Метанокисляющие микроорганизмы донных осадков оказались неожиданно разнообразными

Verrucomicrobium spinosum — представитель группы Verrucomicrobia, которые, как выяснилось, включены в микробные метанокисляющие комплексы. Роль представителей этой группы в природных сообществах чрезвычайно далека от понимания. Фото с сайта microbestiary.org

Американские ученые, применив комплекс новейших методов, исследовали сообщества микроорганизмов в насыщенных метаном донных осадках. Подавляющее большинство этих микроорганизмов относят к некультивируемым, поэтому лабораторное изучение их метаболизма недоступно. Но с помощью предложенных методов оказалось возможно определить, к каким группам принадлежат члены микробного сообщества, какие из них активны и как они связаны друг с другом. Обнаружено, что активное сообщество «метановых» осадков исключительно разнообразно, оно включает все известные группы метанокисляющих архей. Кроме них нашелся известный набор сульфатредукторов, дополненный группой Verrucomicrobia, которую никогда ранее не находили в ассоциации с метанокисляющими комплексами. Но самый важный результат этой работы — то, что новые методы были апробированы на образцах с неизвестным заранее микробным составом. И эти методы прошли «проверку боем».

Публикация в PNAS, выполненная группой специалистов из Калифорнийского технологического института (Пасадена, США) и Объединенного института генома (Уолнат-Крик, США), переносит нас в микробный мир «темной материи». «Темной материей» у микробиологов называется некультивируемая масса микроорганизмов — тех, которые, несмотря на различные ухищрения ученых, не желают расти в лаборатории. Эти микроорганизмы — бактерии, археи, грибы — составляют 80–99% общего микробного разнообразия; с этих позиций мера нашего знакомства с микромиром видится ничтожной.

Любые попытки проникнуть в этот мир исключительно актуальны (см.: Загадочный микроб соглашается расти только в компании, «Элементы», 05.01.2015; Кишечная микрофлора превращает человека в сверхорганизм, «Элементы», 09.06.2006). И если количество слагаемых этой «темной материи» и их облик можно как-то оценить (подсчитать число клеток и увидеть их в микроскоп) и как-то составить представление об их разнообразии (построить деревья по участкам ДНК, полученным с помощью метагеномного анализа), то о функциях и деятельности этих микроорганизмов можно только фантазировать. Представленная на страницах PNAS работа предлагает метод, который отчасти помогает увидеть в том числе и распределение биохимических ролей в этом «тайном обществе» некультивируемых микробов.

Ученые работали с образцами глубоководного океанического грунта из двух разных мест — залива Санта-Моника (Santa Monica Bay) в Калифорнии и Гидратного хребта (Hydrate Ridge) к западу от побережья штата Орегон. В этих районах из глубин к поверхности интенсивно просачивается метан. Нужно отметить, что именно глубоководные осадки вместе с почвами пустынь поставляют наибольшее количество некультивируемых микроорганизмов. Поэтому их исследования практически всегда приносят новую информацию, в особенности если к ним прилагаются новая методика, а также терпение и дотошность самого исследователя.

В данном случае к анализу проб грунта применили три взаимодополняющих метода. Первый из них, получивший наименование BONCAT (Bioorthogonal noncanonical amino acid tagging), был предложен в 2006 году (см. D. C. Dieterich et al., 2006. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)). Его смысл в том, что активная клетка во время синтеза белка может использовать не стандартную аминокислоту, а ее заместитель, приблизительно похожий по химическим свойствам, конфигурации и размерам. В этом случае в клетке появится вещество, угодное ученым, — например, его можно пометить и оценить его количество с помощью тех или иных известных реакций. Это сравнительно новый метод, но он оказался вполне эффективным для определения активности контрольных микробов и их комбинаций.

В данном случае в качестве заместителя аминокислоты метионина подошло вещество HPG (L-homopropargylglycine). Флуоресцентная визуализация этого вещества возможна при взаимодействии алкинов с азидами в присутствии медных катализаторов (см. Азид-алкиновое циклоприсоединение). Таким образом, добавив в пробы HPG и выдержав их в течение часа, а затем соединив с азидными метками, можно под флуоресцентным микроскопом увидеть клетки, которые занимаются синтезом белков, то есть активны. В отличие от имеющихся способов анализа некультивируемых клеток, BONCAT является щадящим методом (клетка остается живой), и это позволяет ее в дальнейшем изучить другими методами.

Второй метод направлен на выяснение принадлежности клеток к той или иной группе микроорганизмов. Для этого часть пробы после BONCAT-процедуры отправляется на метагеномный анализ. Тут можно выяснить общее разнообразие пробы, определив, какие группы в ней представлены.

А часть клеточных агрегатов (а агрегаты отфильтровывались хитрым образом) соединяется со специфическими ДНК-флуоресцентными метками, и это третий метод — флуоресцентная гибридизация in situ. С его помощью определяется, какая из активных клеток к какой группе принадлежит. А какие метки использовать — подсказывают результаты предыдущего анализа, метагеномного. Можно выборочно проверять, имеются ли в агрегатах те или иные интересные бактерии или археи. В результате комбинации этих методов можно выяснить набор активных микроорганизмов в пробе, оценить их количество и то, как активность одних компонентов связана с активностью других.

В принципе, такая комбинация методов была опробована — и вполне успешно — на различных образцах, таких как микробное сообщество полости рта, почвы (см. работу той же лаборатории R. Hatzenpichler et al., 2014. In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry). Что дало возможность публикации в высокорейтинговом журнале новой работы — так это специфичность самих проб. Ученые сделали ставку на исследование микробиоты газогидратов — солидного потенциального источника метанового топлива. То есть эта тема актуальная и важная для налогоплательщиков.

Кроме того, были проведены и эксперименты с пробами осадка. Бактерий из этих проб инкубировали в присутствии метана и без него. Это должно было показать, действительно ли сообщества микроорганизмов занимаются окислением метана и насколько интенсивно.

Результаты, во-первых, показали, что да, метод работает. Действительно, можно увидеть сообщества активных микроорганизмов — архей и бактерий, которые сосуществуют в осадке в местах просачивания метана и окисляют его (см. Анаэробное окисление метана). Все пять групп метанокисляющих архей присутствуют во всех пробах. Вместе с ними определен набор сульфатредукторов, обычных для метанокисляющих сообществ. Во-вторых, в этом консорциуме метанокисляющих микроорганизмов нашлись Verrucomicrobia. Это хорошо известная группа, но она никогда не ассоциировалась с метанокисляющим сообществом. Пока неизвестно, какова роль этих гетеротрофных бактерий в подобных сообществах. Помимо этого, авторы выяснили, что бактерии из проб вполне могут существовать и в отсутствие метана. Однако трудно заключить, является ли это артефактом эксперимента или следствием эврибионтности самого сообщества. Так как ученые не собираются останавливаться после таких успешных результатов, то этот вопрос они наверняка вскоре выяснят.

Методы изучения некультивируемых микроорганизмов пока немногочисленны и мало отработаны. Любое здравое предложение рассматривается и берется в оборот. В этой связи мне хотелось бы упомянуть еще один новаторский подход, потенциально весьма перспективный. Он предложен группой ученых из Калифорнийского Университета (см. С. Brown et al., 2016. In situ replication rates for uncultivated bacteria in microbial communities). Эти специалисты предложили оценивать скорость деления клеток в метагеномной пробе по глубине покрытия геномов отдельных видов. Если клетки делятся быстро, то в пробе ДНК присутствует большое количество полных фрагментов генома этого типа, а если медленно, то фрагментов ДНК немного. В результате в первом случае глубина покрытия окажется высокой, а во втором — низкой, со всеми промежуточными состояниями. Оценив глубину покрытия для разных геномов в пробе, можно понять, какие микроорганизмы в конкретных условиях делятся медленно, а какие быстро. Ученые применили этот метод к ассоциациям микроорганизмов грунтовых вод. Эта работа пока не опубликована, но вынесена для обсуждения в открытых ресурсах, так что у читателей «Элементов» тоже есть возможность обсудить ее.

Источник: elementy.ru

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