[Anaerobic bacteria involved in the degradation of aromatic sulfonates to methane].

An anaerobic microbial consortium that degrades benzene- and p-toluenesulfonate to form methane and fatty acids has been produced. Pure cultures of three strains of anaerobic spore-forming bacteria Clostridium spp., as well as the sulfate-reducing bacteria Desulfovibrio sp., were isolated and characterized. Phylogenetic analysis of 16S rRNA gene sequences of strains showed that pure cultures of clostridia strains 14, 24, and 21 are close to Clostridium lituseburense DSM 797T, C. sartagoforme DSM 1292T, and C. pascui DSM 10365T, and the sulfate-reducing strain SR1 is genotypically closer to Desulfovibrio aminophilus ALA-3T. Preliminary characterization of isolated bacteria makes it possible to assume that these are new species of the genera Clostridium and Desulfovibrio, the distinctive feature of which is the ability to incorporate aromatic sulfonates in their metabolisms.

[Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock].

Heat shock (45 degrees C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37 degrees C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2 for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.

[Interrelation between the pathways of isoprenoid biosynthesis and carbon source catabolism in anaerobic and facultatively anaerobic bacteria].

Data on the interrelation between the pathways of the carbon source catabolism and isoprenoid biosynthesis in anaerobic and facultatively anaerobic bacteria were obtained. Two pathways of isoprenoid biosynthesis (nonmevalonate and mevalonate) were revealed in the representatives of the genus Clostridium. The non-mevalonate pathway of isoprenoid biosynthesis and the glycolytic pathway of substrate oxidation are typical of glucose-grown bacteria, whereas the pentose phosphate cycle operates in xylose-grown bacteria. The mevalonate pathway of isoprenoid biosynthesis was revealed in strain Clostridium thermosaccharolyticum DSM 571 grown in the presence of mevinolin, as well as in a number of lactic acid bacteria. Mevinolin is known to react with the lactate dehydrogenase complex, preventing reduction of pyruvate. The nonmevalonate pathway of isoprenoid biosynthesis was revealed in Bifidobacterium bifidum. The role of different metabolic pathways in isoprenoid biosynthesis is discussed.

[Adaptation of the yeast Yarrowia lipolytica to heat shock].

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).

[Tolerance of the yeast Yarrowia lipolytica to oxidative stress].

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H2O2, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pre-treatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H2O2, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.

[Biosynthesis of naphthoquinone pigments by fungi of the genus Fusarium].

We studied biosynthesis of colored naphthoquinone metabolites by Fusarium decemcellulare, F. graminearum, and F. bulbigenum fungi. F. bulbigenum and F. graminearum synthesized bikaverin and aurofusarin, respectively, which depended on the conditions of cultivation. F. decemcellulare synthesized soluble extracellular naphthoquinones of the naphthazarin structure (javanicin, anhydrojavanicin, fusarubin, anhydrofusarubin, bostricoidin, and novarubin) or extracellular dimeric naphthoquinone aurofusarin. Biosynthesis of naphthoquinone pigments was shown to be the main fungal response to stress exposure. It occurs under conditions of growth inhibition or arrest.

[Isoprenoid pigments in representatives of the family Microbacteriaceae].

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C40-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and beta-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.

[Prevalence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis among bacteria of different systematic groups].

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathway of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulate the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.

Novel psychrophilic anaerobic spore-forming bacterium from the overcooled water brine in permafrost: description Clostridium algoriphilum sp. nov.

A gram-positive, motile, strict anaerobic spore-forming bacterium was isolated from the over-cooled brine in the permafrost. The optimal temperature for isolate growth was 5-6 degrees C at pH 6.8-7.2. The bacterium was growing on the medium rich in saccharides and disaccharides. Out of polysaccharides tested, only xylan sustained the growth. Fermentation of the hexoses led to the formation of acetate, butyrate, lactate, H2,CO2 and some formate and ethanol. Cell wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids of the cell wall were C(14:0) and C(16:1c9). The content of G-C pairs in DNA was 31.4 mol%. As phylogenetic analysis has shown, it is closely linked to the members of cluster 1 of Clostridium. It differs from the other species of the genus by the substrates necessary for the growth, products forming as a result of the fermentation and content of the fatty acids in the cell wall. Thus, it was suggested to describe this strain as a new species named Clostridium algoriphilum. Type strain 14D1 was deposited into the Russian Collection of the Microorganisms VKM B-2271T and German Collection of the Microorganisms DSM 16153T .

[Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate]. / Aktivatsiia tsianidrezistentnoi oksidazy drozhzhei Yarrowia lipolytica adenozin 5'-monofosfatom.

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.

[The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons]. / Al'ternativnaia oksidasa v mitokhondriiakh drozhzhei Yarrowia lipolytica ne sposobna konkurirovat' za élektrony s tsitokhromnym putem.

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.

[Methanogenic sarcina from an anaerobic microbial community degrading p-toluene sulfonate]. / Metanoobrazuiushchaia sartsina iz anaerobnogo soobshchestva, razrushaiushchego p-toluolsul'fonat.

The methanogenic strain MM isolated from an anaerobic microbial community degrading p-toluene sulfonate showed optimal values of temperature and pH for growth equal to 37 degrees C and 6.3-6.9, respectively. The doubling times of the isolate grown on methanol, acetate, and methylamines under the optimal conditions were 8.8, 19.1, and 10.3-28.1 h, respectively. The growth of strain MM was observed only when the cultivation medium contained casamino acids or p-toluene sulfonate. The G + C content of the DNA of the isolate was 40.3 mol%. This, together with DNA-DNA hybridization data, allowed the new isolate to be identified as a strain of the species Methanosarcina mazei. The new isolate differed from the known representatives of this species in that it was resistant to alkylbenzene sulfonates and able to demethylate p-toluene sulfonate when grown on acetate.

[Terminal oxidases in different genera of the family Microbacteriaceae]. / Terminal'nye oksidazy predstavitelei razlichnykh rodov semeistva Microbacteriaceae.

The nature of terminal oxidases in representatives of four different genera of the family Microbacteriaceae was studied. It was found that the late-logarithmic and early-stationary cells of all of the investigated strains of the genera Plantibacter and Okibacterium contain the aa3-type cytochrome oxidase. Bacteria of the genera Rathayibacter and Agreia synthesize three oxidases, the bb3- and aa3-type cytochrome oxidases and nonheme cyanide-resistant oxidase, in proportions dependent on the cultivation conditions and the growth phase. Oxygen deficiency in the cultivation medium induces the synthesis of the bd-type oxidase in all of the microorganisms studied. The data obtained provide evidence that the type of terminal oxidases, along with the known chemotaxonomic characteristics, may serve to differentiate the genera of the family Microbacteriaceae at the phenotypic level.

[Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate]. / Analiz anaérobnogo soobshchestva mikroorganizmov, razrushaiushchego n-toluolsul'fonat.

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.

[Respiratory activity and naphthoquinone synthesis in the fungus Fusarium decemcellulare exposed to oxidative stress]. / Dykhatel'naia aktivnost' i obrazovanie naftokhinonovykh pigmentov u griba Fuzarium decemcellulare v usloviiakh okislitel'nogo stressa.

The effect of oxidants (hydrogen peroxide and juglone) on the growth, respiration, and naphthoquinone synthesis in the fungus Fusarium decemcellulare was studied. The addition of the oxidants to the exponential-phase fungus inhibited cell respiration (either partially or completely, depending on the oxidant concentration), culture growth, and naphthoquinone synthesis. The treatment of fungal cells with nonlethal concentrations of H2O2 (below 0.25 mM) and juglone (below 0.1 mM) induced the resistance of cell respiration to cyanide. The residual respiration in the presence of cyanide could be inhibited by benzohydroxamic acid, indicating the occurrence of alternative oxidase. Increased concentrations of oxidants (0.25 mM juglone and 0.5 mM H2O2) rapidly and irreversibly inhibited cell respiration. These observations suggest that the mitochondrial respiratory chain of fungal cells exposed to oxidative stress is subject to the action of active oxygen species. The treatment of fungal cells with nonlethal concentrations of H2O2 and juglone activated cellular glutathione reductase and glucose-6-phosphate dehydrogenase, which are protective enzymes against oxidative stress.

[Level of cyclic AMP during induction of alternative oxidase in Yarrowia lipolytica yeast cells]. / Soderzhanie tsAMF v usloviiakh induktsii al'ternativnoi oksidazy v kletkakh drozhzhei Yarrowia lipolytica.

The effect of cyanide, antimycin A, ethanol, and acetate on the induction of alternative oxidase in the yeast Yarrowia lipolytica VKM Y-155 sensitive to cyanide, in the presence of the aforementioned compounds led to the development of cyanide-resistant respiration, which could be suppressed by benzohydroxamic acid, an inhibitor of alternative oxidases. The incubation of cells with cyanide, ethanol, or acetate raised the intracellular pool of cAMP, which attained maximal values after a 2- to 3-min incubation, then rapidly decreased to the initial value and did not change over the next three hours of incubation. The possible role of cAMP in the induction of alternative oxidase in yeast cells is discussed.

Involvement of the respiratory chain of gram-negative bacteria in the reduction of tellurite.

The terminal oxidases of the respiratory chain of seven strains of gram-negative bacteria were shown to be involved in the reduction of tellurite. The rate of tellurite reduction correlated with the intensity of respiration. The inhibitors of terminal oxidases, carbon monoxide and cyanide, inhibited the reduction of tellurite. In Pseudomonas aeruginosa PAO ML4262 and P. aeruginosa PAO ML4262 (pBS 10), the respiratory chain was found to contain three types of cytochrome c, one of which (the carbon monoxide-binding cytochrome c) was involved in the reduction of tellurite. Agrobacterium tumefaciens VKM B-1219, P. aeruginosa IBPM B-13, and Escherichia coli G0-102bd++ cells contained oxidases aa3, bb3, and bd, respectively. The respiratory chain of other strains contained two oxidases: E. coli DH5alpha of bb3- and bd-type, and Erwinia carotovora VKM B-567 of bo3- and bd-type. All the strains under study reduced tellurite with the formation of tellurium crystallites. Depending on the position of the active center of terminal oxidases in the plasma membrane, the crystallites appeared either in the periplasmic space [P. aeruginosa PAO ML4262 and P. aeruginosa PAO ML4262 (pBS10)], or on the outer surface of the membrane (A. tumefaciens VKM B-1219 and P. aeruginosa IBPM B-13), its inner surface (E. coli G0-102bd++), or on both surfaces (E. coli DHaalpha and E. carotovora VKM B-567).

Regulation and physiological role of cyanide-resistant oxidases in fungi and plants.

Data on the induction and regulation of cyanide-resistant oxidases in eucaryotic microorganisms and higher plants are reviewed. Expression of an alternative oxidase gene can be caused by a decrease in energy charge in cells. Some evidence exists suggesting that cAMP and Ca2+ act as intracellular signals inducing the expression of the alternative oxidase genes. Under certain conditions cells produce alternative oxidases which remain in an inactive state. Activation of the alternative pathway of cell respiration is usually observed when electron transport via cytochromes is inhibited. The physiological role of the alternative oxidase is discussed.

Toxic effect of surfactants and probable products of their biodegradation on methanogenesis in an anaerobic microbial community.

Surfactants used in household and various industries, are rather toxic; therefore, the accumulation of these compounds in the environment through wastewaters has challenged the problem of their biodegradation. In this research, an attempt was made to assess the toxic effect of various surfactants and the likely products of their biodegradation on the acetoclastic methanogens of an anaerobic microbial community. Among the substances investigated, cationic surfactants were found to be most toxic to methanogens: 154 mg/l alkamon DS and 345 mg/l catamin AB induced a 50% inhibition of methanogenesis. Toxicity studies of some aromatic and cyclic compounds, as the probable products of biodegradation of alkylbenzene sulfonate surfactants, showed that methanogenesis in the microbial community under study are rather tolerant to high concentrations of these compounds.