В активном иле полупромышленного биореактора, очищающего фильтрат сброженного осадка сточных вод Курьяновских очистных сооружений (г. Москва), выявлены и охарактеризованы по физиологическим, морфологическим и молекулярно-биологическим показателям представители нового вида бактерий, окисляющих аммоний нитритом в бескислородных условиях. Клетки бактерий овоидной формы, 0.5 ? 0.8 мкм, имеют внутриклеточные мембранные структуры, характерные для анаммокс-бактерий: анаммоксосому, парифоплазму, а также, в отличие от других анаммокс-бактерий, обширные внутриклеточные мембранные структуры, располагающиеся слоями параллельно цитоплазматической мембране, и никогда – в области анаммоксосомы. Клетки объединены в агрегаты диаметром 5–28 мкм, легко адгезируют к твердым поверхностям. Клетки морфологически лабильны – легко подвергаются плазмолизу, теряют содержимое. Время удвоения – 28 сут.; макс =0.025 сут-1; температурный оптимум роста 20–45°C, оптимум рН 8.0. По результатам филогенетического анализа последовательностей нуклеотидов генов, кодирующих 16S рРНК, бактерия отнесена к новому виду рода-кандидата Jettenia, принадлежащего к порядку Planctomycetales. Для новой бактерии предложено название Candidatus “Jettenia moscovienalis” sp. nov.

A new species of bacteria oxidizing ammonium with nitrite under anoxic conditions was isolated from the activated sludge of a semi-industrial bioreactor treating digested sludge of the Kuryanovo wastewater treatment plant (Moscow, Russia). Physiological, morphological, and molecular genetic characterization of the isolate was carried out. The cells were ovoid (∼0.5 × 0.8 μm), with the intracellular membrane structures characteristic of anammox bacteria (anammoxosome and paryphoplasm); unlike other anammox bacteria, it possessed extensive intracellular membrane structures located in layers parallel to the cytoplasmic membrane, but never close to the anammoxosome. The cells formed aggregates 5–28 μm in diameter and readily attached to solid surfaces. The cells were morphologically labile, easily plasmolyzed, and lost their content. Doubling time was 28 days, μmax = 0.025 day−1; optimal temperature and pH for growth were 20–45°C and 8.0, respectively. Phylogenetic analysis of the 16S rRNA gene sequences suggested its classification as a new species of the candidate genus Jettenia (order Planctomycetales). The name Candidatus “Jettenia moscovienalis” sp. nov. was proposed for the new bacterium.

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.

Методом создания и анализа библиотек клонов гена 16S рРНК изучен видовой состав двух термотолерантных сообществ ацидофильных хемолитотрофных микроорганизмов (АХМ), выделенных из пульпы лабораторных реакторов, в которых проводились процессы окисления концентратов разных золотосодержащих руд. Первое сообщество сформировалось в процессе окисления пирит-арсенопиритного концентрата руды Кючусского месторождения. Клоны бактериального компонента первого сообщества принадлежали к родам Sulfobacillus и Leptospirillum. 32 клона Sulfobacillus были разделены на три группы: Sb. thermosulfidooxidans, Sb. benefaciens и Sb. thermotolerans. 33 клона Leptospirillum были близки к виду Leptospirillum ferriphilum. Все клоны архейного компонента соответствовали виду Ferroplasma acidiphilum. Микроорганизмы данного сообщества были использованы как инокулят для процесса биоокисления другого по составу минерального концентрата – пирротинсодержащего пирит-арсенопиритного концентрата руды Олимпиадинского месторождения, и состав сообщества, сформировавшегося в данном процессе был также исследован. Клоны бактериального компонента второго сообщества также принадлежали к родам Sulfobacillus и Leptospirillum. 14 клонов Sulfobacillus были разделены на две группы: Sb. thermosulfidooxidans (13 клонов) и Sb. thermotolerans (1 клон). 48 клонов рода Leptospirillum были близки к виду L. ferriphilum. Все клоны архейного компонента соответствовали виду F. acidiphilum. В процессе адаптации сообщества к новому окисляемому минеральному субстрату произошли изменения, как в его составе, так и в численном соотношении видов микроорганизмов.

Construction and analysis of the 16S rDNA clone libraries was used to investigate the species composition of two thermotolerant communities of acidophilic chemolithotrophic microorganisms (ACM) isolated from the pulp of laboratory reactors used for oxidation of different gold-containing ore concentrates. The first community was formed during oxidation of the pyrite-arsenopyrite ore concentrate from the Kyuchus deposit. The clones of the bacterial component of this community belonged to the genera Sulfobacillus (32 clones) and Leptospirillum (33 clones). The Sulfobacillus clones belonged to three groups: Sb. thermosulfidooxidans, Sb. benefaciens, and Sb. thermotolerans . All Leptospirillum clones were closely related to L. ferriphilum . All clones of the archaeal component belonged to Ferroplasma acidiphilum . The microorganisms of this community were used as inoculum for biooxidation of a different mineral concentrate, the pyrrhotite-containing pyrite-arsenopyrite ore concentrate from the Olympiadinskoe deposit, and the structure of the community formed in the process was investigated. The clones of the bacterial component of the second community also belonged to the genera Sulfobacillus (14 clones) and Leptospirillum (48 clones). The Sulfobacillus clones belonged to the species Sb. thermosulfidooxidans (13 clones) and Sb. thermotolerans (1 clone). All Leptospirillum clones were closely related to L. ferriphilum . All clones of the archaeal component belonged to Ferroplasma acidiphilum . During the adaptation of the community to a new oxidized mineral substrate, both the composition and the ratio of the constituent microbial species changed.

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.

The species composition of the microbial association involved in industrial tank biooxidation of the concentrate of refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore of the Olympiadinskoe deposit at 39°C was studied by cultural and molecular biological techniques. Pure microbial cultures were isolated, their physiological characteristics were investigated, and their taxonomic position was determined by 16S rRNA gene sequencing. The library of 16S rRNA gene clones obtained from the total DNA isolated from the biomass of the pulp of industrial reactors was analyzed. The diversity of microorganisms revealed by cultural techniques in the association of acidophilic chemolithotrophs (Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, Ferroplasma acidiphilum, Alicyclobacillus tolerans, and Acidiphilium cryptum) was higher than the diversity of the 16S rDNA clone library (At. ferrooxidans, L. ferriphilum, and F. acidiphilum). The combination of microbiological and molecular biological techniques for the investigation of the biodiversity in natural and anthropogenic microbial communities promotes detection of new phylogenetic microbial groups in these communities.

A multiplex method for detection of genetically modified organism (GMO) in various foods has been developed based on PCR-identification of cauliflower mosaic virus (CMV) 35S-promoter. It allows avoiding false positive signals due to contamination of plant raw material with CMV.

Исследовали состав культивируемых аэробных копиотрофных бактерий и грибов, населяющих очищенные от пищи пищеварительные тракты дождевых червей Aporrectodea caliginosa, Lumbricus terrestris и Eisenia fetida, а также почву (компост) и свежие экскременты червей. Микроорганизмы выделяли на питательных средах и идентифицировали, секвенируя фрагменты бактериальных 16S рДНК и грибных 28S рДНК (D1/D2 домен) с последующим филогенетическим анализом. Из пищеварительных трактов червей выделены бактерии из семейств Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, Sphingobacteriaceae (Bacteroidetes), а также из Actinobacteria. Пять штаммов: Ochrobactrum sp. 341-2 ( -Proteobacteria), Massilia sp. 557-1 ( -Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1 и бактерия из семейства Microbacteriaceae, изолят 521-1 (Actinobacteria) имеют идентичность генов 16S рРНК известным генам 93–97%, что позволяет предположить их принадлежность к новым видам и родам. Группировки бактерий, выделяемых из пищеварительных трактов, существенно различаются от группировок из почвы и экскрементов. Ряд таксонов бактерий оказались общими для различных отделов кишечника A. caliginosa и для кишечников червей различных видов, хотя в целом состав бактериальных сообществ этих объектов различен. Предполагается, что существуют группировки бактерий, симбиотически ассоциированных с кишечниками. Среди грибов из очищенных пищеварительных трактов выделялись Bjerkandera adusta и Syspastospora parasitica, представленные стерильным светлоокрашенным мицелием, а также Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), Penicillium spp. Они длительно выживают в пищеварительной среде червей. Для доказательства принадлежности микроорганизмов, ассоциированных с пищеварительным трактом червей, к симбионтам, необходимо изучение их функциональных особенностей и роли в организме-хозяине.

The cultured aerobic copiotrophic bacteria and fungi from food-free digestive tracts of Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida earthworms, soil (compost), and fresh earthworm excrements were investigated. The microorganisms were isolated on nutrient media and identified by sequencing the fragments of bacterial 16S rRNA and fungal 28S rRNA (D1/D2 domain) gene sequences with subsequent phylogenetic analysis. Bacteria isolated from the digestive tracts of earthworms belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Sphingobacteriaceae (Bacteroidetes), as well as Actinobacteria. For five strains, namely Ochrobactrum sp. 341-2 (α-Proteobacteria), Massilia sp. 557-1 (β-Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1, and a bacterium from the family Microbacteriaceae, isolate 521-1 (Actinobacteria), the similarity to known 16S rRNA sequences was 93–97%; they therefore, probably belong to new species and genera. Bacterial groups isolated from the digestive tracts of earthworms were significantly different from those isolated from soil and excrements. Some bacterial taxa occurred in different sections of A. caliginosa intestine and in intestines of different earthworm species; however, the overall composition of bacterial communities in these objects is different. Existence of bacterial groupings symbiotically associated with intestines is proposed. Among the fungi, Bjerkandera adusta and Syspastospora parasitica were isolated from the cleaned digestive tracts as light-colored, sterile mycelium, as well as Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), and Penicillium spp. These fungi survive for a long time in the earthworm’s digestive environment. Investigation of the functional characteristics and role in the host organism is required to confirm the symbiotic status of the microorganisms associated with the earthworm digestive tract.