— The work presents characterization of antibiotic-resistant strains isolated by direct plating of five samples collected at different treatment stages from the Pushchino water treatment facilities in April 2015. Primary analysis of resistance of the collection (~800 strains) to the following antibiotics was carried out: carbenicillin, kanamycin, streptomycin, amikacin, tobramycin, chloramphenicol, rifampicin, gentamicin, tetracycline, ceftazidime, cefepime, and meropenem. Antibiotic-resistant bacteria most common in the Pushchino wastewater treatment facilities were found to belong to the genera Pseudomonas and Alcaligenes . Occurrence of tetracycline resistance genes was investigated, and predominance of the tetA / tetC genes responsible for active transport of this antibiotic from the cell were found to be predominant among the studied strains. The strains containing the genes associated with type I integrons ( intI1, qacE/qacE Δ 1, and sul1 ) constituted 25% of the studied ones. Four Pseudomonas strains were found to contain the IncN plasmids, while seven strains of this genus contained plasmids of the P-9 incompatibility group (ε-subgroup). Three IncP-9 plasmids were conjugative and carried simultaneously the determinants of tetracycline, streptomycin, and gentamicin resistance, which has not been previously reported for the ε-subgroup of IncP-9 plasmids.

A bacterial strain CT3 with a unique ability to degrade anthropogenic toxic compounds ε-caprolactam, toluene, and meta-xylene as sole sources of carbon and energy was isolated from the activated sludge of a municipal wastewater treatment plant. Based on sequencing and phylogenetic analysis of its 16S rRNA gene, the isolate was identified as a Pseudomonas putida strain. P. putida strain СТ3 was found to possess a conjugative megaplasmid (~550 kb) responsible for ε-caprolactam degradation to the TCA cycle intermediates. The genes encoding toluene and meta-xylene oxidation via the TOL pathway are localized on the chromosome.

Naphthalene, as a component of crude oil, is a common environmental pollutant. Biochemical and genetic aspects of naphthalene catabolism have been examined in most detail in the bacteria of Pseudomonas genus. In pseudomonads, the key intermediate in naphthalene degradation is salicylate. In this study, we investigated the ability of Rhodococcus opacus strain 3D to utilize naphthalene as a sole carbon and energy source. The characteristic feature of this strain is the inability to grow in the mineral medium supplemented with salicylate (typical intermediate of naphthalene degradation in Gram-negative bacteria). The absence of salicylate hydroxylase activity and salicylate accumulation in the course of R. opacus 3D cultivation in the mineral medium supplemented with naphthalene indicated existence of an alternative pathway of naphthalene oxidation. At the same time, R. opacus 3D was able to use monoaromatic compounds (salts of gentisic, ortho-phthalic, and 2-hydroxycinnamic acids and coumarin) as growth substrates. Based on the analysis of enzymatic activities, identification of the reaction intermediates, genetic determinants, and growth substrates, we concluded that R. opacus 3D carries out naphthalene degradation through an alternative pathway via formation of ortho-phthalic acid, which is untypical for pseudomonads. Using mass spectrometry, we showed for the first time that salicylic acid associate formed in trace amounts in the process of naphthalene degradation is not further metabolized and accumulated in the growth medium in a form of a dimer.

Исследовано распространение антибиотикоустойчивых микроорганизмов и генов резистентности к антибиотикам в очистных сооружениях г. Пущино. Обнаружены и охарактеризованы плазмиды резистентности к антибиотикам как возможные векторы для распространения генов устойчивости в окружающей среде. Продемонстрирована роль катаболических плазмид в биодеградации углеводородов нефти. Исследован горизонтальный перенос плазмид в лабораторных условиях и в открытой окружающей среде в процессе утилизации полициклических ароматических углеводородов. Исследованы ризосферные бактерии рода Pseudomonas, относящиеся к уникальной группе PGPR. Штаммы продуцируют широкий спектр биологически активных метаболитов, включая сидерофоры, феназины, 2,4-диацетилфлороглюцин, пиолютеорин, пирролнитрин, оомицин А, цианогенные гликозиды, синтезируют фитогормоны, включая индолил-3-уксусную кислоту (ИУК) и стимулируют корнеобразование у растений. На основе одного из таких штаммов P. chlororaphis (ранее P. aureofaciens) BS1393 разработан биопрепарат «Псевдобактерин-2», обладающий высокой эффективностью против целого ряда заболеваний растений. The dissemination of antibiotic-resistant microorganisms and antibiotic resistance genes in treatment facilities of Pushchino was studied. The antibiotic resistance plasmids were isolated and characterized as possible vectors for the spread of resistance genes in the environment. The role of catabolic plasmids in the biodegradation of oil hydrocarbons was demonstrated. The horizontal transfer of plasmids was studied under laboratory conditions and in an open environment during the utilization of polycyclic aromatic hydrocarbons. The rhizospheric bacteria of the genus Pseudomonas belonging to the unique PGPR group were studied. The strains produce a wide range of biologically active metabolites, including siderophores, phenazines, 2,4-diacetylphloroglucin, pyoluteorin, pyrrolnitrin, oomycin A, hydrocyanic acid, synthesize phytohormones, including indolyl-3-acetic acid (IAA), and stimulate root formation in plants. Based on one of these strains of P. chlororaphis (formerly P. aureofaciens) BS1393, the biopreparation Pseudobacterin-2 was developed, which is highly effective against a number of plant diseases.

— Two strains of ultrasmall gram-negative bacteria (USGNB), FM1 and FM2, were isolated from the skin of the smooth clawed frog Xenopus laevis . The cytological, physiological, biochemical, and genotypic characteristics of the isolates were studied. Based on the sequencing of their 16S rRNA genes and on their phenotypic properties, the isolates were assigned to the genus Chryseobacterium . The cells were extremely small, with cell volumes of ~0.06 and ~0.015 µm 3 for developing cultures of strains FM1 and FM2, respectively. Since the USGNB cells were firmly attached to the skin surface and could not be removed by repeated washing with water, these bacteria may be classified as epibionts. Adhesive properties of the fimbria-like appendages revealed in strains FM1 and FM2 by electron microscopy could probably contribute to tight binding of USGNB cells to the skin. Localization of ultrasmall gram-negative bacteria on skin surface of the frogs may indicate their action as a protective bacterial filter; skin surface of Xenopus laevis is thus characterized for the first time as a specific habitat of ultrasmall Chryseobacterium strains. Isolation and characterization of two ultrasmall Chryseobacterium strains, FM1 and FM2, improves our understanding of diversity of the cellular structural and functional characteristics and of the ecological niches of this bacterial genus.

Diversity of the oil-degrading microbial strains isolated from the water and sediments of the Gulf of Finland (Baltic Sea) in winter and in summer was studied. Substrate specificity of the isolates for aliphatic and aromatic hydrocarbons was studied. The isolates belonged to 32 genera of the types Proteobacteria (alpha-, beta-, and gammaproteobacteria), Actinobacteria,Firmicutes, and Bacteroidetes. Seasonal variations of the oil-degrading microbial communities was revealed. The presence of the known genes responsible for the degradation of oil aliphatic and aromatic hydrocarbons was determined. The alkB sequence of the alkane hydroxylase gene was found in ~16% of the studied strains. The sequence of the phnAc phenanthrene 3,4- dioxygenase was found in Sphingobacterium sp. and Arthrobacter sp. isolates retrieved in winter and summer. In five Pseudomonas sp. strains from winter samples, the classical operons of naphthalene degradation (nah) were localized in catabolic plasmids, of which three belonged to IncР-9, one, to IncР-7, and two to an unidentified incompatibility group. Burkholderia and Delftia strains contained the operons for naphthalene degradation via salicylate and gentisate (nag). The presence of nag genes has not been previously reported for Delftia spp. strains. The sequences of the nagG salicylate 5-hydroxylase gene were also found in Achromobacter, Sphingobacterium, and Stenotrophomonas strains.

A facultative methylotrophic bacterium, strain Lp-1, which was isolated from root nodules of lupine (Lupinus polyphyllus L.) on the medium with methanol as a carbon and energy source, exhibited high similarity of the 16S rRNA gene sequences to Delftia strains (94‒99.9%). The cells of Delftia sp. Lp-1 were motile gram-negative rods dividing by binary fission. Predominant fatty acids were C16:0 (34.2%), C16:1ω9 (14.5%), and C18:1ω7c (17.3%). Phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol were the dominant phospholipids. Q8 was the major ubiquinone. Optimal growth occurred at 24‒26°C and pH 7.1‒7.3; growth was inhibited by 1% NaCl. The organism oxidized methanol with the classical methanol dehydrogenase and used the ribulose bisphosphate pathway of C1 metabolism. Analysis of translated amino acid sequence of the large subunit of the MxaF methanol dehydrogenase revealed 85.5‒94% similarity to the sequences of such autotrophic methylotrophs of the class Alphaproteobacteria as Angulomicrobium, Starkeya, and Ancylobacter, indicating the possible acquisition of the mxaF gene via horizontal gene transfer. Delftia sp. Lp-1 (VKM B-3039, DSM 24446), the first methylotrophic member of the genus Delftia, was shown to be a plant symbiont, stimulating plant growth and morphogenesis, increasing the level of photosynthetic pigments and specific leaf weight. It possesses the nifH gene of nitrogen fixation, is capable of phosphate solubilization, synthesis of auxins and siderophores, and is antagonistic to plant pathogenic fungi and bacilli.

We isolated 50 strains of free-living ultrasmall bacteria with a cell volume that varies from 0.02 to 1.3 μm3 from a range of natural biotopes, namely permafrost soils, oil slime, soils, lake silt, thermal swamp moss, and the skin integuments of the clawed frog, Xenopus laevis. Of them, 23 isolates, characterized by a cell size of less than 0.1 μm3 and a genome size from 1.5 to 2.4 Mb, were subsumed to ultramicrobacteria belonging to different philogenetic groups (Alphaproteobacteria, Bacteroidetes, Actinobacteria) and genera (Kaistia, Chryseobacterium, Microbacterium, Leucobacter, Leifsonia, and Agrococcus) of the Bacteria domain. They are free-living mesophilic heterotrophic aerobic bacteria. The representatives of Kaistia and Chryseobacterium genera were capable of facultative parasitism on other species of chemo-organotrophic bacteria and cyanobacteria. The ultramicrobacteria differed in their morpholgy, cell ultrastructural organization, and physiological and biochemical features. According to the fine structure of their cell walls, the isolates were subdivided into two groups, namely Gram-positive and Gram-negative forms.

The number of spores formed in a single cell of Anaerobacter polyendosporus PS-1 T is significantly influenced by the composition of nutrient media. Depending on carbohydrate concentration in synthetic medium, the number of spores may vary from one or two to as many as five to seven. Investigation of spore formation process by fluorescence and electron microscopy revealed that on media with 0.5–1.0% glucose or galactose most of vegetative cells remained rod-shaped after cessation of cell division in the culture. The nucleoids of these cells were localized at cell poles close to the polar site of the cytoplasmic membrane. Fore-spores were formed at one or both of these poles. A satellite nucleoid (operator) was observed close to each forespore. In the variant with bipolar organization of mother cells, only one or two spores per cell were formed. In the second variant of culture development, when the cells were grown at low galactose concentrations (0.1–0.3%), most of vegetative cells increased in volume and became oval or spherical after cessation of cell division in the culture. Epifluorescence microscopy with nucleic acid-specific fluorochromes (DAPI and acridine orange) revealed the presence of multiple (six to nine) nucleoids in these cells. The nucleoids were located at the cell periphery in close contact with the cytoplasmic membrane. These nucleoids became the centers (poles) for forespore formation. Thus, in the early stationary phase transversion from bipolar to multipolar cells occurred. Cessation of cell division combined with continuing replication of the nucleoids resulted in formation on multinuclear cells. The multiplicity of nucleoides and multipolarity of these cells were prerequisites determining endogenous polysporogenesis, occurring as synchronous formation of three to seven twin spores in many of the oval and spherical cells.