— 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.

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.

Mass spectrometry was applied to identify metabolites and estimate the efficiency of phenanthrene biodegradation and transformation by rhizosphere bacteria Pseudomonas aureofaciens BS1393. Strains P. aureofaciens BS1393(pOV17) and P. aureofaciens BS1393(NPL-41) bearing various naphthalene biodegradation plasmids were used in the work. The strain BS1393(pOV17) contains the pOV17 wild type naphthalene biodegradation plasmid that determines the oxidation of naphthalene to Krebs cycle metabolites. The strain BS1393(NPL-41) contains the mutant plasmid NPL-41 governing the initial stages of naphthalene oxidation into salicylic acid. The limiting stages of phenanthrene biodegradation in bacteria with various plasmids have been identified according to the accumulation of intermediates. When bacteria were grown on phenanthrene, the main metabolites were as follows: (a) 2-hydroxy-2H-benzo[h]chromene-2-carboxylic acid/trans-4-(1-hydroxynaph-2-yl)-2-oxobut-3-enoic acid, (b) 1-hydroxy-2-naphthoic acid, and (c) salicylic acid. In the strain BS1393(pOV17), metabolite (а) was observed during 1–14 days of cultivation. Unlike it, in the strain BS1393(NPL-41), an insignificant amount of this metabolite was found after only 14 days. The availability of metabolite (b) in the growth of both strains was an evidence of the limited rate of its further decarboxylation. Metabolite (c) as a final product was found in the growth of the strain BS1393(NPL-41). Contrastingly, in the strain BS1393(pOV17), this metabolite was not found, which indicates the complete oxidation of phenanthrene.

Major metabolites formed by Pseudomonas bacteria bearing various naphthalene degradation plasmids were identified using high performance liquid chromatography and mass spectrometry in combination with electrospray ionization. During the growth of P. aureofaciens BS1393(NPL-41) bacteria bearing the plasmid NPL-41, partial oxidation of naphthalene was shown to occur to form the major exometabolite (salicylate) and minor exometabolite (phenol). The growth of P. aureofaciens BS1393(pOV17) bacteria bearing the plasmid pOV17 was accompanied by complete oxidation of naphthalene and formation of trace amounts of salicylate and epoxybenzene. Based on the data of molecular mass spectrometry, we determined the dynamics of CO2 production and oxygen uptake in the process of bacterial growth. The specific quantity of CO2 per unit of cell amount for P. aureofaciens BS1393(NPL-41) bacteria was shown to exceed fivefold that for P. aureofaciens BS1393(pOV17). The respiratory quotient (RQ) representing the ratio of the molar concentrations of metabolic CO2 to consumed oxygen depends on the type of plasmids that determine naphthalene degradation. The value of RQ during the growth of P. aureofaciens BS1393(pOV17) bacteria was almost 1.5 times greater than in the case of P. aureofaciens BS1393(NPL-41). The ratio of O2 uptake to CO2 formed by the first culture was 4.5 mol O2/mol CO2; by the second culture, 6.5 mol O2/mol CO2.

By the example of glucose uptake by the soil bacteria Pseudomonas aureofaciens BS1393(pBS216) and Rhodococcus sp. 3–30 immobilized on a solid-phase surface (quartz sand), their growth parameters were determined: growth rate (doubling time), total CO2 production, CO2 production per cell, lag period with respect to substrate uptake, respiratory quotient. The growth of P. aureofaciens and Rhodococcus sp. on glucose revealed (1) differences of the lag period with respect to substrate (lag time of ∼4 h for P. aureofaciens and ∼26 h for Rhodococcus sp.); (2) differences between the maximal rates of CO2 production (∼50 μg C-CO2 g−1 sand h−1 for P. aureofaciens and ∼8.5 μg C-CO2 g−1 sand h−1 for Rhodococcus sp.); (3) differences in CO2 production per cell (∼1.94 × 10−9 μM CO2/CFU for P. aureofaciens and more than ∼3.4 × 10−9 μM CO2/CFU for Rhodococcus sp.). The kinetics of the metabolic CO2 isotopic composition was shown to be determined by the difference in the carbon isotopic characteristics of products in the cell. Upon introduction of glucose into the medium (the preparatory stage of the metabolism), the uptake of intracellular 13C-depleted products (lipids) is noted; at the stage of the maximal cell growth rate, introduced glucose is mainly metabolized; and at the final stage, upon exhaustion of substrate, the “stored” products—the lipid fraction—get involved in the metabolism. At the maximal rate of glucose uptake, the CO2 carbon isotopic fractionation coefficient relative to organic products of microbial biosynthesis was determined to be α = 1.009 ± 0.002.

Using molecular and isotopic mass spectrometry, we investigated the toxic effect of naphthalene as a representative of polycyclic aromatic hydrocarbons (PAHs) on plants growing under sterile conditions and plants inoculated with microorganisms capable and incapable of naphthalene degradation. Tobacco plants of the Samsun variety were grown in a closed gas-nutrient system on a mineral medium with sucrose as a carbon source. Naphthalene used as a toxicant at a concentration of 5.2 × 10 −4 % contained 13 C isotope whose amount was characterized by the value δ 13 C = +281.4 ± 0.6‰ relative to the PDB standard and differed from that of sucrose, the main source of carbon (δ 13 C = −12.0 ± 0.1‰). Degradation of naphthalene was determined by the inclusion of its carbon in metabolic CO 2 and plant tissues (the root, stem, leaves). The effect of naphthalene on plants was indicated by the rates of O 2 production and CO 2 uptake during the light period as compared with the dark period of exposure. A decrease of the toxic effect of naphthalene on plants was observed only at the inoculation of plants with Pseudomonas aureofaciens BS1393 rhizosphere bacteria bearing plasmid pBS216, which controls the naphthalene biodegradation ability. The occurrence of other heterotrophic microorganisms incapable of naphthalene degradation had no similar protective effect.

На примере потребления глюкозы почвенными бактериями Pseudomonas aureofaciens BS1393(pBS216) и Rhodococcus sp. 3-30, иммобилизованными на твердофазной поверхности (кварцевый песок), определены их ростовые показатели: скорость роста бактерий (время удвоения клеток), суммарная продукция СО2 и продукция из расчета на бактериальную клетку, лаг-период относительно потребления субстрата, дыхательный коэффициент. При росте бактерий P. aureofaciens и Rhodococcus sp. на глюкозе выявлены различия штаммов по параметрам: (1) лаг-периодов относительно внесенного субстрата (лаг-период около 4 ч для P. aureofaciens и около 26 ч для Rhodococcus sp.); (2) максимальной скорости продукции СО2 (около 50 мкг СО2 г-1 песка ч-1 для P. aureofaciens и около 8.5 мкг СО2 г-1 песка ч-1 для Rhodococcus sp.); (3) продукции СО2 из расчета на одну клетку (около 1.94 ? 10-9 мкмоль СО2 для P. aureofaciens и около 3.4 ? 10-9 мкмоль СО2 для Rhodococcus sp.). Показано, что кинетика изотопного состава метаболической СО2 определяется различием в изотопных характеристиках углерода продуктов в клетке: после внесения глюкозы в среду (подготовительная стадия метаболизма) отмечено использование внутриклеточных обедненных 13 продуктов (липидов), на стадии максимальной скорости роста клеток метаболизируется, главным образом, внесенная глюкоза, и на завершающей стадии после исчерпания субстрата в обмен включаются “запасенные” продукты – липидная фракция. При максимальной скорости потребления глюкозы определен коэффициент фракционирования изотопов углерода СО2 относительно органических продуктов микробного биосинтеза, который составляет величину = 1.009 ± 0.002.

Исследован углеводородокисляющий потенциал почвенной микробиоты и интродуцированных в почву углеводородокисляющих микроорганизмов на основе количественных и изотопных характеристик углерода продуктов, образующихся при микробной деградации нефти. Из сравнения скоростей продукции СО2 в нативной почве и почве, загрязненной сырой нефтью, обнаружено, что интенсивность микробной минерализации почвенного органического вещества (ПОВ) в присутствии нефти выше по сравнению с незагрязненной почвой, т.е., обнаруживается затравочное влияние (прайминг-эффект) углеводородов нефти. Показано, что количество углерода вновь синтезированных органических продуктов за счет потребленной нефти (биомасса клеток и экзометаболиты) значительно превосходит количество ПОВ, израсходованное на продукцию СО2. Обнаружено, что в результате микробиологических процессов в почве, загрязненной нефтью, наблюдается мощный поток углекислоты, поступающей в атмосферу.

The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO2 production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect of oil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO2. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.

Plasmid pBS501 was detected in the strain Comamonas sp. BS501. This plasmid specifies high level of induced resistance (5 mM) to cobalt/nickel both in the host strain and in related strains C. testosteroni B-1241 and C. acidovorans B-1251. Hybridization analysis revealed a homology of pBS501 restriction fragments with the only well-characterized operon cnrXYHCBAT that resides in plasmid pMOL28 from Cupriavidus metallidurans CH34. Essential differences in the structural organization of the cobalt/nickel resistance determinant were found between plasmid pBS501 and the cnr operon.

Совмещение генетических систем деградации полиароматических углеводородов, резистентности к тяжелым металлам и стимуляции роста/защиты растений является одним из подходов в создании полифункциональных штаммов для фиторемедиации почв с комплексным загрязнением органическими поллютантами и тяжелыми металлами. Получен ризосферный штамм Pseudomonas chlororaphis PCL1391(pBS216*, pBS501), обладающий способностью стимулировать рост растений, в котором nah оперон плазмиды pBS216 обеспечивает биодеградацию нафталина, а cnr-подобный оперон плазмиды pBS501 – устойчивость к никелю/кобальту, связанную с выводом катионов тяжелых металлов из клетки. В присутствии 100 мкМ никеля жизнеспособность, скорость роста и эффективность биодеградации нафталина устойчивого штамма PCL1391(pBS216*, pBS501) были значительно выше по сравнению с чувствительным PCL1391(pBS216). Во время роста устойчивого штамма никель (100 мкМ) не оказывал ингибирующего влияния на активность ключевых ферментов биодеградации нафталина в отличие от чувствительного штамма.

Combination of genetic systems of degradation of polyaromatic hydrocarbons, resistance to heavy metals, and promotion of plant growth/protection is one of the approaches to the creation of polyfunctional strains for phytoremediation of soils after co-contamination with organic pollutants and heavy metals. A plant-growth-promoting rhizosphere strain Pseudomonas chlororaphis PCL1391 (pBS216 * , pBS501) has been obtained, in which the nah operon of plasmid pBS216 provides naphthalene biodegradation and the cnr -like operon of plasmid pBS501 provides resistance to cobalt and nickel due to the extrusion of heavy metal cations from the cells. In the presence of 100 µM of nickel, the viability, growth rate, and naphthalene biodegradation efficiency of the resistant strain PCL1391 (pBS216 * , pBS501) were much higher as compared with the sensitive PCL1391 (pBS216). During the growth of the resistant strain, in contrast to the sensitive strain, nickel (100 µM) had no inhibiting effect on the activity of the key enzymes of naphthalene biodegradation.

Plasmid pBS501, responsible for the resistance of the wild-type Pseudomonas sp. BS501(pBS501) to cobalt and nickel ions, was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393(pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501(pBS501) (1.2 versus 0.2 μg Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393(pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393(pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici 1818 in a medium containing 0.5 mM cobalt or nickel.

The effect of heavy metal cations (cobalt, nickel, and copper) and the anion detergent sodium dodecyl sulfate (SDS) on the barrier properties of the plasma membrane (PM) of the Co-sensitive strain Pseudomonas putida BS394 and the Co-resistant strains Pseudomonas sp. BS501 (wild-type strain) and Pseudomonas putida BS394 (pBS501) (transformed strain) was studied by high-frequency electro-orientational spectroscopy. The cations were found to rank, in order of decreasing damage inflicted on the PM, as copper > cobalt > nickel. The strains studied were found to rank, in order of increasing resistance of the PM to damage inflicted by copper and cobalt cations, as P. putida BS394 < P. putida BS394 (pBS501) < Pseudomonas sp. BS501. In order of increasing resistance to SDS, the strains ranked inversely. The strains did not differ in sensitivity to nickel cations. Investigation of the surface of intact and trypsin-treated cells by microelectrophoresis showed that the surface layers of the cell wall of wild-type and transformed cells contained increased amounts of proteins. The surface proteins of Co-resistant cells had molecular masses of 49, 40, and 32 kDa. Exposure of Co-resistant cells to trypsin considerably reduced their resistance to cobalt cations. It is assumed that the resistance of the PM of the wild-type and transformed pseudomonads to heavy metal cations is determined by plasmid pBS501 and is related to the synthesis of protective surface proteins of the cell wall.