylamin depending on growth conditions. All three uptake systems are energy-dependent, the methylamine uptake was inhibited by oxidative phosphorylation uncoupler and respiratory inhibitors. The first active transport system for methanol in the cells of obligate methylotroph was also demonstrated. The parameters of this system were measured, their dependence on energy, presence of respiratory inhibitors and uncoupler was shown.Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexyl-carbodiimide     

Transformation of a new Gram-positive methylotroph,Brevibacterium methylicum,by plasmid DNA

Summary Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD⁺-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Sm^r/Sp^r, Km^r/Gm^r) and pEC71 (7.1 kb; Km^r/Nm^r) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments. Offprint requests to: J. Nevera     

Oxidative phosphorylation in membrane vesicles of a gram-positive methylotroph

Membrane preparations, capable of high rates of respiration-linked ATP synthesis, have been obtained from a gram-positive methylotrophic bacterium Bacillus sp. MGA3. NADH, succinate, reduced TMPD and methanol were shown to be suitable substrates for the oxidative phosphorylation. Esterification of orthophosphate was dependent on electron transfer, as evidenced by the requirement for both substrate and oxygen. Phosphorylation was also dependent on ADP and was destroyed by boiling the membrane preparation. The phosphorylation was markedly uncoupled by carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone (CCCP) and was inhibited by N,N-dicyclohexylcarbodiimide (DCCD). KCN caused strong inhibition of substrate oxidation as well as phosphorylation for all substrates tested. Rotenone, amytal and antimycin A caused inhibition when NADH or methanol were used as substrates. Antimycin A inhibited respiration and ATP synthesis with succinate as substrate and had no effect on ascorbate —N,N,N,N-tetramethyl-p-phenylenediimide (TMPD) oxidation by membrane preparations of Bacillus sp. MGA3. P/O ratios determined were 2.4 with NADH, 1.7 with succinate and 0.8 with reduced TMPD. The measured P/O ratio with methanol-oxidizing system was similar to that with NADH (about 2.4).Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - TMPD N,N,N,N-tetramethyl-p-phenylenediimide - Q ubiquinone Q     

Enzyme activity of the formate hydrogenlyase complex in Citrobacter freundii

Citrobacter freundii 62 can grow in the absence of oxygen in media containing glucose, peptone, fumarate or malate. When the medium contained fumarate or malate, the culture could grow under anaerobic conditions only in the presence of molecular hydrogen, formate or nitrate. The highest activity of formatehydrogenlyase and hydrogenase was found when C. freundii grew in a medium with glucose and formate. The activity was lower in media with other organic substrates, particularly, in the absence of formate or H2. The activity of hydrogenase was very low in cells grown under aerobic conditions or in the presence of nitrates while the activity of formatehydrogenlyase was not found at all for all practical purposes. The activity of formate dehydrogenase assessed in the presence of methylene blue was rather high irrespective of the conditions under which the culture was grown. However, when the activity of formate dehydrogenase was determined in the presence of benzyl viologen, it was high only in cells grown in the medium with glucose and formate.     

Taxonomic studies on methylotrophic bacteria by 5S ribosomal RNA sequencing

Nucleotide sequences of 5S ribosomal RNA (rRNA) isolated from 19 strains of Gram-negative methylotrophic bacteria were determined. Comparison of these sequences allowed construction of a tentative phylogenetic tree and showed that the bacteria analysed belong to the Proteobacteria and fell into several clusters, including obligate methanotrophs, obligate methylotrophs and several groups of facultative methylotrophs. Taxonomic relations between methylotrophic and non-methylotrophic bacteria are discussed, and the polyphyletic nature of methylotrophy as a taxonomic feature is highlighted.     

Integrated membrane systems for gas separation in biotechnology: potential and prospects

Integrated non-porous membrane systems were applied for microbial combustible gas separation processes. Methane/CO₂ mixtures of various concentrations from methane fermentation processes (biogas) were separated using a membrane-separation complex of permabsorber type into individual components of technical grade (more than 95% purity). In experiments with three-component mixtures, using a selective membrane valve with various liquid carriers, all the gases of interest (H₂, CH₄ and CO₂) were obtained at greater than 90% purity in one separation step. The perspectives for the further application of non-porous membrane separating devices for various gaseous mixtures from different microbial processes are discussed.V. Teplyakov and E. Sostina are with the A.V. Topchiev institute of Petrochemical Synthesis, Russian Academy of Sciences, Membrane Research Center, Moscow 117912, Russia. E. Sostina is also, and A. Netrusov is with the Microbiology Department, Moscow University, Moscow 119899, Russia. I. Beckman is with the Chemistry Department, Moscow University, Moscow 119899, Russia.     

Turning Cellulose Waste Into Electricity: Hydrogen Conversion by a Hydrogenase Electrode

Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L^-1 h^-1 and 1 mM L^-1 h^-1, respectively. An enzymatic fuel cell (EFC) with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h) to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value.     

Components of antioxidant systems in the cells of aerotolerant sulfate-reducing bacteria of the genus <Emphasis Type="Italic">Desulfovibrio</Emphasis> (strains A2 and TomC) isolated from metal mining waste

Two strains of sulfate-reducing bacteria of the genus Desulfovibrio (A2 and TomC) isolated from metal mining waste were able to grow on agar Postgate C nutrient medium under microaerobic conditions. Since their growth in liquid nutrient medium was just slightly affected by 1% O₂ (initial concentration in the gas phase) and 0.05–0.1 mM H₂O₂, these strains were relatively oxygen-tolerant. Only the presence of oxidants in high concentrations (5–10% О₂ or 0.3–1.0 mM H₂O₂) resulted in practically complete inhibition of their growth. Strain A2 was more resistant to oxidative stresses than strain TomC. Activities of the key enzymes of antioxidant defense—superoxide dismutase (SOD), catalase, and peroxidase—were revealed in the cell-free extracts of strain A2 grown under strict anaerobic conditions. While strain TomC was found to possess no peroxidase activity, its catalase activity was much higher than that of strain A2 (36 and 2 U/mg protein, respectively). SOD activity of both strains was almost the same (5 U/mg protein). Sublethal H₂O₂ doses (concentration of 0.05–0.15 mM and exposure for 45–240 min) resulted in a drastic increase of catalase activity, especially in strain A2. Sublethal О₂ doses (1–2% in the gas phase) had no significant effect on activities of the antioxidant enzymes of both strains. The cytochrome composition determined from the absolute absorption spectra of the whole cells of strains TomC and A2 revealed the presence of the c heme (438 and 831 pmol/mg protein) and the d heme (336 and 303 pmol/mg protein, respectively). The presence of the d heme indicated the presence of the bd heme–heme quinol oxidase, which together with the c heme may provide for the functioning of the electron transport segment of the antioxidant defensive system, which is responsible for aerotolerance of sulfate-reducing bacteria.     

Methylobacillus flagellatus KT contains a novel cbo-type cytochrome oxidase

The o-type oxidase from the methanol-grown obligate methylotroph Methylobacillus flagellatus KT has been purified to homogeneity. The complex is composed of four subunits (57, 40, 35 and 30 kDa). It contains six haems (4C:1B:1O) and one copper atom per molecule. It is proposed that the haem O-Cu(B) binuclear centre and a low-spin haem B are located in subunit I (57 kDa), two haems C reside in the cytochrome c homodimer (35 kDa), two haems C belong to the dihaem cytochrome c (30 kDa). The presented data provide evidence that cytochrome cbo is a novel representative of the haem-copper oxidase superfamily.