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4-2017 А Химия.15.15 Изв. Сарат. ун-та. Нов. сер. Сер. Химия. Биология. Экология. 2017. Т. 17, вып. 4 УДК 579.64: 579.84 КОНСЕРВАТИВНЫЕ И ВАРИАБЕЛЬНЫЕ УЧАСТКИ ГЕНА МЕТАНОЛДЕГИДРОГЕНАЗЫ У ГАЛОТОЛЕРАНТНЫХ ПРЕДСТАВИТЕЛЕЙ РОДА METHYLOPHAGA Д. Ю. Шаравин, А. П. Соломенный Шаравин Дмитрий Юрьевич, инженер лаборатории водной ми- Род Methylophaga (класс Gammaproteo- кробиологии, Институт экологии и генетики микроорганизмов bacteria, порядок Thiotrichales, семейство Pisciric- Уральского отделения РАН (Пермь), кандидат биологических kettsiaceae) был первоначально описан группой наук. E-mail: [email protected] исследователей [5] на основании изучения Соломенный Александр Петрович, старший научный сотрудник двух морских метанол-утилизирующих видов лаборатории водной микробиологии, Институт экологии и гене- M. marina и M. thalassica и к настоящему времени тики микроорганизмов Уральского отделения РАН (Пермь), кан- насчитывает десять видов. Представители рода дидат биологических наук. E-mail: [email protected] существенно отличаются от остальных метило- трофов низким содержанием Г+Ц в тотальной В работе изучены консервативные и вариабельные участки в ДНК (большинство укладывается в диапазон структуре гена mxaF и транслированной аминокислотной по- 43–46 мол.%), тогда как содержание Г+Ц пар у следовательности у выделенного из микробно-растительной ассоциации галотолерантного метилотрофного изолята М1К, от- метилотрофов из класса Alphaproteobacteria на- несённого к роду Methylophaga. В ходе проведённого исследова- ходится в диапазоне 54–72 мол.%, метилотрофов ния с использованием метода минимума эволюции обнаружена Betaproteobacteria – 50–69 мол.%, а метилотрофы гетерогенность mxaF среди исследованных родов метило- и ме- класса Gammaproteobacteria, за исключением танотрофов. Высокая солеустойчивость и зафиксированная спо- рода Methylophaga, имеют содержание Г+Ц собность синтезировать ауксин из L-триптофана делает штамм 58–69 мол.%. Почти все представители рода М1К перспективным для использования в агротехнологии. Methylophaga способны выдержать концентра- Ключевые слова: галотолерантность, метилотрофы, метано- цию NaCl 10%, а некоторые до 20%, что доста- трофы, фитосимбиоз, микроэволюция. точно редко встречается среди метилобактерий DOI: 10.18500/1816-9775-2017-17-4-452-457 [3, 6]. Цель данной работы – изучение гетероген- Введение ности структуры МДГ у галотолерантных мети- Бактерии, использующие окисленные про- лотрофных представителей рода Methylophaga. изводные метана (метанол и метиламин) в ка- честве источников углерода и энергии, играют Материалы и методы глобальную роль в круговороте С1-соединений Объектом исследования являлся галото- [1]. Некоторые из них способны фиксировать лерантный метилотрофный штамм М1К, изо- молекулярный азот, синтезировать фитогормоны, лированный из ризосферы солеустойчивого осмопротекторы и рассматриваются в качестве растения Suaeda prostrata Pall., в районе Верх- перспективных объектов биотехнологии. Фито- некамского месторождения калийно-магниевых симбиотические метилобактерии, колонизируя солей (г. Соликамск, Пермский край, сентябрь поверхность и внутренние ткани растений, могут 2012 г.). Изолят культивировался на жидкой сре- оказывать положительное влияние на рост, раз- де «К» с добавлением 5% NaCl и 1% метанола витие и продуктивность культурных растений [1, в качестве субстрата [3]. Способность штамма 2]. Метилотрофные микроорганизмы окисляют М1К синтезировать индолил-3-уксусную кис- метанол до формальдегида при помощи фер- лоту (ИУК) определена при помощи реактива мента метанолдегидрогеназы (МДГ), который Сальковского после добавления в культураль- представляет собой тетрамер, состоящий из двух ную среду L-триптофана, измерения осущест- больших и двух малых субъединиц, двух молекул вляли в 3–5-кратной повторности [7]. пирролохинолинхинона (PQQ) и одного иона С помощью ПЦР амплифицирована по- Ca2+. Ген mxaF кодирует большую α-субъединицу следовательность гена 16S рРНК со стандарт- МДГ, считается, что последовательность mxaF ными праймерами [8]. Путём компьютерно- достаточно консервативна среди протеобактерий го геномного анализа удалось определить и может отражать их филогенетическое родство, специ фичный фрагмент гена mxaF, подобрать подобно гену 16S рРНК [3, 4]. фланкирующие последовательности, при- ©452 Шаравин Д. Ю., Соломенный А. П., 2017 Научный отдел Д. Ю. Шаравин, А. П. Соломенный. Консервативные и вариабельные участки гена годные в качестве праймеров (MFGmxa2-f mxaF и филогенетически близкие представите- 5’-GGAACGAAACCATGCGTCCTGG и лям рода Methylophaga. MFGmxa2-r 5’-CCCTGGTTGTGGAAACCCAT) и амплифицировать вариабельный фрагмент Результаты и их обсуждение размером 368 пн. Характеристика штамма М1К приведена в Сравнительный анализ нуклеотидных и таблице в сравнении с другими представителями аминокислотных последовательностей mxaF рода Methylophaga. По основным параметрам проводили с помощью компьютерной програм- штамм М1К схож с представителями рода мы MEGA 6.0. Диаграммы построены методом Methylophaga (морфология клетки, допустимое минимума эволюции, показатель достоверности содержание солей в среде культивирования, оп- определен на основании анализа 1000 альтерна- тимальная температура роста, состав клеточных тивных деревьев. В качестве внегрупповых ви- жирных кислот), но отличается по диапазону pH дов представлены метанотрофы (Methylomonas и спектру ростовых субстратов. В эксперименте paludis DSM 24973T, Methylomarinum vadi T2-1T, по оценке синтеза ИУК содержание ауксина Methylococcus capsulatus BathT, Methylohalobius в культуральной жидкости достигало 10 мкг/ crimeensis 10KiT, Methylomicrobium alcaliphilum мл, что является типичным для непатогенных 20ZT, Methylomicrobium japanense NIT, фитосимбиотических метилотрофных микро- Methylomicrobium buryatense 5GT), содержащие организмов [9]. Дифференциальные характеристики штамма М1К (данные этого исследования) и штаммов родственных видов рода Methylophaga. 1, MIK; 2, M. nitratireducenticrescens JAM1T [6]; 3, M. frappieri JAM7T [6]; 4, M. alcalica M39T [10]; 5, M. aminisulfi divorans MPT [11]; 6, M. lonarensis MPLT [12]; 7, M. muralis Kr3T [13] Параметр 123456 7 Размер клетки, 0,6–0,8× 0,6–0,8× 0,2–0,4× 1,2–2,0× 0,7× 0,6×1,5 0,7×1,5 мкм 0,9–1,3 1,4–2,8 0,8–1,2 0,2 1,7–2,0 Подвижность ++++– + + Диап. NaCl, % 0,5–10,0 0,5–8,0 0,5–8,0 0,05–10,0 1,5–9,0 0,05–10,0 0,3–20,0 Оптим. NaCl , % 3,0 3,0 3,0 3–4 3,0 0,5–2,0 3,0–9,0 Диап. pH 7,0–9,0 6,0–11,0 6,0–11,0 7,0–11,0 6,0–8,0 7,0–10,0 6,0–11,0 Оптим. pH 8,0 8,0 8,0 9,0–9,5 6,8–7,0 9,0–10,0 8,0–9,0 Темп. диап., °C 15–37 15–37 15–37 4–35 20–37 20–37 0–42 Оптим. темп., °C 25 30 34 25–29 30 28–30 20–32 Восст. нитратов –+–++ + + Метанол 1% + + + + + + + Метанол 2% + н.д. н.д. н.д. н.д. н.д.+ Метиламин ±––++ – + Диметиламин ± н.д. н.д. н.д.+ н.д. н.д. Триметиламин – н.д. н.д. н.д.+ н.д.+ C16:0, C16:0, C16:0, C16:0, C16:0 C16:0 Доминирующие C16:0 C16:1, C16:1, C16:1 C16:1 C16:1, C16:1 ЖК C16:1 C18:1 C18:1 C18:1 C18:1 C18:1 C18:1 Примечание. н.д. – нет данных. BLAST анализ фрагмента последователь- M. frappieri JAM7T и 85.3% – с M. lonarensis ности гена 16S рРНК (1406 пн) показал, что MPLT. Нуклеотидные последовательности генов штамм M1K имеет 98,3–98,7% сходства с груп- 16S рРНК и mxaF штамма М1К депонированы в пой некультивируемых представителей рода базе данных GenBank под номерами KM192265 Methylophaga и обладает наибольшим сходством и KT728194 соответственно. (98,3%) с Methylophaga nitratireducenticrescens Кластирование последовательностей ге- JAM1T (GenBank № CP003390). Последо- нов 16S рРНК и mxaF (рис. 1) обнаружило вательность гена mxaF у штамма M1K об- группирование пар между штаммами M1K ладает высокой степенью сходства (97,6%) с и JAM1T вида M. nitratireducenticrescens, а так- M. nitratireducenticrescens JAM1T, 90,4% – с же между M. aminisulfi divorans MPT и M. thio- Биология 453 Изв. Сарат. ун-та. Нов. сер. Сер. Химия. Биология. Экология. 2017. Т. 17, вып. 4 oxydans DMS010T. Обнаруженная дивергенция ванием алгоритма минимальной эволюции от M. lonarensis MPLT совпадает в обоих по- графов, построенных с применением метода строениях. Полученные диаграммы в целом максимального правдоподобия, когда класте- сходны по порядку объединения в кластеры, ризация последовательностей генов mxaF и однако наблюдаются расхождения в положении 16S рРНК происходила идентично. Это позво- M. alcalica M39T и M. frappieri JAM7T, что от- ляет корректно обсуждать параметры эволюци- личает филограммы, построенные с использо- онной изменчивости гена mxaF. 92 Methylophaga nitratireducenticrescens M1K (KM192265) 58 Methylophaga nitratireducenticrescens JAM1T (CP003390) 91 Ⱥ Methylophaga alcalica M39T (AF384373) 45 Methylophaga frappieri JAM7T (CP003380) 54 Methylophaga thiooxydans DMS010T (DQ660915) 100 Methylophaga aminisulfidivorans MPT (NR043871) Methylophaga lonarensis MPLT (JF330773) Methylomarinum vadi T2-1T (AB453958) 100 Methylomonas paludis DSM 24973T (HE801216) 95 Methylomicrobium buryatense 5GT (AF096092) 100 T 97 Methylomicrobium alcaliphilum 20Z (NR 074649) Methylomicrobium japanense NIT (NR 043450) T 100 Methylococcus capsulatus Bath (AE017282) Methylohalobius crimeensis 10KiT (AJ581837) а 0.01 100 Methylophaga nitratireducenticrescens M1K (KT728194) Ȼ 87 Methylophaga nitratireducenticrescens JAM1T (CP003390) 79 Methylophaga frappieri JAM7T (CP003380) Methylophaga aminisulfidivorans MPT (EF378629) 85 88 Methylophaga thiooxydans DMS010T (EU001860) 99 Methylophaga alcalica M39T (EU001862) Methylophaga lonarensis MPLT (JF795948) Methylomonas paludis DSM 24973 T (HE801218) 89 Methylohalobius crimeensis 10Ki T (NZ ATXB01000001) 57
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