vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2023-68-2-154-162vestib-872Research ArticleСтатьиМолекулярно-генетическая характеристика мутантного штамма Pseudomonas chlororaphis subsp. аurantiaca с повышенной устойчивостью к пероксиду водородаMolecular and genetic characterization of the Pseudomonas chlororaphis subsp. aurantiaca mutant strain with increased resistance to hydrogen peroxideВеремеенкоЕ. Г.VerameyenkaK. G.

Веремеенко Екатерина Геннадьевна – канд. биол.наук, доцент

пр. Независимости, 4, 220030, г. Минск

Katsiaryna G. Verameyenka – Ph. D. (Biol.), AssociateProfessor

4, Nezavisimosti Ave., 220030, Minsk

veremeenkoKatya@yandex.ruБондареваК. С.BondaravaK. S.

Бондарева Кристина Савельевна – магистрант

Krystsina S. Bondarava – Master student

4, Nezavisimosti Ave., 220030, Minsk

BKristinaSav@yandex.ruЛевданскаяА. И.LiaudanskayaA. I.

Левданская Анастасия Игоревна – ст. преподаватель

Anastasia I. Liaudanskaya – Senior Lecturer

4, Nezavisimosti Ave., 220030, Minsk

liaudanskaya@bsu.byМаксимоваН. П.MaximovaN. P.

Максимова Наталья Павловна – д-р биол. наук, профессор, заведующий кафедрой

Natalia P. Maximova – D. Sc. (Biol.), Professor, Headof the Department

4, Nezavisimosti Ave., 220030, Minsk

nataliamaximova@yahoo.comБелорусский государственный университетBelarusian State University202305052023682154162Copyright © Веремеенко Е.Г., Бондарева К.С., Левданская А.И., Максимова Н.П., 20232023Веремеенко Е.Г., Бондарева К.С., Левданская А.И., Максимова Н.П.Verameyenka K.G., Bondarava K.S., Liaudanskaya A.I., Maximova N.P.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/872

Химический мутагенез, сопровождающийся тщательно продуманной стратегией селекции, представляет собой эффективный способ получения микробных продуцентов разнообразных биологически активных соединений. Однако существенным минусом данного метода является множественность изменений генома, в результате чего впоследствии сложно идентифицировать гены, продукты которых вносят наибольший вклад в образование целевого метаболита. Современные технологии секвенирования и анализа геномов позволяют преодолеть данный недостаток и открывают новые перспективы в идентификации метаболических путей, задействованных в образовании биологически активных соединений.

Целью данной работы являлся геномный анализ и молекулярно-генетическая характеристика мутантного штамма Рseudomonas chlororaphis subsp. aurantiaca B-162/15 для обнаружения потенциальных генов-кандидатов, продукты которых могут принимать участие в обеспечении сверхпродукции феназиновых соединений.

В рамках данного исследования были осуществлены полногеномное секвенирование и аннотация генома мутантного штамма B-162/15 бактерий Р. chlororaphis subsp. aurantiaca. В ходе аннотации было идентифицировано 6493 последовательности, кодирующие белки, и 66 последовательностей, кодирующих транспортные и рибосомальные РНК. При сравнении генома мутантного штамма с ранее отсеквенированным геномом штамма дикого типа B-162 выявлено 16 мутаций. Три из обнаруженных мутаций локализованы в межгенных областях, остальные 13 – в кодирующих областях. Шесть из идентифицированных в кодирующих областях мутаций привели к радикальным заменам аминокислот в структуре белков, что потенциально может оказать влияние на функциональную активность этих белков. Выявлены аминокислотные замены с высоким показателем расстояния Grantham, например, в таких белках, как FliD, железосодержащий редокс-белок и β-субъединица аргинин N-сукцинилтрансферазы. Установлено присутствие в геноме штамма В-162/15 регионов, содержащих фаговые гены.

A whole genome sequencing of natural and mutant producer strains is the best way to analyze the genome and to search for mutations that could cause the acquisition of a number of properties valuable for biotechnological and pharmaceutical industry.

The main goal of current research was to identify mutations that had been induced by chemical mutagenesis in the genome of the mutant strain Рseudomonas chlororaphis subsp. aurantiaca B-162/15 resistant to hydrogen peroxide. It would give an opportunity to discover new genes potentially participating in phenazine compounds biosynthesis. Such an approach also makes it possible to identify genes, whose products do not directly participate in the phenazine synthesis, but influence the phenazine detoxification, excretion, and optimization of antioxidant system activity. Most of all, it could help us to discover new unpredicted enzyme systems that might be involved into this process.

The genome size of P. chlororaphis subsp. aurantiaca B-162/15 was 7109863 b. p. It contained 6493 open reading frames and 66 sequences encoding transport and ribosomal RNA. Comparison of a wild-type strain and B-162/15 mutant genomes revealed 16 mutations, 13 of which were located in coding sequences and 3 were located in intergenic regions. Six mutations led to radical replacements in amino acid sequences of coded proteins (with a Grantham distance of more than 80). We managed to identify four potential gene-candidates, which could influence the phenazine metabolism and provided the ability of mutant strain to superproductivity. They were arginine N-succinyltransferase, phosphoenolpyruvate synthase, iron-contain-ing redox enzyme family protein, membrane-associated proteins in eicosanoid and glutathione metabolism. Three prophage regions were identified, two regions of which were intact and one region was incomplete. The prophage genes, as well as the bacterial genes were inside these regions. We also managed to identify two genes of Agrobacterium tumefaciens inside prophage region 2. It was possible that these regions were introduced into the genome of studied strain by viral transduction. 

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The authors declare that there are no conflicts of interest present.