I. A. Bryantseva

Thiorhodospira sibirica gen. nov., sp. nov., a new alkaliphilic purple sulfur bacterium from a Siberian soda lake.

A new purple sulfur bacterium was isolated from microbial films on decaying plant mass in the near-shore area of the soda lake Malyi Kasytui (pH 9.5, 0.2% salinity) located in the steppe of the Chita region of south-east Siberia. Single cells were vibrioid- or spiral-shaped (3-4 microns wide and 7-20 microns long) and motile by means of a polar tuft of flagella. Internal photosynthetic membranes were of the lamellar type. Lamellae almost filled the whole cell, forming strands and coils. Photosynthetic pigments were bacteriochlorophyll a and carotenoids of the spirilloxanthin group. The new bacterium was strictly anaerobic. Under anoxic conditions, hydrogen sulfide and elemental sulfur were used as photosynthetic electron donors. During growth on sulfide, sulfur globules were formed as intermediate oxidation products. They were deposited outside the cytoplasm of the cells, in the peripheral periplasmic space and extracellularly. Thiosulfate was not used. Carbon dioxide, acetate, pyruvate, propionate, succinate, fumarate and malate were utilized as carbon sources. Optimum growth rates were obtained at pH 9.0 and optimum temperature was 30 degrees C. Good growth was observed in a mineral salts medium containing 5 g sodium bicarbonate l-1 without sodium chloride. The new bacterium tolerated up to 60 g sodium chloride l-1 and up to 80 g sodium carbonates l-1. Growth factors were not required. The DNA G + C composition was 56.0-57.4 mol%. Based on physiological, biochemical and genetic characteristics, the newly isolated bacterium is recognized as a new species of a new genus with the proposed name Thiorhodospira sibirica.

Heliorestis daurensis, gen. nov. sp. nov., an alkaliphilic rod-to-coiled-shaped phototrophic heliobacterium from a Siberian soda lake

Abstract A novel alkaliphilic heliobacterium was isolated from microbial mats of a low-salt alkaline Siberian soda lake. Cells of the new organism were tightly coiled when grown in coculture with a rod-shaped bacterium, but grew as short filaments when finally obtained in pure culture. The new phototroph, designated strain BT-H1, produced bacteriochlorophyll g and a neurosporene-like pigment, and lacked internal photosynthetic membranes. Similar to other heliobacteria, strain BT-H1 grew photoheterotrophically on a limited range of organic compounds including acetate and pyruvate. Sulfide was oxidized to elemental sulfur and polysulfides under photoheterotrophic conditions; however, photoautotrophic growth was not observed. Cultures of strain BT-H1 were alkaliphilic, growing optimally at pH 9, and unlike other heliobacteria, they grew optimally at a temperature of 25 °C rather than at 40 °C or above. Analysis of the 16S rRNA gene sequence of the new organism showed that it groups within the heliobacterial clade. However, its branching order was phylogenetically basal to all previously investigated species of heliobacteria. The G+C content of the DNA of strain BT-H1 (44.9 mol%) was also quite distinct from that of other heliobacteria. This unique assemblage of properties implicates strain BT-H1 as a new genus and species of the heliobacteria, Heliorestis daurensis, named for its unusual morphology (“restis” is Latin for “rope”) and for the Daur Steppe in Russia in which these soda lakes are located.

Ectothiorhodospira variabilis, sp. nov., an alkaliphilic and halophilic purple sulfur bacterium from soda lakes

During studies of moderately halophilic strains of Ectothiorhodospira from steppe soda lakes, we found a novel group of bacteria related to Ectothiorhodospira haloalkaliphila with salt optima at 50-80 g NaCl l(-1). Phylogenetic analysis using 16S rRNA gene sequences of strains from soda lakes in Mongolia, Egypt and Siberia revealed separation of the group of new isolates from other Ectothiorhodospira species, including the closely related Ect. haloalkaliphila. DNA-DNA hybridization studies demonstrated that the new isolates form a homogeneous group at the species level, but at the same time are distinct from related species such as Ect. haloalkaliphila, Ect. vacuolata, Ect. shaposhnikovii and Ect. marina. The new isolates are considered to be strains of a novel species, for which the name Ectothiorhodospira variabilis sp. nov. is proposed, with the type strain WN22(T) (=VKM B-2479(T) =DSM 21381(T)). Photosynthetic pigments of the novel species are bacteriochlorophyll a and carotenoids of the spirilloxanthin series with spirilloxanthin and derivatives thereof, together with small amounts of lycopene and rhodopin. Gas vesicles are formed by most of the strains, particularly in media containing yeast extract (0.5 g l(-1)) and acetate (0.5-2.0 g l(-1)). Sequence analysis of nifH (nitrogenase) and cbbL (RuBisCO) confirmed the assignment of the strains to the genus Ectothiorhodospira and in particular the close relationship to Ect. haloalkaliphila. The novel species Ect. variabilis is found in soda lakes separated by great geographical distances and is an alkaliphilic and halophilic bacterium that tolerates salt concentrations up to 150-200 g NaCl l(-1).

Microbial communities of the stratified soda Lake Doroninskoe (Transbaikal region)

The physicochemical properties, species composition, and vertical distribution of microorganisms in the water column, shoreline microbial mat, and small shoreline mud volcanoes of the stratified soda Lake Doroninskoe were investigated in September 2007. The lake is located in the Transbaikal region, in the permafrost zone (51°25′N; 112°28′E). The maximal depth of the contemporary lake is about 6 m, the pH value of the water is 9.72, and the water mineralization in the near-bottom horizon is 32.3 g l−1. In summer, the surface oxygen-containing horizon of the water column becomes demineralized to 26.5 g l−1; at a depth of 3.5–4.0 m, an abrupt transition occurs to the aerobic zone containing hydrosulfide (up to 12.56 g l−1). Hydrosulfide was also detected in trace quantities in the upper water horizons. The density stratification of the water column usually ensures stable anaerobic conditions until the freezing period (November and December). The primary production of oxygenic phototrophs reached 176–230 μg l−1. High rates of dark CO2 assimilation (61–240 μg l−1) were detected in the chemocline. Within this zone, an alkaliphilic species of sulfur-oxidizing bacteria of the genus Thioalkalivibrio was detected (104 cells ml−1). Lithoheterotrophic bacteria Halomonas spp., as well as bacteriochlorophyll a-containing aerobic anoxygenic phototrophic bacteria (AAP) Roseinatronobacter sp. capable of thiosulfate oxidation, were isolated from samples collected from the aerobic zone (0–3 m). The water transparency in September was extremely low; therefore, no visible clusters of anoxygenic phototrophic bacteria (APBs) were detected at the boundary of the hydrosulfide layer. However, purple sulfur bacteria which, according to the results of the 16S rRNA gene analysis, belong to the species Thioalkalicoccus limnaeus, Ectothiorhodospira variabilis, “Ect. magna,” and Ect. shaposhnikovii, were isolated from samples of deep silt sediments. Ect. variabilis and Ect. shaposhnikovii were the major APB species in the shoreline algo-bacterial mat. The halotolerant bacterium Ect. shaposhnikovii, purple nonsulfur bacteria of the genus Rhodobacter, and AAP of Roseococcus sp. were isolated from the samples collected from mud volcanoes. All these species are alkaliphiles, moderate halophiles, or halotolerant microorganisms.

Ectothiorhodospira magna sp. nov., a New Large Alkaliphilic Purple Sulfur Bacterium

Two strains of purple sulfur bacteria of the family Ectothiorhodospiraceae were isolated from moderately saline steppe lakes (with pH above 9.0) of the Transbaikal region (strain B7-7) and Mongolia (strain M10). The cells of the novel strains were spiral-shaped, 2.0–3.2 × 9.6–20.0 μm, motile due to a polar tuft of flagella. Photosynthetic pigments were represented by bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Photosynthetic membranes were represented by long strands of lamellae distributed throughout the whole cell; unlike most Ectothiorhodospiraceae species, the membranes were not packed into regular stacks. Bacteria were capable of weak growth on sulfide and slow grow on hydrogen under photoautotrophic conditions. The best growth was noted on sulfide in the presence of acetate and bicarbonate. Thiosulfate did not stimulate phototrophic growth, even in the presence of organic substrates. The new isolates were alkaliphiles growing at a pH optimum of 9–10. Growth was possible within a salinity range of 0–80 g/l NaCl, with an optimum at 5–15 g/l NaCl. The morphology, the structure of the photosynthetic apparatus (strands of lamellae), and the physiology of the new strains were similar to those of Thiorhodospira sibirica. However, analysis of the 16S rRNA gene sequences demonstrated that the studied isolates were closely related to the type strain Ectothiorhodospira shaposhnikovii (99% similarity) of the family Ectothiorhodospiraceae, whereas the level of similarity between the new strains and Thiorhodospira sibirica was only 94–95%. According to the results of DNA-DNA hybridization, the DNA-DNA homology level between the tested strains was almost 100%; the similarity between the new isolates and the type strain Ectothiorhodospira shaposhnikovii was only 58%. The isolates differed from other representatives of the genus Ectothiorhodospira in the structure of the gene encoding the key enzyme of autotrophic CO2 fixation, ribulose-1,5-bisphosphate carboxylase (RuBisCo), which was similar to the RuBisCo genes of members of another family of sulfur bacteria, Chromatiaceae. The new isolates of purple bacteria were described as a new species of the genus Ectothiorhodospira, Ect. magna sp. nov. with the type strain B7-7T (= VKM B-2537 = DSM 22250).

Application of ribulose-1,5-bisphosphate carboxylase/oxygenase genes as molecular markers for assessment of the diversity of autotrophic microbial communities inhabiting the upper sediment horizons of the saline and soda lakes of the Kulunda Steppe

The genes encoding the key metabolic reactions are often used as functional markers for phylogenetic analysis and microbial ecology studies. The composition and structure of the genes encoding ribulose-1,5-bisphosphate carboxylase (RuBisCO) of various photoautotrophic bacteria, representatives of the order Chromatiales, including collection strains and the strains isolated from saline and soda lakes, were studied in detail. The green-like form I RuBisCO was detected in the majority of the studied strains. In some strains, the genes encoding both form I and form II RuBisCO were present, which has not been previously known for the representatives of this group of bacteria. Moreover, RuBisCO genes were used as functional markers to investigate the autotrophic microbial community inhabiting the upper horizons of bottom sediments of two saline soda lakes and two hypersaline neutral lakes of the Kulunda Steppe. In general, the diversity of autotrophic bacteria in the studied sediment horizons was low. In soda lakes, haloalkaliphilic cyanobacteria and sulfuroxidizing bacteria (SOB) of the genus Halorhodospira were predominant. In saline lakes, halophilic chemoautotrophic SOB Halothiobacillus and Thioalkalivibrio were found, as well as photoautotrophic bacteria of the genus Ectothiorhodosinus and cyanobacteria. Many phylotypes remained unidentified, which indicates the presence of groups of microorganisms with an unknown type of metabolism.

Rhodobaculum claviforme gen. nov., sp. nov., a new alkaliphilic nonsulfur purple bacterium

Two alkaliphilic strains of nonsulfur purple bacteria (NPB), B7-4 and B8-2, were isolated from moderately saline alkaline steppe lakes in southeast Siberia with pH values above 9.0. The isolates were motile, polymorphous cells (from short rods to long spindly cells) 1–2.5 × 2.5–7 μm. Intracellular membranes of vesicular type were mostly located at the cell periphery. The microorganisms contained bacteriochlorophyll a and carotenoids of the spheroidene and spirilloxanthin series. The photosynthetic apparatus was represented by LH2 and LH1 light-harvesting complexes. In the presence of organic compounds, the strains grew aerobically in the dark or anaerobically in the light. Capacity for photo- and chemoautotrophic growth was not detected. The cbbL gene encoding RuBisCO was not revealed. Optimal growth of both strains occurred at 2% NaCl (range from 0.5 to 4%), pH 8.0–8.8 (range from 7.5 to 9.7), and 25–35°C. The DNA G+C content was 67.6–69.8 mol %. Pairwise comparison of the nucleotides of the 16S rRNA genes revealed that strains B7-4 and B8-2 belonged to the same species (99.9% homology) and were most closely related to the aerobic alkaliphilic bacteriochlorophyll a-containing anoxygenic phototrophic bacterium (APB) Roseibacula alcaliphilum DeT (95.2%) and to NPB strains Rhodobaca barguzinensis VKM B-2406T (94.2%) and Rbc. bogoriensis LBB1T (93.9%). The isolates were closely related to the NPB Rhodobacter veldkampii DSM 11550T (94.8%) and to aerobic bacteriochlorophyll a-containing bacteria Roseinatronobacter monicus ROS 35T and Roseicitreum antarcticul ZS2-28T (93.5 and 93.9%, respectively). New strains were described as a new NPB genus and species of the family Rhodobacteriaceae, Rhodobaculum claviforme gen. nov., sp. nov., with B7-4T (VKM B-2708, LMG 28126) as the type strain.

Candidatus ‘Chloroploca asiatica’ gen. nov., sp. nov., a new mesophilic filamentous anoxygenic phototrophic bacterium

Five phylogenetically similar monocultures of mesophilic filamentous anoxygenic phototrophic bacteria (FAPB) were isolated from microbial mats of low-mineral (5–28 g/L) alkaline lakes in Buryat Republic, Transbaikalia and Mongolia, as well as from biofilms of an alkaline sulfide spring (3 g/L) of the Umhei hydrothermal system (Buryat Republic). New isolates were characterized by short trichomes (15–30 μm long and ∼1 μm in diameter), straight, curved, or wavy, surrounded by a thin iron-sorbing mucous sheath. Gliding motion of the trichomes was not observed. The trichomes formed bunches consisting of several filaments. Trichomes multiply by the separation of short fragments or single cells from the parental trichome. The cells in the filaments were elongated; they contained chlorosomes, gas vesicles, poly-β-hydroxybutyrate granules, and small polyphosphate inclusions. Bacteria contained bacteriochlorophylls c and a and γ-carotene. Absorption maxima of the pigments in the cells were observed at 462, (shoulder at 515), 742, 805, and 863 nm. The organisms were strict anaerobes capable of photoautotrophic growth with sulfide as an electron donor. Elemental sulfur emerged into the medium as a result of sulfide photooxidation. The organisms were tolerant to sulfide (up to 8 mM). Best growth occurred at pH 8.0, 3–15 g/L NaCl, and 1–5 g/L sodium bicarbonate. According to phylogenetic analysis, the 16S rRNA gene sequences of the FAPB isolates formed a separate cluster most closely related to the species cluster of the family Oscillochloridaceae, suborder Chloroflexinae, order Chloroflexales, class Chloroflexi. The differences with the closest 16S rRNA gene sequences of the known FAPB were 9–10%. The formal description of a new taxon, Candidatus’ Chloroploca asiatica’ gen. nov., sp. nov., is provided.

Benthic phototrophic community from Kiran soda lake, south-eastern Siberia

Phototrophic bacterial mats from Kiran soda lake (south-eastern Siberia) were studied using integrated approach including analysis of the ion composition of water, pigments composition, bacterial diversity and the vertical distribution of phototrophic microorganisms in the mats. Bacterial diversity was investigated using microscopic examination, 16S rRNA gene Illumina sequencing and culturing methods. The mats were formed as a result of decomposition of sedimented planktonic microorganisms, among which cyanobacteria of the genus Arthrospira predominated. Cyanobacteria were the largest part of phototrophs in the mats, but anoxygenic phototrophs were significant fraction. The prevailing species of the anoxygenic phototrophic bacteria are typical for soda lakes. The mats harbored aerobic anoxygenic phototrophic bacteria, purple sulfur and non-sulfur bacteria, as well as new filamentous phototrophic Chloroflexi. New strains of Thiocapsa sp. Kir-1, Ectothiorhodospira sp. Kir-2 and Kir-4, Thiorhodospira sp. Kir-3 and novel phototrophic Chloroflexi bacterium Kir15-3F were isolated and identified.

Phylogenetic position of the purple sulfur bacterium Lamprobacter modestohalophilus determined based on the data on new strains of the species

Lamprobacter, the genus of halophilic purple sulfur bacteria (PSB) with the single species Lpb. modestohalophilus was described in 1979. Rod-shaped Lamprobacter cells contained gas vesicles during the nonmotile growth phase; motile cells without gas vesicles were sometimes formed. Bacteria contained bacteriochlorophyll a and a carotenoid okenone. The names of this genus and species were included in the list of approved microbial names in 1988. Since the type strain Lpb. modestohalophilus RO1T has been lost, its 16S rRNA gene sequences have not been obtained. Based on analysis of the 16S rRNA genes, a new genus Halochromatium comprising the motile extremely halophilic Chromatium-like species was proposed in 1998. Members of this genus never contain gas vesicles. In spite of the phenotypic differences between the genera Lamprobacter and Halochromatium, phylogenetic boundaries between these taxa remained undetermined. Description of a marine bacteria formerly belonging to Lamprobacter according to its morphological and physiological properties as a new Halochromatium species, Hch. roseum, resulted in additional complication of the taxonomic situation. The present work provides evidence for the preservation of two phenotypically and phylogenetically different genera, Lamprobacter and Halochromatium, Lpb. modestohalophilus is proposed as the type species of the genus Lamprobacter. Characteristics of two Lpb. modestohalophilus strains were extensively investigated, and one of them (strain Sivash) was proposed as the neotype strain of the species. It was suggested to retain the genus Halochromatium as containing extremely halophilic species Hch. salexigens and Hch. glycolicum, while transfer of the weakly halophilic species Hch. roseum to the genus Lamprobacter is proposed, resulting in a new combination Lamprobacter roseus comb. nov.