[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.

[Use of genes of carbon metabolism enzymes as molecular markers of Chlorobi Phylum representatives].

This work examined the feasibility of using certain genes of carbon metabolism enzymes as molecular markers adequate for studying phylogeny and ecology of green sulfur bacteria (GSB) of the Chlorobi phylum. Primers designed to amplify the genes of ATP citrate lyase (aclB) and citrate synthase (gltA) revealed the respective genes in the genomes of all of the newly studied GSB strains. The phylogenetic trees constructed based on nucleotide sequences of these genes and amino acid sequences of the conceptually translated proteins were on the whole congruent with the 16S rRNA gene tree, with the single exception of GltA of Chloroherpeton thalassium, which formed a separate branch beyond the cluster comprised by other representatives of the Chlorobi phylum. Thus, the aclB genes but not gltA genes proved to be suitable for the design of primers specific to all Chlorobi representatives. Therefore, it was the aclB gene that was further used asa molecular marker to detect GSB in enrichment cultures and environmental samples. AclB phylotypes of GSB were revealed in all of the samples studied, with the exception of environmental samples from soda lakes. The identification of the revealed phylotypes was in agreement with the identification based on the FMO protein gene (fmo), is a well-known Chlorobi-specific molecular marker.

[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.

[Enzymes of the citramalate cycle in Rhodospirillum rubrum].

Rhodospirillum rubrum is among the bacteria that can assimilate acetate in the absence of isocitrate lyase, the key enzyme of glyoxylate shunt. Previously we have suggested the functioning of a new anaplerotic cycle of acetate assimilation in this bacterium: citramalate cycle, where acetyl-CoA is oxidized to glyoxylate. This work has demonstrated the presence of all the key enzymes of this cycle in R. rubrum extracts: citramalate synthase catalyzing condensation of acetyl-CoA and pyruvate with the formation of citramalate, mesaconase forming mesaconate from L-citramalate, and the enzymes catalyzing transformation of propyonyl-CoA + glyoxylate <--> 3-methylmalyl-CoA <--> mesaconyl-CoA. At the same time, R. rubrum synthesizes crotonyl-CoA carboxylase/reductase, which is the key enzyme of ethylmalonyl-CoA pathway discovered recently in Rhodobacter sphaeroides. Physiological differences between the citramalate cycle and the ethylmalonyl-CoA pathway are discussed.

[Transport systems for carbonate in the extremely natronophilic cyanobacterium Euhalothece sp].

The effect of carbonate concentration, pH of the medium, and illumination intensity on the major physiological characteristics (growth rate and the intensities of CO2 assimilation and oxygen photoproduction) of the natronophilic cyanobacterium Euhalothece sp. Z-M001 have been studied. It was established that the investigated microorganism has at least two transport systems (TS) for CO2, which differ in both the pH optimum and substrate affinity: TS I has a pH, 9.4-9.5 and a K(S) 0.5 of 13-17 mM, whereas TS II has a pH(opt) 9.9-10.2 and a K(S) 0.5 of 600-800 mM. The substrate affinity of these transport systems is several orders of magnitude lower than the substrate affinity of the transport systems of freshwater cyanobacteria. It is suggested that they are unique for extremely alkaliphilic cyanobacteria and reflect their adaptation to the seasonal cycles of the lake hydrochemistry.

[Chlorobaculum macestae sp. nov., a new green sulfur bacterium].

The investigated green sulfur bacterium, strain M, was isolated from a sulfidic spring on the Black Sea Coast of the Caucasus. The cells of strain M are straight or curved rods 0.6-0.9 x 1.8-4.2 microm in size. According to the cell wall structure, the bacteria are gram-negative. Chlorosomes are located along the cell periphery. Strain M is an obligate anaerobe capable of photoautotrophic growth on sulfide, thiosulfate, and H2. It utilizes ammonium, urea, casein hydrolysate, and N2 as nitrogen sources and sulfide, thiosulfate, and elemental sulfur as sulfur sources. Bacteriochlorophyll c and the carotenoid chlorobactene are the main pigments. The optimal growth temperature is 25-28 degrees C; the optimal pH is 6.8. The strain does not require NaCl. Vitamin B12 stimulates growth. The content of the G+C base pairs in the DNA of strain M is 58.3 mol %. In the phylogenetic tree constructed on the basis of analysis of nucleotide sequences of 16S rRNA genes, strain M forms a separate branch, which occupies an intermediate position between the phylogenetic cluster containing representatives of the genus Chlorobaculum (94.9-96.8%) and the cluster containing species of the genus Chlorobium (94.1-96.5%). According to the results of analysis of the amino acid sequence corresponding to the fmo gene, strain M represents a branch which, unlike that in the "ribosomal" tree, falls into the cluster of the genus Chlorobaculum (95.8-97.2%). Phylogenetic analysis of the amino acid sequence corresponding to the nifH gene placed species of the genera Chlorobaculum and Chlorobium into a single cluster, whereas strain M formed a separate branch. The results obtained allow us to describe strain M as a new species of the genus Chlorobaculum. Chlorobaculum macestae sp. nov.

[Ecophysiology and polymorphism of the unicellular extremely natronophilic cyanobacterium Euhalothece sp. Z-M001 from Lake Magadi].

Strain Z-M001 of a unicellular cyanobacterium, assigned by analysis of the 16S rRNA gene sequence to the phylogenetic group of the generic level Euhalothece, was isolated from soda Lake Magadi. It was shown that strain Z-M001, unlike all other known cultured and uncultured organisms of the Euhalothece group, is extremely natronophilic, and it was named accordingly "Euhalothece natronophila". In its ecophysiological characteristics, it is comparable to extremely alkaliphilic organotrophic natronobacteria, which is essential for soda ecosystems, because cyanobacteria belong to primary producers. "E. natronophila" exhibits considerable morphological variability depending on the concentration of carbonates in the medium. The polymorphism of "E. natronophila" is primarily connected to limitation by utilizable forms of carbon.

[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.

[The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: enzymes of the citramalate cycle in Rhodobacter sphaeroides].

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shortcut, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shortcut, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl-CoA and occurs via the following reaction sequence: propionyl-CoA (+ CO2) --> methylmalonyl-CoA --> succinyl-CoA --> succinate --> fumarate --> malate --> oxalacetate (- CO2) --> phosphoenolpyruvate --> pyruvate. The independence of the cell growth and the acetate assimilation of CO2 is due to the accumulation of CO2/HCO3- (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.

[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.

[The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells].

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.

[An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups]. / Sistema oligonukleotidnykh praimerov dlia amplifikatsii genov ribulozo-1,5-bisfosfatkarboksilazy/oksigenazy u bakterii razlichnykh taksonomicheskikh grupp.

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.

[Participation of inorganic phosphates as electron donors in the primary reactions of photosynthesis in Rhodobacter sphaeroides]. / Issledovanie uchastiia neorganicheskogo fosfata kak donora élektronov v pervichnykh reaktsiiakh fotosinteza u Rhodobacter sphaeroides.

The formation of ATP during photophosphorylation in chromatophores from purple nonsulfur bacterium Rhodobacter sphaeroides in the presence of phenazine methosulfate and without exogenous electron carriers under constant illumination and by the action of single light flashes was studied. It was shown that the photoinduced transport of electrons to the exogenous electron acceptor depends on phosphate. It was assumed that phosphate ions are electron donors in the reaction center P870; by the action of light, P870 converts the phosphate ion HPO4(2-) into anion radical HPO4-.. In the difference EPR spectra "light minus darkness" at 77 K, an asymmetrical doublet signal with a weak low-field line was observed. The signal had a g-tensor of about 2.014 and a hyperfine coupling constant of about 2.5 mT and belongs probably to the phosphate anion radical.

[A comparative study of the isocitrate dehydrogenases of Chlorobium limicola forma Chlorobium thiosulfatophilum and Rhodopseudomonas palustris]. / Sravnitel'noe issledovanie uzotsitratdegidrogenazy u Chlorobium limicola forma thiosulfatophilum i Rhodopseudomonas palustris.

The carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle in the obligate photolithotroph Chlorobium limicola forma thiosulfatophilum and the oxidation of isocitrate in the tricarboxylic acid cycle in the photoheterotroph Rhodopseudomonas palustris are catalyzed by isocitrate dehydrogenases. A comparative study of these enzymes isolated from the two bacteria showed that they virtually do not differ in the enzymatic and kinetic properties.

[Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions]. / Issledovanie temnovogo metabolizma atsetata u kletok Rhodospirillum rubrum, vyrosshikh v fotogeterotrofnykh usloviiakh.

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.

[On the mechanism of autotrophic fixation of carbone dioxide by Chloroflexus aurantiacus]. / O mekhanizme avtotrofnoi fiksatsii uglekisloty u Chloroflexus aurantiacus.

The activity of two carboxylating enzymes was studied in the green filamentous bacterium Chloroflexus aurantiacus. The carboxylation reaction involving pyruvate synthase was optimized using 14CO2 and cell extracts. Pyruvate synthase was shown to be absent from cells of Cfl. aurantiacus OK-70 and present (in a quantity sufficient to account for autotrophic growth) in cells of Cfl. aurantiacus B-3. Differences in the levels of acetyl CoA carboxylase activity were revealed between cells of the strains studied grown under different conditions. The data obtained confirm the operation of different mechanisms of autotrophic CO2 assimilation in Cfl. aurantiacus B-3 and Cfl. aurantiacus OK-70: in the former organism, it is the reductive cycle of dicarboxylic acids, and in the latter one, it is the 3-hydroxy-propionate cycle.