G. A. Dubinina

Azospirillum thiophilum sp. nov., a diazotrophic bacterium isolated from a sulfide spring.

A novel nitrogen-fixing strain, designated BV-S(T), was isolated from a sulfur bacterial mat collected from a sulfide spring of the Stavropol Krai, North Caucasus, Russia. Strain BV-S(T) grew optimally at pH 7.5 and 37°C. According to the results of phylogenetic analysis, strain BV-S(T) belonged to the genus Azospirillum within the family Rhodospirillaceae of the class Alphaproteobacteria. Within the genus Azospirillum, strain BV-S(T) was most closely related to Azospirillum doebereinerae GSF71(T), A. picis IMMIB TAR-3(T) and A. lipoferum ATCC 29707(T) (97.7, 97.7 and 97.4 % 16S rRNA gene sequence similarity, respectively). DNA-DNA relatedness between strain BV-S(T) and A. doebereinerae DSM 13131(T), A. picis DSM 19922(T) and A. lipoferum ATCC 29707(T) was 38, 55 and 42 %, respectively. Similarities between nifH sequences of strain BV-S(T) and members of the genus Azospirillum ranged from 94.5 to 96.8 %. Chemotaxonomic characteristics (quinone Q-10, major fatty acid C(18 : 1)ω7c and G+C content 67 mol%) were similar to those of members of the genus Azospirillum. In contrast to known Azospirillum species, strain BV-S(T) was capable of mixotrophic growth under microaerobic conditions with simultaneous utilization of organic substrates and thiosulfate as electron donors for energy conservation. Oxidation of sulfide was accompanied by deposits of sulfur globules within the cells. Based on these observations, strain BV-S(T) is considered as a representative of a novel species of the genus Azospirillum, for which the name Azospirillum thiophilum sp. nov. is proposed. The type strain is BV-S(T) (=DSM 21654(T) =VKM B-2513(T)).

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.

Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov., gammaproteobacteria isolated from sulfide springs

Five strains of filamentous, sulfur-oxidizing bacteria were isolated from sulfur mats of different sulfide springs from various regions of the Northern Caucasus, Russia. A phylogenetic analysis based on 16S rRNA gene sequence comparison showed that all of the isolates are affiliated with the filamentous, colourless, sulfur-oxidizing bacteria of the genus Thiothrix within the Gammaproteobacteria and are closely related to Thiothrix fructosivorans. All strains are capable of growing heterotrophically, lithoautotrophically with thiosulfate or sulfide as the sole energy source and mixotrophically. Strains G1(T), G2, P and K2 are able to fix molecular nitrogen, but strain BL(T) is not. Randomly amplified polymorphic DNA (RAPD)-PCR analysis was used to assess the level of genetic relationships among the Thiothrix isolates. The Nei and Li similarity index revealed high genetic similarity among strains G1(T), G2, P and K2 (above 75 %), indicating that they are closely related. In combination with physiological and morphological data, strains G1(T), G2, P and K2 can be considered as members of the same species. The lowest genetic similarity (approx. 20 %) was reached between strain BL(T) and the other isolated Thiothrix strains. Strains BL(T) and G1(T) shared 35 % DNA-DNA relatedness and showed 51 and 53 % relatedness, respectively, to Thiothrix fructosivorans ATCC 49749. On the basis of this polyphasic analysis, strains G1(T), G2, P and K2 represent a novel species within the genus Thiothrix, for which the name Thiothrix caldifontis sp. nov. is proposed, with strain G1(T) (=DSM 21228(T) =VKM B-2520(T)) as the type strain. In addition, strain BL(T) represents a second novel species, Thiothrix lacustris sp. nov., with strain BL(T) (=DSM 21227(T) =VKM B-2521(T)) as the type strain.

Lithoautotrophic Growth of the Freshwater Colorless Sulfur Bacterium Beggiatoa “leptomitiformis” D-402

The freshwater colorless sulfur bacterium Beggiatoa “leptomitiformis”D-402 was shown to be capable of lithoautotrophic growth in a batch culture under microoxic conditions at O2concentrations in the medium of no higher than 0.5 mg/l. The cell yield was maximum at a dissolved oxygen concentration of 0.15 mg/l. A high activity level of key enzymes of the Calvin cycle and of enzymes involved in dissimilatory oxidation of thiosulfate was recorded in the cells. The high rate of CO2assimilation (112–139 nmol/(min mg protein)) and the cell yield (12 mg dry cells/mmol thiosulfate oxidized), 91–92% of which was accounted for by CO2carbon, were close to those typical of autotrophic bacteria. Thiosulfate was oxidized almost completely to sulfate, and the fraction of intracellular sulfur in the final products did not exceed 0.2–1.7% of the thiosulfate sulfur. The cell membrane fraction contained cytochromes (b + o) and two cytochromes cwith Mrof 23 and 26 kDa; the soluble fraction contained cytochrome cwith Mrof 12 kDa.

Microorganisms in Heat Supply Systems and Internal Corrosion of Steel Pipelines

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 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 a water pH ranging from 8.1 to 9.6 and a temperature lower than 90°C. Magnetite, lepidocrocite, goethite, and X-ray amorphous ferric oxide were the corrosion products identified on the steel-3 plates, as well as siderite, aragonite, and S0. The accumulation of corrosion deposits and variation in the total and local corrosion of the steel plates in a testing unit were considered in terms of the influence of microbial processes.

Phylogenetic in situ/ex situ Analysis of a Sulfur Mat Microbial Community from a Thermal Sulfide Spring 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 spring (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 Sphaerotilus 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.

Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium 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 that defend against 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 midexponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 μg/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.

The Role of Malate Dehydrogenase Isoforms in the Regulation of Anabolic and Catabolic Processes in the Colorless Sulfur Bacterium 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 use of inhibition analysis, the tetrameric isoenzyme was shown to operate in the glyoxylate cycle and the dimeric form 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.

Hoeflea siderophila sp. nov., a new neutrophilic iron-oxidizing bacterium

A new representative of neutrophilic iron-oxidizing bacteria was isolated from the iron-containing sediments of the brackish low-temperature iron-rich spring of the Staraya Russa Resort (Novgorod region, Russia). The cells of strain Hf1 were thin, slightly curved rods, motile by means of a single polar flagellum. The bacterium reproduced by binary division and was capable of producing rosettes. Optimal growth was observed within the pH range of 6.2–8.5 (with an optimum at 7.5), at 9–38°C (with an optimum at 30°C), and in the salinity range of 0.1–8.5% NaCl (with an optimum at 1%). The organism was a facultative anaerobe. The strain was capable of mixotrophic and organoheterotrophic growth. Fe(II) oxidation occurred under anaerobic conditions via reduction of NO3− and N2O, or under microaerobic conditions with oxygen as an electron acceptor. According to phylogenetic analysis based on the comparison of the 16S rRNA gene sequences, the strain was closest to the organotrophic marine bacterium Hoeflea phototrophica (98.5% similarity). The level of DNA-DNA homology with the type species of the genus Hoeflea was 19%. The DNA G + C base content was 57.5 mol %. According to its phenotypic and chemotaxonomic properties, as well as to the results of phylogenetic analysis, strain Hf1 was classified into the genus Hoeflea of the family Phyllobacteriaceae, order Rhizobiales of the phylum Alphaproteobacteria as a novel species, Hoeflea siderophila sp. nov. The type strain is Hf1T (=DSM 21587 = VKM A7094). The GenBank accession number for the 16S rRNA gene sequences of strain Hf1T is EU670237.

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, nitratedependent 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 fractionation of 56/54Fe in pure cultures and model experiments revealed a 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 during Precambrian era until the 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.