[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.

[Specific proteolysis of Clostridium thermocellum endoglucanase upon heterologous expression in Escherichia coli cells]. / Spetsificheskii proteoliz éndogliukanazy Clostridium thermocellum pri geterologichnoi ékspressii v kletkakh Escherichia coli.

The cel(1) gene of Clostridium thermocellum encoding endoglucanase cloned earlier in Escherichia coli was deleted at specific sites and fused with the E. coli gene lacZ'. Analysis of heterologous expression of the produced variants was performed. It was established that the post-translational modification of the clostridial endoglucanase in E. coli involves a membrane-linked N-terminal cleavage of 6 kDa polypeptide during secretion through the cytoplasmic membrane and a C-terminal cleavage of 4 kDa protein not related to the secretion. A comparative study of the authors' and literature data supports the suggestions that: (i) the cel(1) cloned in this laboratory is a deleted variant of the cloned cel(1) gene of C. thermocellum described previously by other authors, and (ii) the C-terminal proteolysis of the endoglucanase precursor takes place in a linker region connecting the catalytic and cellulose binding domains. An account of similar kind of proteolysis responsible for multiple forms of cellulolytic enzymes is given.

Analysis of pH-dependent protein interactions with gel filtration medium.

A prepacked Superose 12 HR 10/30 column was used to study the effects of elution ionic strength and pH on the chromatographic behaviour of a strong hydrophobic Clostridium thermocellum endoglucanase (1) and two weak hydrophobic proteins, Clostridium thermocellum endoglucanase C and egg white lysozyme. Ion-exclusion or ion-exchange interactions between weakly hydrophobic proteins and the gel matrix were observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the pI values of the proteins. These interactions were due to the presence of negatively charged groups on the surface of Superose and could be eliminated at any pH by adding electrolyte at a concentration determined by its chemical identity. The optimum results were observed with sodium sulphate at a concentration of 100 mM. The chromatographic behaviour of strong hydrophobic endoglucanase (1) on a Superose column as a function of pH was much more complex because of two interplaying effects, electrostatic and hydrophobic. Ideal size-exclusion chromatography could be achieved only in a narrow range of the conditions: first, the mobile phase must contain a weak salting-out electrolyte such as NaCl, and second, the mobile phase pH must be high enough that hydrophobic interactions between the solute and support are balanced by their electrostatic repulsion. At pH greater than pI, the retardation of endoglucanase (1) gradually increased with decreasing pH as a result of lowering of repulsive electrostatic interactions whether or not the buffer ionic strength was high. At pH less than pI a drastic increase in the capacity factor k' was observed owing to the additivity of hydrophobic and ion-exchange effects.(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation and certain properties of the thermostable endoglucanase from E. coli C600 (pKNE-102) coded by a Clostridium thermocellum gene]. / Vydelenie i nekotorye svoistva termostabil'noi éndogliukanazy iz E. coli C 600 (pKNE-102), kodiruemoi genom Clostridium thermocellum.

A previously unknown endoglucanase encoded by the C. thermocellum gene was isolated from the recombinant strain of E. coli (pKNE-102). The purification procedure included ammonium sulfate precipitation, heat treatment, chromatography on a polyvinyl matrix (Toyopearl HW-50F) and chromatofocusing on a high performance Mono P HR 5/20 column. Sodium dodecyl sulfate electrophoresis analysis of the Toyopearl HW-50F effluent revealed two protein bands with Mr of 41 kDa and 42 kDa. These protein components differed also by their pI values (4.45, and 4.40, respectively) and could be separated by chromatofocusing. Both components were found to be active and exhibited similar enzymatic properties as well as high thermal stability.

[Cloning and expression of Clostridium thermocellum F7 cellulase genes in Escherichia coli and Bacillus subtilis cells]. / Klonirovanie i ékspressiia genov tselliulaz Clostridium thermocellum F7 v kletkakh Escherichia coli i Bacillus subtilis.

The Clostridium thermocellum total DNA Sau 3A fragments' library was constructed on the basis of shuttle vector pMK4 for the Escherichia coli - Bacillus subtilis. 14 clones with endoglucanase activity and one with beta-glucosidase activity were selected in E. coli cells. Recombinant plasmids pCE were characterized by structural instability of various degree in B. subtilis cells. The results of the physical mapping, analysis of gene products in E. coli mini-cells as well as the DNA-DNA blot hybridization have led to conclusion on cloning of 7 individual genes for endoglucanases. Up to 3 polypeptides of various molecular weight corresponding to the products of cel gene were revealed in E. coli mini-cells containing the recombinant plasmids. The hybridization analysis demonstrated considerable homology of the majority of cel genes.

[Cloning and expression of Clostridium thermocellum F7 endoglucanase gene in gram-negative bacteria]. / Klonirovanie i ékspressiia gena éndogliukanazy Clostridium thermocellum F7 v gramotritsatel'nykh bakteriiakh.

The endoglucanase gene of Clostridium thermocellum F7 was cloned in Escherichia coli cells using pKM4 vector. Both the physical mapping and analysis of the gene products in the E. coli mini-cells system suggest cloning of a new cel gene different from those described earlier. The activity of endoglucanase in E. coli cells is localized in the periplasm, which correlates with secretion of enzymes of this type in C. thermicellum. Apart from 2 major components with Mr 42.5 and 43 kDa, corresponding to mature protein forms, we observed the formation of minor products of various electrophoretic motilities. Cloning of the endoglucanase gene on bhr vector pBS954 controlled by its own regulatory signal yielded high level of the endoglucanase activity in the recombinant strains of Klebsiella pneumoniae, Serratia marcescens and Erwinia carotovora comparable with the level of the gene expression in E. coli cells.

[Various kinetic properties of beta-glucosidase cloned from Clostridium thermocellum in E. coli]. / Nekotorye kineticheskie svoistva beta-gliukozidazy, klonirovannoi iz Clostridium thermocellum v E. coli.

The kinetics of pNPG, pNPX and cellobiose hydrolysis by beta-glucosidase cloned from C. thermocellum into E. coli was studied. The V values for these substrate hydrolysis are 102, 357 and 6.7 mumols/min/mg protein, respectively; Km are 0.44 mM, 50 mM and 100 mM, respectively, sigma-Gluconolactone inhibits the hydrolysis of all substrates according to a competitive mechanism with Ki of 0.032 mM, 6.0 mM and 0.25 mM, respectively. Glucose inhibits the hydrolysis of pNPG and pNPX also via a competitive mechanism with Ki of 10 mM and 37 mM, while cellobiose--via a mixed type mechanism with Ki of 110 mM and 350 mM. The existence of separate adsorption sites for each substrate and of a common catalytic site for pNPG and pNPX hydrolysis is supposed.

[Minimal synthetic nutrient medium for Clostridium stricklandii bacteria]. / Minimal'naia sinteticheskaia pitatel'naia sreda dlia bakterii Clostridium stricklandii.

A composition of minimal culture medium for the anaerobic bacterium Clostridium sticklandii strain CSG was determined. A fully synthetic culture medium promoting the cell yield up to 1 g dry biomass per 1 1 was found. It is composed of 13 amino acids, sodium formiate, four vitamins, microelements and salts. The strain under study does not utilize glucose as a carbon and energy source.

[Glucose metabolism in Clostridium sporogenes and Clostridium sticklandii bacteria]. / Metabolizm gliukozy u bakterii Clostridium sporogenes i Clostridium sticklandii.

Clostridium sporogenes 272 has a high rate of glucose fermentation. Its cell-free extract contains all glycolytic enzymes catalysing glucose degradation to pyruvate and shows the phosphoroclastic activity. C. sticklandii CSG has a low rate of glucose fermentation. Hence, the activity of the following enzymes is lower in this organism comparing to C. sporogenes: phosphohexoisomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12). Moreover, it is possible that the system of glucose transport into the cell is damaged in C. sticklandii.

[Catabolism of threonine in the bacterium Clostridium sticklandii]. / Katabolizm treonina u bakterii Clostridium sticklandii.

Catabolism of L-threonine in the anaerobic bacteria Cl. sticklandii has been studied. Degradation of this amino acid was shown to occur with participation of the following enzymes: NAD-dependent L-threonine-3-dehydrogenase (EC 1.1.1.103), glycine acetyltransferase (EC 2.3.1.29), phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1). The presence of the first two enzymes in Clostridia has been shown for the first time. A scheme of threonine oxidation down to acetic acid and glycine by Cl. sticklandii was proposed. The oxidation of one threonine molecule is coupled with phosphorylation of one ADP molecule and reduction of one NAD+ molecule.

[Synthetic medium for culturing Clostridium sporogenes]. / Sinteticheskaia sreda dlia kul'tivirovaniia Clostridium sporogenes.

Clostridium sporogenes was shown to be capable of good stable-growth and biomass accumulation up to 5 mg/ml (dry weight) in a chemically defined medium. Along with glucose, the culture required the following amino acids for its growth: arginine, phenylalanine, glycine tyrosine, leucine, isoleucine, valine, tryptophan, and methionine. The culture could grow in the absence of inorganic nitrogen but required such elements as phosphorus, iron and magnesium. Addition of cysteine to the medium accelerated the growth and increased the biomass yield. Leucine at a concentration over 0.1% inhibited the growth. Two vitamins, viz. biotin and p-aminobenzoic acid, are sufficient for the growth of the culture.

[Cyanide-resistant respiration of Pseudomonas aeruginosa bacteria]. / Izuchenie tsianidrezistentnogo dykhaniia bakterii Pseudomonas aeruginosa.

The transition of the bacterial culture into the stationary growth phase is accompanied by an appearance of cyanide-resistant respiration. Chloramphenicol inhibits the development of cyanide-resistant respiration. The cyanide-resistant oxidase is localized in the bacterial membrane. Its appearance is not due to the quantitative and qualitative changes of flavins, non-heme iron, ubiquinone and cytochromes of the b and c types, but is accompanied by an increase in the copper content of the membrane preparations. Neither cyanide-sensitive, nor cyanide-resistant chains of the bacterial electron transfer contain cytochromes of the a type. The cyanide-resistant oxidase accepts electrons at the ubiquinone--cytochrome b level of the main respiratory chain. The cyanide-resistant respiration is not accompanied by a formation of hydrogen peroxide. Cytochrome o performs the function of cyanide-sensitive oxidase. The nature of cyanide-resistant oxidase still remains obscure.

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria.

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.

[Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]. / Obratnyi perenos elektronov na urovne tsitokhroma c v tsianidrezistentnykh mitokhondriiakh drozhzhei Candida lypolytica.

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.

[The absence of coupling between the formation of membrane potential and electron transfer via the alternative pathway of cyanide-resistant mitochondria]. / Ob otsutstvii sopriazheniia mezhdu perenosom elektronov po al'ternativnomu puti tsianidrezistentnykh mitokhondrii i obrazovaniem membrannogo potentsiala.

The estimation of membrane potential of cyanide-resistant mitochondria of Candida lipolytica yeast was carried out using positively charged dye phenosafranine. The electron transfer via alternative pathway of cynide resistant mitochondria was shown not to be coupled with the formation of potential on membrane mitochondria.

[Changes in the electron transport chain in Escherichia coli depending on the cultivation conditions and growth phase]. / Izmeneniia tsepi perenosa élektronov u Eschirichia coli v zavisimosti ot uslovii kul'tivirovaniia i fazy rosta.

Changes in the electron transport chain of E. coli K-12 were studied as a function of the growth phase and the nature of a terminal electron acceptor in the growth medium. The content of flavins in the preparations of bacterial membranes hardly changed in all cases. The highest concentration of quinones was observed in the bacterial membranes at the stationary growth phase under anaerobic conditions of growth in the presence of nitrate. These membranes contained also the greatest amount of cytochrome beta1. The concentration of cytochrome alpha2 in all the membranes was low and varied among different preparations. All the membranes contained a CO-binding pigment whose content was maximal in the membranes of "nitrate" cells. The membranes of cells grown under aerobic conditions oxidized malate, apart from NADH and lactate, whereas the membranes of cells cultivated under anaerobic conditions in the presence of nitrate oxidized formiate. In most cases, the oxidase activity of the membranes of cells collected at the stationary growth phase was higher cf. the exponential phase.

[Mechanism of action of some quinoline alkaloids on respiratory chain of mitochondria]. / Mekhanizm deistviia alkaloidov riada khinolina na dykhatel'nuiu tser mitokhondrii

The mechanism of action of some quinoline alkaloids and their derivatives on respiratory chain of rat liver and Candida lipolytica yeast mitochondria was studied. The alkaloids were shown to inhibit electron transfer in the respiratory chain. The site of their action is localized between b and c cytochromes. Besides their ability to inhibit electron transfer in the respiratory chain, alkaloids are shown to be specific inhibitors of "exogenous" NADH-dehydrogenase of C. lipolytica yeast mitochondria. In addition to their inhibiting properties alkaloids can stimulate ATPase activity of mitochondria. O-alkylation of pseudane-IX permits to differentiate the inhibiting and uncoupling properties of this alkaloid.