M. V. Sukhacheva

Methanogenesis at extremely haloalkaline conditions in the soda lakes of Kulunda Steppe (Altai, Russia)

Microbial methanogenesis at extreme conditions of saline alkaline soda lakes has, so far, been poorly investigated. Despite the obvious domination of sulfidogenesis as the therminal anaerobic process in the hypersaline soda lakes of Kulunda Steppe (Altai, southwestern Siberia), high concentrations of methane were detected in the anaerobic sediments. Potential activity measurements with different substrates gave results significantly deviating from what is commonly found in hypersaline habitats with neutral pH. In particular, not only a non-competitive methylotrophic pathway was active, but also lithotrophic and, in some cases, even acetate-dependent methanogenesis was found to be present in hypersaline soda lake sediments. All three pathways were functioning exclusively within the alkaline pH range between 8 and 10.5, while the salt concentration was the key factor influencing the activity. Methylotrophic and, to a lesser extent, lithotrophic methanogenesis were active up to soda-saturating conditions (4 M total Na(+)). Acetate-dependent methanogenesis was observed at salinities below 3 M total Na(+). Detection of methanogens in sediments using the mcrA gene as a functional marker demonstrated domination of methylotrophic genera Methanolobus and Methanosalsum and lithotrophic Methanocalculus. In a few cases, acetoclastic Methanosaeta was detected, as well as two deep lineage methanogens. Cultivation results corresponded well to the mcrA-based observations. Enrichments for natronophilic methylotrophic methanogens resulted in isolation of Methanolobus strains at moderate salinity, while at salt concentrations above 2 M Na(+) a novel member of the genus Methanosalsum was dominating. Enrichments with H2 or formate invariably resulted in domination of close relatives of Methanocalculus natronophilus. Enrichments with acetate at low salt concentration yielded two acetoclastic alkaliphilic Methanosaeta cultures, while at salinity above 1 M Na(+) syntrophic associations were apparently responsible for the observed acetate conversion to methane. Overall, the results indicated the presence of functionally structured and active methanogenic populations in Siberian hypersaline soda lakes.

Draft Genome Sequence of the Anoxygenic Filamentous Phototrophic Bacterium Oscillochloris trichoides subsp. DG-6

Oscillochloris trichoides is a mesophilic, filamentous, photoautotrophic, nonsulfur, diazotrophic bacterium which is capable of carbon dioxide fixation via the reductive pentose phosphate cycle and possesses no assimilative sulfate reduction. Here, we present the draft genome sequence of Oscillochloris trichoides subsp. DG-6, the type strain of the species, which has permitted the prediction of genes for carbon and nitrogen metabolism and for the light-harvesting apparatus.

Methanosalsum natronophilum sp. nov., and Methanocalculus alkaliphilus sp. nov., haloalkaliphilic methanogens from hypersaline soda lakes.

Two groups of haloalkaliphilic methanogenic archaea were dominating in enrichments from hypersaline soda lake sediments at pH 10. At moderate salt concentrations with formate or H2 as electron donor, methanogens belonging to the genus Methanocalculus were enriched, while at high salt concentrations with methylated substrates, a group related to Methanosalsum zhilinae was dominating. For both groups, several pure cultures were obtained including the type strains AMF2T for the Methanocalculus group and AME2T for the Methanosalsum group. The Methanocalculus group is characterized by lithoheterotrophic growth with either formate (preferable substrate) or H2 at moderate salinity up to 1.5-2 M total Na+ and obligate alkaliphilic growth with an optimum at pH 9.5. According to phylogenetic analysis, the group also includes closely related strains isolated previously from the low-salt alkaline Lonar Lake. The novel Methanosalsum group is characterized by high salt tolerance (up to 3.5 M total Na+) and obligate alkaliphilic growth with an optimum at pH 9.5. It has a typical methylotrophic substrate profile, utilizing methanol, methylamines and dimethyl sulfide (at low concentrations) as methanogenic substrates. On the basis of physiological and phylogenetic data, it is proposed that the two groups of soda lake methanogenic isolates are assigned into two novel species, Methanocalculus alkaliphilus sp. nov. (type strain AMF2T = DSM 24457T = UNIQEM U859T) and Methanosalsum natronophilum sp. nov. (type strain AME2T = DSM 24634T = NBRC 110091T).

Filamentous anoxygenic phototrophic bacteria from cyanobacterial mats of Alla hot springs (Barguzin Valley, Russia)

Alkaline hydrotherms of the Baikal rift zone are unique systems to study the diversity of thermophilic bacteria. In this study, we present data on the phototrophic bacterial community of cyanobacterial mats from the alkaline Alla hot spring. Using a clonal analysis approach, this study evaluated the species diversity, the proportion of oxygenic and anoxygenic phototrophs and their distribution between various areas of the spring. Novel group-specific PCR primers were designed and applied to detect representatives of the Chloroflexus and Roseiflexus genera in mat samples. For the first time, the presence of Roseiflexus-like bacteria was detected in the Baikal rift zone.

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.

Hydrocarbon-oxidizing potential and the genes for n-alkane biodegradation in a new acidophilic mycobacterial association from sulfur blocks

The capacity of AGS10, a new aerobic acidophilic (growing within the pH range from 1.3 to 4.5 with the optimum at 2.0–2.5) bacterial association from sulfur blocks of the Astrakhan gas-processing complex (AGC), for oxidation of hydrocarbons of various chemical structure was investigated. A broad spectrum of normal (C10-C21) and iso-alkanes, toluene, naphthalene, and phenanthrene, as well as isoprenoids resistant to microbial degradation, pristane and phytane (components of paraffin oil), and 2,2,4,4,6,8,8,-heptamethylnonane, a branched hydrocarbon, were biodegraded under acidic conditions. Microbiological investigation revealed the dominance of mycobacteria in the AGS10 association, which was confirmed by analysis of the 16S rRNA gene clone library. In the phylogenetic tree, the 16S rRNA sequences formed a branch within the cluster of slow-growing mycobacteria, with 98% homology to the closest species Mycobacterium florentinum. Genomic DNA of AGS10 culture grown on C14-C17n-alkanes at pH 2.5 was found to contain the genes of two hydroxylase families, alkB and Cyp153, indicating their combined involvement in hydrocarbon biodegradation. The high hydrocarbon-oxidizing potential of the AGS10 bacterial association indicated that further search for the genes responsible for degradation of various hydrocarbons in acidophilic mycobacteria could be promising.

Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenase enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNA gene tree to a considerable degree, which made it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterial communities in sediments of hyper saline and soda lakes. Although diazotrophs were revealed in all environmental samples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophic gammaproteobacteria were predominant in integrated samples. Analysis of the upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria, and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.

Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area, Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, and salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54°C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected in the samples from both the thermophilic and mesophilic mats. Cultures of nonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolated from the mats developed at high (50.6–49.4°C) and low temperatures (45–20°C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealed in low-temperature mats. Truly thermophilic purple and green sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfur communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophyll a-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20°C) mat is of interest.

Characterization of a New Strain of a Purple Nonsulfur Bacterium from a Thermal Spring

A new budding purple nonsulfur bacterium of the genus Rhodobacter (strain Ku-2) was isolated from a mat of a moderately thermal spring (Baikal rift zone, Buryatia, Russia). The bacterium had lamellar photosynthetic membranes, which are typical of only one Rhodobacter species, Rba. blasticus. The cells contained spheroidene carotenoids and bacteriochlorophyll a (Bchl a). In vivo absorption spectrum of the cells, with the major maximum at 863 nm and an additional peak at 887 nm, is characteristic of the pigment-protein complexes of Bchl a-containing membranes. The previously described Rba. blasticus strains do not exhibit the 887-nm maximum. The new isolate was photoheterotrophic, with optimal growth occurring at 35°C, 3 g/L NaCl, and pH 7–8. The DNA G+C content was 64.4 mol %. The similarity between the 16S rRNA gene sequences of strain Ku-2 and the Rba. blasticus type strain was 98.7%. The PufM amino acid sequences of strain Ku-2 and the earlier studied Rba. blasticus type strain were 89.5 % identical. Thus, strain Ku-2 belongs to the genus Rhodobacter and is phylogenetically close to Rba. blasticus.

Reconstruction of iron metabolism pathways of bacteria Magnetospirillum aberrantis SpK spp. based on sequenced genome analysis

ISSN 00124966, Doklady Biological Sciences, 2012, Vol. 444, pp. 202–205.