[Poly- ß -Hydroxybutyrate Accumulation by Rhodobacter sphaeroides Phase Variants].

The Rand M variants of a purple photosynthetic bacterium Rhodobacter sphaeroides 2R grown on a medium with acetate accumulate poly- ß-hydroxybutyrate (PHB). Accumulation of this polymer occurs in the cells grown either anaerobically on the light or aerobically in the dark. On the medium with C/N imbalance (C/N = 4), PHB content during the stationary growth phase under aerobic conditions in the dark was 40 and 70% of the dry biomass of the R and M variant, respectively. The Rba. sphaeroides M variant is therefore a promising culture for large-scale PHB production. Investigation of activity of the TCA cycle enzymes revealed that decreased activity of citrate synthase, the key enzyme for acetate involvement in the reactions of the tricarboxylic acids cycle, was primarily responsible for enhanced PHB synthesis by Rba. sphaeroides. Moreover, the Rba. sphaeroides M variant grown under aerobic conditions in the dark exhibited considerably lower activity of NADH oxidase, which participates in the oxidation of reduced NADH produced in the TCA cycle during acetate oxidation. The combination of these two factors increases the possibilities for acetate assimilation via an alternative mechanism of PHB synthesis.

[metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides].

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobactersphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2-3 times lower in the cells of the M. variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa3, acting as. cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient al the cytochrome aa3 level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.

[Comparative study of metabolism of the purple photosynthetic bacteria grown in the light and in the dark under anaerobic and aerobic conditions].

For three species of anoxygenic phototrophic alphaproteobacteria differing in their reaction to oxygen and light, physiological characteristics (capacity for acetate assimilation, activity of the tricarboxylic acid (TCA) cycle enzymes, respiration, and the properties of the oxidase systems) were studied. Nonsulfur purple bacteria Rhodobacter sphaeroides, Rhodobaca bogoriensis, and aerobic anoxygenic phototrophic bacteria Roseinatronobacter thiooxidans were the subjects of investigation. All these organisms were able to grow under aerobic conditions in the dark using the respiratory system with cytochrome aa3 as the terminal oxidase. They differed, however, in their capacity for growth in the light, bacteriochlorophyll synthesis, and regulation of activity of the TCA cycle enzymes. Oxygen suppressed bacteriochlorophyll synthesis by Rha. sphaeroides and Rbc. bogoriensis both in the dark and in the light. Bacteriochlorophyll synthesis in Rna. thiooxidans occurred only in the dark and was suppressed by light. The results on acetate assimilation by the studied strains reflected the degree of their adaptation to aerobic growth in the dark. Acetate assimilation by light-grown Rha. sphaeroides was significantly higher than by the dark-grown ones. Unlike Rha. sphaeroides, acetate assimilation by Rbc. bogoriensis in the light under anaerobic and aerobic conditions was much less dependent on the growth conditions. Aerobic acetate assimilation by all studied bacteria was promoted by light. In Rha. sphaeroides, activity of the TCA cycle enzymes increased significantly in the cells grown aerobically in the dark. In Rbc. bogoriensis, activity of most of the TCA cycle enzymes under aerobic conditions either decreased or remained unchanged. Our results confirm the origin of modern chemoorganotrophs from anoxygenic phototrophic bacteria. The evolution from anoxygenic photoorganotrophs to aerobic chemoorganotrophs included several stages: nonsulfur purple bacteria --> nonsulfur purple bacteria similar to Rbc. bogoriensis --> aerobic anoxygenic phototrophs --> chemoorganotrophs.

[Factors controlling the biosynthesis of chlorosome antenna bacteriochlorophylls in green filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae].

We determined the concentrations of bacteriochlorophylls (BChl) in the light-harvesting antennae of Oscillochloris trichoides (of the family Oscillochloridaceae belonging to green filamentous mesophilic bacteria) cultivated either with gabaculine, an inhibitor of the C-5 pathway of BChl biosynthesis in a number of bacteria, or at various illumination intensities. We determined the BChl c: BChl a molar ratios in intact cells, in chlorosome-membrane complexes, and in isolated chlorosomes. We revealed that BChl c synthesis in Osc. trichoides was more gabaculine-sensitive than BChl a synthesis. Accordingly, an increase in gabaculine concentrations in the medium resulted in a decrease in the BChl c: BChl a ratio in the tested samples. We suggest that BChl synthesis in Osc. trichoides proceeds via the C-5 pathway, similar to representatives of other families of green bacteria (Chlorobium limicola and Chloroflexus aurantiacus). We demonstrated that the BChl c: BChl a ratio in the chlorosomes varied from 55 : 1 to 110 : 1, depending on light intensity. This ratio is, therefore, closer to that of Chlorobiaceae, and it significantly exceeds the BChl c: BChl a ratio in Chloroflexaceae.

[Phylogeny of anoxygenic filamentous phototrophic bacteria of the family Oscillochloridaceae as inferred from comparative analyses of the rrs, cbbL, and nifH genes].

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose- 1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the "red-like" form I RuBPC were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their identification as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.

[Carbon metabolism of filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae].

The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO2 assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains, CO2 fixation occurs via the operation of the Calvin cycle. Phosphoenolpyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+ CO2) --> oxalacetate --> malate --> fumarate --> succinate --> succinyl-CoA (+ CO2) --> 2-oxoglutarate (+ CO2) --> isocitrate. Acetate, utilized as and additional carbon source, can be carboxylated to pyruvate by pyruvate synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.

[A comparative study of the fluorescence properties of the chlorosomal antenna of the green bacterium from the family Oscillochloridaceae and the members from two other families of green bacteria].

The fluorescence properties of bacteriochlorophylls (BChl) of the chlorosomal light-harvesting antenna of Oscillochloris trichoides (strain DG-6) from a new family of green filamentous bacteria Oscillochloridaceae were investigated in comparison with green bacteria from two other families. A strong dependence of the fluorescence intensity of chlorosomal bacteriochlorophyll c of Osc. trichoides on the redox potential of medium was found, which previously was observed only in green sulfur bacteria. The presence of BChl a in chlorosomes did not appear in their absorption spectra but was visualized by fluorescence spectroscopy at 77 K. From the comparative analysis of fluorescence spectral data for the chlorosomal light-harvesting antenna of Osc. trichoides and similar spectral data for green bacteria from two other families, it was concluded that, in some fluorescence spectral features (spectral position of bacteriochlorophyll c/a fluorescence bands; shape and full width at half maximum fluorescence band of chlorosomal bacteriochlorophyll c; the Stokes shift value of bacteriochlorophyll c band; a high molar ratio of bacteriochlorophyll c : bacteriochlorophyll a in chlorosomes that makes the bacteriochlorophyll a fluorescence band unresolved at room temperature; and highly redox-dependent fluorescence intensity of chlorosomal bacteriochlorophyll c), Osc. trichoides chlorosomes are close to the chlorosomal antenna of Chlorobiaceae species.

[A study of the content of pigments in the light-harvesting antenna of the green bacterium from the new family Oscillochloridaceae]. / Izuchenie sostava pigmentov svetosobiraiushchei antenny zelenykh bakterii novogo semeistva Oscillochloridaceae.

The properties of the light-harvesting superantenna of the photosynthesizing bacteria from the new family of green filamentous bacteria Oscillochloridaceae were investigated by optical spectroscopy. The antenna of Oscillochloris trichoides consists of peripheral chlorosomal and membrane subantennas. A method of isolation of Osc. trichoides chlorosomal antenna was developed using the chaothropic agent sodium thiocyanate, which simultaneously acts to stabilize chlorosomal activity. An analysis of the second derivatives of the absorption spectra of isolated chlorosomes and their acetone-methanol extracts suggested that BChl c was a predominant light-harvesting pigment in Osc. trichoides chlorosomes. Besides, it was found that, in addition to the BChl c-antenna, chlorosomes contain a minor BChl a-antenna. It was shown that the membrane BChl a-subantenna is a light-harvesting complex with absorption maxima in the near infrared region at 805 and 860 nm. Analysis of the spectral data obtained suggested that the Osc. trichoides chlorosomal antenna resembles those from Chlorobiaceae species, whereas the membrane B805-860 BChl a antenna of Osc. trichoides is close to the membrane B808-866 BChl a antenna of Chloroflexaceae species.

[Photometric assay for methanol in the presence of ethanol]. / Fotometricheskii metod opredeleniia metanola v prisutstvii étanola.

A new enzymatic photometric assay for determination of methanol and ethanol in solutions containing both alcohols is described. The assay allows detecting methanol in the concentration range of tens ppm of in the presence of tens per cent of ethanol. The lower determination threshold for methanol is at least 0.002% in the presence of 45% ethanol, with a coefficient of variation of 0.02-0.05. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements.

[Photometric method for determining ethanol]. / Fotometricheskii metod opredeleniia étanola.

A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.

Proposal of Oscillochloridaceae fam. nov. on the basis of a phylogenetic analysis of the filamentous anoxygenic phototrophic bacteria, and emended description of Oscillochloris and Oscillochloris trichoides in comparison with further new isolates.

The nucleotide sequences of the genes of 16S rRNAs were determined for the type strain Oscillochloris trichoides DG-6T and three new strains of Oscillochloris-like mesophilic filamentous green bacteria. Two major clusters have been found within the family Chloroflexaceae by phylogenetic-analysis: one cluster includes thermophilic species of Chloroflexus and the second includes mesophilic strains of Oscillochloris. The degree of relatedness of these clusters was below an intergeneric level, having only 82.5-86.5% of 16S rDNA sequence similarity. These phylogenetic data correlate well with the significant physiological, biochemical and chemotaxonomical differences between members of both groups. Therefore, the Oscillochloris and Chloroflexus clusters should be considered as two separate families. The description of the new family, Oscillochloridaceae fam. nov., and emended descriptions of the genus Oscillochloris and the species Oscillochloris trichoides are presented.

Oscillochloris trichoides neotype strain DG-6.

The new strain of filamentous green bacterium strain DG-6 was isolated in pure culture from the spring of Caucuses. The study of this bacterium allows to suggest that it is a member of the familyChloroflexaceae and may be considered asOscillochloris trichoides neotype strain. The description of this green bacterium is given.

[Thiosulfate oxidation by nonsulfur purple bacteria]. / Okislenie tiosul'fata purpurnymi nesernymi bakteriiami.

The capability to oxidize thiosulfate was studied in 11 cultures of purple bacteria belonging to Rhodomicrobium vannielii, Rhodopseudmonas viridis, Rh. sphaeroides, Rh. capsulata, and Rhodospirillum rubrum. All the bacteria oxidized thiosulfate under aerobic conditions in the dark. The strains 2R, 8259, A1, A2 and D1 of Rh. sphaeroides oxidized thiosulfate under anaerobic conditions in the light, and the process was coupled with carbon dioxide fixation. All the strains contained thiosulfate reductase, and the majority of them possessed also the activity of thiosulfate oxidase and sulfite oxidase.

[Morphological differentiation in non-sulfur purple bacteria]. / Morfologicheskaia differentsiatsiia u nesernykh purpurnykh bakterii

Cell differentiation in non-sulphur purple bacteria is complicated, as compared to binary fission, during bud formation with production of hyphae and special resting cells of the exospore type, and can be demonstrated in the folowing series of microorganisms: Rhodopseudomonas sulfidophila leads to Rh. capsulata leads to Rh. acidophila leads to Rh. viridis and Rh. palustris leads to Rh. sulfoviridis leads to Rhodom. vannielii. Contrary to phototrophic bacteria multiplying by division, a distinct sequence in appearance of various morphological forms was found in bud-forming bacteria. Formation of specialized cells is regulated by the substrate concentration, accumulation of metabolites, and the population density. Mobile cells are not formed at all or partly, resting cells are formed sometimes at a higher rate, and vegetative cells contain reserve substances in all bud-forming phototrophic bacteria if conditions of the environment are not optimal.

[Dark metabolism of Rhodospeudomonas sulfidophila]. / Temnovoi metabolizm Rhodopseudomonas sulfidophila

A new strain of phototrophic purple bacterium Rhodopseudomonas sulfidophila RP-6 has been isolated from samples of the salty meromictic lake Repnoye. The bacterium utilizes thiosulphate yielding sulphates and assimilates molecular hydrogen in the darkness under aerobic conditions. Thiosulphate stimulates fixation of 14C-bicarbonate by the cells both in the light and darkness. Organic compounds are necessary for growth of the bacterium in the darkness. R. sulphidophila RP-6, R. palustus, and Rhodomicrobium vanniellii CO-1 do not grow in the presence of carbon monoxide neither in the light or in the darkness.

[Characteristics of a new species of budding purple bacteria containing bacteriochlorophyll b]. / Kharakteristika novogo vida pochkuiushchikhsia purpurnykh bakterii, soderzhashchikh bakteriokhlorofill b

A culture of phototrophic purple bacteria containing bacteriochlorophyll b has been isolated. The bacterium forms a sessile bud and, along with producing motile swarmer cells, it produces immobile oval cells surrounded with a slime capsule. It grows on media with simple organic substances in the presence of yeast extract or vitamins, under anaerobic conditions in the light or under microaerophilic conditions in the darkness. Besides organic substances, the culture assimilates hydrogen sulphide and thiosulphate as electron donors; it is incapable of assimilative sulphate reduction. In the light, the bacterium oxidizes thiosulphate to sulphates, without accumulation of molecular sulphur. The bacterium is classed as a new species of the genus Rhodopseudomonas--Rhodopseudomonas sulfoviridis nov. sp.