Thioflexithrix psekupsensis gen. nov., sp. nov., a filamentous gliding sulfur bacterium from the family Beggiatoaceae.

A sulfur-oxidizing, filamentous, gliding micro-organism, strain D3T, was isolated from a sulfidic spring in Goryachy Klyuch, Krasnodar, Russia. The cell walls were Gram-negative. The new isolate was a microaerophilic facultative anaerobe and an obligate chemolithoautotroph. The pH range for growth was pH 6.8-7.6, with an optimum at pH 7.2. The temperature range for growth was 10-46 °C, with an optimum at 32 °C. The G+C content of DNA was 42.1 mol%. Phylogenetic analysis of the 16S rRNA gene showed that strain D3T belongs to the family Beggiatoaceae, order Thiotrichales and was distantly related to the genera of the family Beggiatoaceae(86-88 % sequence similarity). The major respiratory quinone was ubiquinone-6. Major fatty acids were C18:1 ω7 (37.6 %), C16 : 0 (34.7 %) and C16: 1 ω7 (27.7 %). On the basis of its physiological properties and the results of phylogenetic analysis, strain D3T is considered to represent a novel species of a new genus, for which the name Thioflexithrix psekupsensis gen. nov., sp. nov. is proposed. The type strain is D3T (=KCTC 62399=UNIQEM U981).

[Neutrophilic lithotrophic iron-oxidizing prokaryotes and their role in the biogeochemical processes of the iron cycle].

Biology of lithotrophic neutrophilic iron-oxidizing prokaryotes and their role in the processes of the biogeochemical cycle of iron are discussed. This group of microorganisms is phylogenetically, taxonomically, and physiologically heterogeneous, comprising three metabolically different groups: aerobes, nitrate-dependent anaerobes, and phototrophs; the latter two groups have been revealed relatively recently. Their taxonomy and metabolism are described. Materials on the structure and functioning of the electron transport chain in the course of Fe(II) oxidation by members of various physiological groups are discussed. Occurrence of iron oxidizers in freshwater and marine ecosystems, thermal springs, areas of hydrothermal activity, and underwater volcanic areas are considered. Molecular genetic techniques were used to determine the structure of iron-oxidizing microbial communities in various natural ecosystems. Analysis of stable isotope fractioning of 56/54Fe in pure cultures and model experiments revealed predominance of biological oxidation over abiotic ones in shallow aquatic habitats and mineral springs, which was especially pronounced under microaerobic conditions at the redox zone boundary. Discovery of anaerobic bacterial Fe(II) oxidation resulted in development of new hypotheses concerning the possible role of microorganisms and the mechanisms of formation of the major iron ore deposits in Precambrian and early Proterozoic epoch. Paleobiological data are presented on the microfossils and specific biomarkers retrieved from ancient ore samples and confirming involvement of anaerobic biogenic processes in their formation.

[Detection and analysis of sulfur metabolism genes in Sphaerotilus natans subsp. sulfidivorans representatives].

The lithotrophic capacity of the betaproteobacteria Sphaerotilus natans subsp. sulfidivorans was confirmed at genetic level: functional genes of sulfur metabolism were detected (aprBA, soxB, and sqr, coding for adenylyl phosphosulfate reductase, thiosulfate-cleaving enzyme, and sulfide:quinone oxidoreductase, respectively), and the expression of aprA and soxB genes was demonstrated. An evolutionary scenario for soxB genes in Sphaerotilus representatives is suggested based on comparative analysis of codon occurrence frequency, DNA base composition (G + C content), and topology of phylogenetic trees. The ancestor bacterium of the Sphaerotilus-Leptothrix group was capable of lithotrophic growth in the presence of reduced sulfur compounds. However, in the course of further evolution, the sulfur metabolism genes, including the soxB gene, were lost by some Sphaerotilus strains. As a result, the lithotrophic Sphaerotilus-Leptothrix group split into two phylogenetic lineages, lithotrophic and organotrophic ones.

Biodiversity and monitoring of colorless filamentous bacteria in sulfide aquatic systems of North Caucasus region.

Bacterial mats in sulfide aquatic systems of North Caucasus are basically composed by the species of genera Thiothrix and Sphaerotilus. Additionally, several non-filamentous sulfur-oxidizing bacteria were isolated from the mats and several minor 16S rRNA phylotypes were found in clone libraries from these mats. The minor components were affiliated with Proteobacteria, Chlorobia, Cyanobacteria and Firmicutes. Even in an individual mat population heterogeneity of Thiothrix spp. was revealed by analysis of 16S rRNA gene and RAPD-PCR. Five Thiothrix isolates were described as new species Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov. In the Thiothrix-Sphaerotilus type of bacterial mat the proportion of dominant organisms might be influenced by sulfide concentration in the spring water. The higher sulfide concentration (more than 10 mg/l) in the spring water is more favorable for the development of bacterial mats with dominant Thiothrix organisms than for Thiothrix-Sphaerotilus type of sulfur mat.

[Ecophysiology of lithotrophic sulfur-oxidizing Sphaerotilus species from sulfide springs in the Northern Caucasus].

Six strains of sulfur-oxidizing bacteria of the known organotrophic species Sphaerotilus natans were isolated from two North Caucasian sulfide springs. Similar to known colorless sulfur bacteria, all the strains accumulated elemental sulfur when grown in media with sulfide. Unlike previously isolated S. natans strains, new isolates had higher temperature growth optima (33-37 degrees C) and variable metabolism. All the strains were capable of organotrophic, lithoheterotrophic, and mixotrophic growth with sulfur compounds as electron donors for energy metabolism. Variable metabolism of new Sphaerotilus isolates is a highly important adaptation mechanism which facilitates extension of their geographic range and supports their mass development in new habitats, e.g. sulfide springs. Within the cluster of new isolates, the physiological heterogeneity was shown to result from the inducible nature of the enzymes of oxidative sulfur metabolism and from their resistance to aerobic cultivation.

[Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide stream in the North Caucasus].

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide stream (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Spahaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism's population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or "threads" of natural sulfide springs.

[Lithotrophic microorganisms of the oxidative cycles of sulfur and iron].

The review deals with sulfur bacteria (the first chemolithotrophs ever studied) and with the acidophilic bacteria of sulfur and iron cycles which were investigated as a result of Winogradsky's discovery. The diversity of these organisms and the factors and mechanism of its origin are emphasized; their metabolic functions and nutritional regulation are discussed.

[Two new species of microaerophilic sulfur spirilla, Spirillum winogradskii sp. nov. and Spirillum kriegii sp. nov].

New microaerophilic sulfur-oxidizing spirilla were isolated from hydrogen sulfide sludge of wastewater treatment plants. Strains D-427 and D-430 have spiral cells that are highly motile due to bipolar flagellum bundles covered with mucous sheaths. Under a phase-contrast microscope, these bundles are visible as single polar flagella. Spheroplasts are formed in the stationary growth phase. Both strains are obligate organotrophs able to oxidize a number of reduced sulfur compounds. The oxidation of sulfide and polysulfide leads to the formation of intracellular globules of elemental sulfur; thiosulfate oxidation results in tetrathionate accumulation in the medium. The cells are unable to utilize reduced sulfur compounds in the energy metabolism; their oxidation is caused by a chemical interaction with H2O2 and O2, synthesized in the electron transport chain. Both strains are obligate microaerophiles with an optimal oxygen concentration in the gas phase of 2 and 0.8% for strains D-427 and D-430, respectively. The strains utilize a limited number of organic acids as growth substrates, mainly tricarboxylic-acid-cycle intermediates. The DNA G+C content is 38.0 mol % (T(m)) for strain D-427 and 38.9 mol % for strain D-430. Phylogenetic analysis, based on the comparison of 16S rRNA gene sequences, revealed that the new isolates of sulfur spirilla are the most closely related to Spirillum volutans, the type species of the genus (97.4% similarity). They were assigned to the genus Spirillum within the class Beta-proteobacteria as two new species, S. winogradskii sp. nov. (D-427T = DSM 12756T) and S. kriegii sp. nov. (B-430T = BKM B-2372T). The emended description of the genus Spirillum is provided.

[Regulation of metabolic and electron transport pathways in the freshwater bacterium Beggiatoa leptomitiformis D-402].

The biomass yield of freshwater filamentous sulfur bacteria of the genus Beggiatoa, when grown lithoheterotrophically or mixotrophically, has been shown to increase 2 to 2.5 times under microaerobic conditions (0.12 mg/l oxygen) as compared to aerobic conditions (9 mg/l oxygen). The activity of the glyoxylate cycle key enzymes have been found to increase two to three times under microaerobic conditions (at an O2 concentration of 2 mg/l), and the activities of the sulfur metabolism enzymes increased three to five times (at an O2 concentration of 0.1-0.5 mg/l). It has also been found that, under microaerobic conditions, thiosulfate was almost completely oxidized to sulfate by the bacteria, without accumulation of intermediate metabolites. At the same time, a 2- to 15-fold decrease in the activities of the tricarboxylic acid cycle enzymes involved in the reduction of NAD and FAD was observed. Reorganization of the respiratory chain after changes in aeration and type of nutrition was also observed. It has been found that, in cells grown heterotrophically, the terminal part of the respiratory chain contained an aa3-type oxidase, whereas, during mixotrophic, lithoheterotrophic, and autotrophic growth, aa3-type oxidase synthesis was inhibited, and the synthesis of a cbb3-type oxidase, which is induced under microaerobic conditions, was activated. The gene of the catalytic subunit CcoN of the cbb3-type oxidase was sequenced and proved to be highly homologous to the corresponding genes of other proteobacteria.

[The functional role of reduced inorganic sulfur compounds in the metabolism of the microaerophilic bacterium Spirillum winogradskii].

Oxidation of reduced sulfur compounds by microaerophilic sulfur bacterium Spirillum winogradskii was found to occur only concomitantly with consumption of an organic substrate and was not linked to their utilization as electron donors in energy metabolism. No enzymes of dissimilatory sulfur metabolism were found in the cells of the sulfur bacterium oxidizing thiosulfate to tetrathionate; oxidation of thiosulfate and sulfide was caused by their reaction with reactive oxygen species (ROS), mostly H2O2 produced in the course of aerobic growth. Decreased lytic effect of ROS in the presence of thiosulfate resulted in a twofold increase in the cell yield under aerobic conditions and more efficient substrate utilization. The latter effect was caused by decreased expense of energy for the biosynthesis of oxygen-protecting polysaccharides. The stimulatory effect of thiosulfate on the growth processes was due to the activation of a number of TCA cycle enzymes producing the intermediates for constructive metabolism, especially of the NADP-dependent malic enzyme. As a result of thiosulfate-induced synthesis of SH-containing cell components, the integral antioxidative activity increased 1.5-fold.

[Microbiological processes in the Lost City vent field, mid-Atlantic ridge].

Microbiological and biogeochemical measurements showed that the intensities of CO2 assimilation, methane oxidation, and sulfate reduction in the Lost City vent field (30 degrees N) reach 3.8 microg C/(1 day), 0.06 microg C/(1 day), and 117 microg S/(1 day), respectively. On the surface of the carbonate structures occurring in this field, two varieties of bacterial mats were found. The first variety, which is specific to the Lost City alkaline vent field, represents jelly bacterial mats dominated by slime-producing bacteria of several morphotypes. This mat variety also contains chemolithotrophic and heterotrophic microorganisms, either microaerobic or anaerobic. The intensities of CO2 assimilation, methane oxidation, and sulfate reduction in this variety reach 747 microg C/(dm3 day), 0.02 microg C/(dm3 day), and 28,000 microg S/(dm3 day), respectively. Bacterial mats of the second variety are formed by nonpigmented filamentous sulfur bacteria, which are close morphologically to Thiothrix. The intensities of CO2 assimilation, methane oxidation, and sulfate reduction in the second mat variety reach 8.2 microg C/(dm3 day), 5.8 microg C/(dm3 day), and 17,000 microg S/(dm3 day), respectively. These data suggest the existence of subsurface microflora in the Lost City vent field.

[Role of malate dehydrogenase isoforms in the regulation of anabolic and catabolic processes in the colorless sulfur bacterium Beggiatoa leptomitiformis D-402]. / Rol' izoform malatdegidrogenazy v reguliatsii anabolicheskikh i katabolicheskikh protsessov u bestsvetnykh serobakterii Beggiatoa leptomitiformis D-402.

The functional role of tetrameric and dimeric isoforms of malate dehydrogenase in the carbon metabolism of the colorless sulfur bacterium Beggiatoa leptomitiformis, strain D-402, was studied. This strain can grow both lithotrophically and organotrophically. By using the inhibition analysis, the tetrameric isoenzyme was shown to operate in the glyoxylate cycle and the dimeric one was found to be involved in the TCA cycle. The dynamics of the dimeric isoenzyme conversion to the tetrameric isoform was found to be determined by the rate of thiosulfate oxidation. The regulation of the carbon metabolism in Beggiatoa leptomitiformis is supposed to be accomplished by means of structural and functional changes in the protein molecule of malate dehydrogenase.

[The role of oxygen in the regulation of the metabolism of aerotolerant spirochetes, a major component of "Thiodendron" bacterial sulfur mats]. / Rol' kisloroda v reguliatsii metabolizma aérotolerantnykh spirokhet--osnovnogo komponenta bakterial'nykh sernykh matov "Thiodendron".

Two spirochete strains isolated earlier from "Thiodendron" bacterial sulfur mats grew better under microaerobic (0.3-0.5 mg O2/l) than under anaerobic conditions. The microaerobic growth of these strains was accompanied by a twofold increase in the cell yield and the efficiency of glucose utilization, despite an amount of ATP (and hence glucose) was spent in this case for the synthesis of exopolysaccharides. Glucose metabolism under microaerobic conditions gave rise to more oxidized products (acetate and carbon dioxide) than under anaerobic conditions (formate, ethanol, pyruvate, and hydrogen). The paper considers two putative mechanisms implemented by aerotolerant spirochetes: adaptive (the use of a more efficient pathway of glucose catabolism) and protective (an enhanced synthesis of exopolysaccharides and the reduction of hydrogen peroxide by the reduced sulfur compounds thiosulfate and sulfide, yielding elemental sulfur). The formation of "Thiodendron" bacterial sulfur mats in saltwater environments is also discussed.

[Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium Spirillum winogradskii]. / Okislitel'nyi stress i sistemy antioksidantnoi zashchity kletok u mikroaérofil'nykh bakterii Spirillum winogradskii.

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O2) aerobic conditions (21% O2 of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes protecting from toxic forms of O2--peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)--massive lysis of cells starts in the mid-exponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 micrograms/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H2O2, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H2O2 accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H2O2, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.

[Microorganisms in heat supply lines and internal corrosion of steel pipes]. / Mikroorganizmy v teplovykh setiakh i vnutrenniaia korroziia stal'nykh truboprovodov.

In laboratory experiments with batch cultures of thermophilic microorganisms isolated from urban heat supply systems, the growth of sulfate-reducing, iron-oxidizing, and iron-reducing bacteria was found to accelerate the corrosion rate of the steel-3 plates used in the pipelines. In the absence of bacteria and dissolved oxygen, minimal, corrosion was determined. The aforementioned microorganisms, as well as sulfur-oxidizing bacteria, were found to be widespread in water and corrosion deposits in low-alloy steel pipelines (both delivery and return) of the Moscow heat networks, as well as in the corrosion deposits on the steel-3 plates in a testing unit supplied with the network water. The microorganisms were found in samples with water pH ranging from 8.1 to 9.6 and a temperature lower than 90 degrees C. Magnetite, lepidocrocite, goethite, X-ray amorphous ferric oxide were the corrosion products identified on the steel-3 plates, as well as siderite, aragonite, and S0. The effect of microbiological processes on the rate of electrochemical corrosion was evaluated from the accumulation of corrosion deposits and from variation in total and local corrosion of the steel plates in a testing unit.