[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.

[Detection of C-P-lyase activity in a cell-free extract of Escherichia coli].

A system has been developed for in vitro testing of E. coli C-P-lyase (the enzyme cleaving C-P bonds in phosphonates). NADH, ATP, and the system of ATP regeneration were necessary but not sufficient for expression of the C-P-lyase activity in cell-free extracts of E. coli. Experimental evidence suggests that glucose 6-phosphate and (or) glucose activate C-P-lyase, serving as precursors in the formation of (alkylphosphono)ribose, an intermediate in the reaction. Guanine is the most likely acceptor of the phosphate group.

[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.

[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.

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).

[Effect of the artificial electron acceptor reumycin on the nature of an intermediate accumulated by Pseudomonas aeruginosa bacteria]. / Vliianie reumitsina--iskusstvennogo aktseptora élektronov na prirodu intermediata, nakaplivaemogo bakteriiami Pseudomonas aeruginosa.

We investigated the effect of the autooxidizable electron acceptor reumicin on the oversynthesis of intermediates by the bacterium Pseudomonas aeruginosa during its growth limitation by a nitrogen source. Addition of reumicin which accepted the reducing equivalents from NADH(+)-dehydrogenase of the respiratory chain was shown to reduce the rate of alpha-ketoglutarate formation and yield, and to induce the synthesis and excretion of pyruvate into the cultivation medium. The maximal accumulation of pyruvate was observed when reumicin was added at a concentration of 0.4-0.5 mM. It is concluded that the nature of the overproduced intermediate, the rate of its biosynthesis and yield depend on the extent of reducibility of the pyridine nucleotide pool. The oversynthesis of alpha-ketoglutarate is suppressed by chloramphenicol, a protein synthesis inhibitor, which points to a possible involvement of inducible systems including the carriers of the acids via the cytoplasmic membrane.

[The physiologic role of pyocyanine synthesized by Pseudomonas aeruginosa]. / Fiziologicheskaia rol' piotsianina, sinteziruemogo Pseudomonas aeruginosa.

The physiological role of pyocyanine for Pseudomonas aeruginosa was studied. Its synthesis was shown to commence at the retardation growth phase. Pyocyanine was accumulated only in the growth medium. The addition of 2,6-dichlorophenolindophenol accepting the reducing equivalents from coenzyme Q and transferring them to cytochrome c inhibited the pigment accumulation. This was indicative of the connection between pyocyanine synthesis and the level of the reducing equivalents in the cells. Pyocyanine did not accept the reducing equivalents from coenzyme Q in the respiratory chain of P. aeruginosa. Only reduced pyridine nucleotides served as substrates for pyocyanine in the reaction of autooxidation. The kinetic parameters of this reaction and the affinity of NADH dehydrogenase for the substrate were measured. The kinetic data were analysed to show that, under the physiological conditions, pyocyanine could not apparently compete with the respiratory chain for the reducing equivalents and hence directly regulate the level of NAD(P)H in P. aeruginosa cells. In order to keep the oxidising activity at a level necessary for the cells, the latter decreased the content of the reducing equivalents either by synthesizing pyocyanine or owing to the activity of cyanide-resistant oxidase. These processes of releasing the reducing equivalents are in a reciprocal relationship.

[Effect of limitation of bacterial growth with carbon, sulfur and iron on the synthesis of cytochromes, development of cyanide-resistant respiration and super-synthesis of metabolites]. / Vliianie limitirovaniia rosta bakterii uglerodom, seroi i zhelezom na sintez tsitokhromov, razvitie tsianidrezistentnogo dykhaniia i sverkhsintez metabolitov.

The work is concerned with the effect produced by limiting the growth of various bacteria with carbon, sulfur and iron on cytochrome synthesis, development of cyanide-resistant respiration and oversynthesis of metabolites. The cessation of bacterial growth due to the exhaustion of a carbon source was shown to be accompanied with the development of cyanide-resistant respiration though the oversynthesis of metabolites did not occur. If the growth was limited by a sulfur or iron source, the concentration of cytochromes a, b and c fell down as compared with that when the growth was limited by a carbon source, and metabolites were produced and accumulated in the medium. In that case, the respiration of virtually all the bacteria was inhibited by cyanide to a great extent. As was demonstrated for Pseudomonas aeruginosa, the development of cyanide-resistant respiration was inhibited when metabolites accumulated and then the respiration became completely resistant to cyanide as soon as the oversynthesis ceased. Apparently, whatever limits the bacterial growth, the process of oversynthesis inhibits cyanide-resistant oxidase.

On the absence of correlation between cyanide-resistant respiration and cytochrome d content in bacteria.

The regularity of appearance of cyanide-resistant respiration and cytochrome d in various bacteria as well as the relationship between the degree of resistance of respiration to cyanide and cytochrome d content was studied. Bacteria able to synthesize cyanide-resistant respiration were shown to appear during transition of culture to the stationary phase of growth caused by the exhaustion of carbon source. No regulatory of appearance of cytochrome d was observed. There is no correlation between the degree of resistance to cyanide and cytochrome d content. It was concluded that the cyanide-resistant respiration of bacteria and eukaryotic microorganisms may be associated with the functioning of a non-cytochrome nature oxidase.

[Absence of a correlation between the respiratory resistance of bacteria to cyanide and their cytochrome d content]. / Ob otsutstvii korreliarsii mezhdu rezistentnost'iu dykhaniia bakterii k tsianidu i soderzhaniem v nikh tsitokhroma d.

The object of this work was to study whether a correlation existed between the content of cytochrome d in different bacterial cells and the inhibition of their respiration by cyanide. No correlation was found between the concentration of cytochrome d and the degree of resistance to cyanide of the bacterial respiration. Just as the cyanide-resistant respiration of eukaryotic microorganisms, the cyanide-resistant respiration of bacteria was concluded to involve the operation of oxidase whose nature was not cytochromic .

[Cyanide-resistant respiration of bacteria and its relation to metabolite oversynthesis]. / Tsianidrezistentnoe dykhanie bakterii i ego vzaimosviaz' s izbytochnym sintezom metabolitov.

The phenomenon of cyanide-resistant respiration was studied in bacteria as well as its relation to the oversynthesis of metabolites. Cyanide-resistant respiration was shown to be common of aerobic and facultative anaerobic bacteria. The resistance of respiration to cyanide was observed when the growth of a microbial culture was inhibited or ceased entirely, provided the culture lacked the oversynthesis of metabolites. When a culture was capable of metabolites oversynthesis under such conditions, cyanide-resistant respiration (if any) declined. Therefore, there is a competition between metabolites oversynthesis and cyanide-resistant respiration, which is typical of many microorganisms.

Cyanide-resistant respiration in Streptomyces citreofluorescens.

The capacity of Streptomycetes to carry out cyanide-resistant respiration was investigated. With the model strain, Streptomyces citreofluorescens, it was shown that deprivation of glucose followed by transition from exponential to stationary growth was coupled with declining sensitivity of cellular respiration to cyanide ions. Cyanide-resistant oxidase is located within the cytoplasmic membrane. The occurrence of the cyanide-resistant oxidase did not correspond to qualitative changes of cytochrome spectrum. Cytochrome d is involved neither in cyanide-sensitive nor in cyanide-resistant respiratory chain.

[Effect of glutamic acid oversynthesis on the development of cyanide-resistant respiration in the bacterium Corynebacterium glutamicum]. / Vliianie protsessa sverkhsinteza glutaminovoi kisloty na razvitie tsianidrezistentnogo dykhaniia u bakterii corynebacterium glutamicum.

The composition of the respiratory chains of the wild stain Corynebacterium glutamicum and of its mutant differing in their ability for the glutamic acid oversynthesis in a medium with melassa was studied. Under excess of biotine and the parent strain is incapable of acid oversynthesis, while the mutant forms and excretes the acid. Both bacterial strains contain menaquinone and equal sets of cytochromes C550, b556, b563, and a600. The membrane-bound dehydrogenases of the parent strain are represented by NADH-, NADPH- and succinate dehydrogenases. Unlike the parent strain, the mutant membrane preparation does not oxidize NADPH. Both strains do not practically differ in their menaquinone content. The cyanide-resistant oxidase of a non-cytochrome nature appears in the wild strain during its transfer to the stationary growth phase. Induction of glutamic acid oversnythesis by addition of penicilline prevents the formation of the cyanide-resistant oxidase. On the contrary, the mutant transfer to the stationary growth phase is not accompanied by a formation of cyanide-resistant oxidase, which appears only after cessation of glutamic acid oversynthesis. Induction of the cyanide-resistant respiration by addition of cyanide inhibits the acid oversynthesis. Oxidation of substrates by membrane preparations of both bacterial strains in the absence and presence of cyanide is not followed by the hydrogen peroxide formation. It is assumed that there exist competitive interactions between the supersynthesis of glutamic acid and the cyanide-resistant respiration. The possible structure of the respiratory chain of Cor. glutamicum is discussed.

[Membrane potential of Pseudomonas aeruginosa spheroplasts having a cyanide-resistant respiration]. / Membrannyi potentsial sferoplastov Pseudomonas aeruginosa, obladaiushchikh tsianidresistentnym dykhaniem.

The membrane potential (delta psi) of Ps. aeruginosa spheroplasts was measured by a hydrophobic cation--tetraphenylphosphonium (TPP+) under conditions of simultaneous and separate action of the main respiratory chain and cyanide-resistant oxidase. In all cases, oxidation of endogenous substrates by spheroplasts was followed by generation of a membrane potential of the value of 127--156 mv. An addition of 1 mM cyanide did not practically affect the potential value. The discharge of the potential was attained by further addition of dicyclohexylcarbodiimide (DCCD) or valinomycin. The oxidation of the substrates added by the main respiratory chain was accompanied by an additional increase in the potential by 20--30 mv. In this case, an addition of cyanide decreased the potential by the same value. Oxidation of all substrates, except for TMPD+ ascorbate under conditions of functioning of cyanide-resistant oxidase also increased the potential by 15--50 mv. However, in this case cyanide did not practically affect the increase of the potential. The gain in the potential under TMPD + ascorbate oxidation by cyanide-resistant spheroplasts is prevented by cyanide. A question is discussed as to whether the transfer of reducing equivalents directly via cyanide-resistant oxidase is associated with the generation of a membrane potential or its functioning is not connected with the accumulation of energy in the form available for the cell.

[Effect of sulfur, copper, and iron deficiency on the development of cyanide resistant respiration and the cytochrome composition of Pseudomonas aeruginosa]. / Vliianie defitsita sery, medi i zheleza na razvitie tsianidrezistentnogo dykhaniia i tsitokhromnyi sostav u Pseudomonas aeruginosa i Candida lipolytica.

The effect of deficiency in sulfur, copper and iron in the growth medium on cyanide resistant respiration and cytochrome composition was studied in Pseudomonas aeruginosa and Candida lipolytica. It has been shown that: cyanide resistant respiration was observed at the stationary growth phase when the two microorganisms were cultivated in a complete medium; this respiration was detected already at the phase of decelerated growth in the case of copper deficiency; iron deficiency inhibited cyanide resistant respiration in the bacterium but stimulated its appearance in the yeast; sulfur deficiency inhibited the manifestation of cyanide resistant respiration in the both microorganisms; limitation of the bacterial growth with iron resulted in the accumulation of an iron complex (identical to pyoverdin in its spectral characteristics) in the cultural broth; the deficiency of sulfur, copper and iron inhibited the synthesis of all cytochromes in the bacterium; copper deficiency inhibited only the synthesis of a + a3 in the yeast; iron deficiency inhibited the synthesis of all cytochromes in the yeast; sulfur deficiency had virtually no effect on the content of cytochromes in the yeast. A possible nature of cyanide resistant oxidases in these microorganisms is discussed.

[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.

[Conditions for the manifestation of cyanide-resistant respiration in Pseudomonas aeruginosa]. / Usloviia poiavleniia tsianidrezistentnogo dykhaniia u Pseudomonas aeruginosa.

Conditions for manifestation of cyanide-resistant respiration were studied in Pseudomonas aeruginosa. Transition of the culture from the logarithmic to the stationary phase of growth, once the source of carbon (hexadecane, glucose, citrate, succinate) or nitrogen was exhausted, was accompanied with a decrease in the sensitivity of bacterial respiration to cyanide. As soon as the limiting factor was added, the culture started to grow again and its respiration became more sensitive to the inhibitor. Changes in the sensitivity of respiration to cyanide were observed when the bacterial growth was not limited with oxygen, and cyanide was not accumulated in the cultural broth. The manifestation of cyanide-resistant respiration was caused by changes in the physiological state of the bacterium induced by the cessation of growth. The culture formed phenazine pigments while growing on all of the studied substrates. However, no correlation was established between the resistance of respiration to cyanide and the concentration of the pigments in the medium. Therefore, the resistance of respiration of Ps. aeruginosa to cyanide should be attributed to changes in the electron transport chain.