The aim of our work was to study the colonization of potato, tomato, rapeseed and camelina by associative microorganisms Methylobacterium mesophilicum, Pseudomonas aureofaciens BS1393 and Pseudomonas putida BS3701; examine the resistance of colonized plants to biotic (phytopathogens Erwinia carotovora and Sclerotinia sclerotiorum) and abiotic (naphthalene, oil) stressors. Colonized plants were characterized by an increased growth rate (1.5–2.0 times higher) compared to non-colonized ones; flower-bud formation, flowering and fructification of the colonized plants also started earlier. An increased resistance of colonized plants to phytopathogens, naphthalene (100 mµ/ml) and oil (0.7 %) was noted, too. The level of superoxide dismutase (SOD) in control plants on a medium with naphthalene or oil increased by 160–150%; in colonized plants – by 20–18 %. Colonized plants were more viable because of the presence of P. putida BS3701 on the roots.

The in vivo and in vitro interactions between tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) and the bacteria Pseudomonas fluorescens, Acinetobacter baumannii, Rhodococcus erythropolis,Pseudomonas aureofaciens, Pseudomonas putida, Methylovorus mays have been studied. These microorganisms were shown to be in stable associations with plants. The colonized plants were characterized by more rapid growth, a higher yield, and better adaptation to in vivo conditions. The colonized plants were more resistant to bacterial phytopathogens Erwinia carotovora and Pseudomonas syringae. Plants colonized by naphthalene-resistant bacteria can grow steadily on a medium containing this compound. The results demonstrate the prospects of the use of beneficial associative microorganisms in the development of technologies for plant protection against biotic and abiotic stressors.

Изучено влияние концентрации фосфата в среде культивирования на рост и процесс деградации нафталина Pseudomonas putida BS3701. В среде с глюкозой лимитирование роста бактерии наблюдали при концентрации фосфата 0.1 мМ, а в среде с нафталином – при 0.4 мМ. При недостатке фосфата снижалась активность нафталиндиоксигеназы и салицилатгидроксилазы, а также в среде культивирования накапливался салицилат. При деградации нафталина на среде с фосфатом в клетках P. putida BS3701 накапливалось втрое больше полифосфатов, чем на среде с глюкозой. Полученные данные свидетельствуют о том, что при дефиците фосфата нарушается регуляция экспрессии генов “верхнего” и “нижнего” пути окисления нафталина. Обсуждается участие полифосфатов в регуляции метаболизма нафталина.

The non-homologous salicylate hydroxylases NahG and NahU of the strains Pseudomonas fluorescens 142 NF and P. putida BS3701 were extracted and purified by ion-exchange and hydrophobic and gel permeation chromatography. The purified enzymes differed in kinetic and catalyst characteristics during salicylate hydrolysis. For NahU salicylate hydroxylase, K m and V max were found to be higher (3.1 ± 0.6 μM and 7.7 ± 0.4 μM/min, respectively) than for NahG salicylate hydroxylase (1.3 ± 0.1 μM and 4.7 ± 0.1 μM/min, respectively). The activity of both enzymes toward substituted salicylates was higher in cases where the substituent groups were in para-position than in cases with those in meta-position. The activity toward substituted salicylates with substituent groups in meta position was different. The activity of salicylate hydroxylase NahG was higher toward salicylates with substituent groups in position 3; salicylate hydroxylase NahU activity was higher toward those with substituent groups in position 5. This suggests about a difference in the spatial configuration of active sites of the purified non-homologous salicylate hydroxylases.

The effect of phosphate concentration in the culture medium on the growth and naphthalene degradation by Pseudomonas putida BS 3701 was studied. The limiting concentration of phosphate was 0.4 mM and 0.1 mM under cultivation in media with naphthalene and glucose, respectively The phosphate deficiency correlated with a decrease in the activities of naphthalene dioxygenase and salicylate hydroxylase and with salicylate accumulation in the culture medium. We suggest that this fact indicates the impaired regulation of gene expression of "upper" and "lower" pathways of naphthalene oxidation. Under naphthalene degradation, the cells accumulated three times more inorganic polyphosphates as compared with the consumption of glucose. The involvement of polyphosphates in the regulation of naphthalene metabolism has been considered.

The unrelated salicylate hydroxylases NahG and NahU of the strains Pseudomonasfluorescens 142 NF and P. Putida BS3701 were extracted and purified by ion-exchange and hydrophobic and gel permeation chromatography. The extracted enzymes differed in kinetic and catalyst performance during salicylate hydrolysis. For NahU salicylate hydroxylase, Km and Vmax were found to be higher (3.1 +/- 0.6 microM and 7.7 +/- 0.4 microM/min, respectively) than for NahG salicylate hydroxylase (1.3 +/- 0.1 microM and 4.7 +/- 0.1 microM/min, respectively). The activity of both enzymes toward substituted salicylates was higher in cases where the substituent groups were in para position than in cases with those in meta position. The activity toward substituted salicylates with substituent groups in meta position was different. The activity of salicylate hydroxylase NahG was higher toward salicylates with substituent groups in position 3; salicylate hydroxylase NahU activity was higher toward those with substituent groups in position 5. This suggests a difference in the spatial configuration of active sites in extracted unrelated salicylate hydroxylases.

Из штаммов Pseudomonas fluorescens 142NF и P. putida BS3701 методами ионообменной, гидрофобной хроматографии и гель-фильтрации выделены и очищены до гомогенного состояния негомологичные салицилатгидроксилазы NahG и NahU. Выделенные ферменты различались по кинетическим и каталитическим характеристикам при гидролизе салицилата. Показано, что салицилатгидроксилаза NahU характеризовалась более высокими значениями Км и Vмах (3.1 ± 0.6 мкМ и 7.7 ± 0.4 мкмоль/мин соответственно) по сравнению с салицилатгидроксилазой NahG (1.3 ± 0.1 мкМ и 4.7 ± 0.1 мкмоль/мин). Активность обоих ферментов по отношению к замещенным салицилатам, у которых группа заместителя находилась в пара-положении, была выше, чем по отношению к салицилатам с группой заместителя в мета-положении. Активность ферментов по отношению к замещенным салицилатам, содержащим группу заместителя в мета-положении, была различной. При этом активность салицилатгидроксилазы NahU была выше по отношению к салицилатам, содержащим группу заместителя в положении 3, а салицилатгидроксилазы NahG – по отношению к салицилатам, имеющим группу заместителя в положении 5. Это может свидетельствовать о различной пространственной конфигурации активного центра выделенных негомологичных салицилатгидроксилаз.