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"10.29235/1029-8940-2022-67-1-91-104vestib-790Research ArticleСтатьиПротеомное профилирование штаммов-продуцентов феназиновых соединений Pseudomonas chlororaphis subsp. aurantiacаProteomic analysis of Pseudomonas chlororaphis subsp. aurantiacа strains capable of phenasine compounds overproductionВеремеенкоЕ. Г.VerameyenkaK. G.

Веремеенко Екатерина Геннадьевна - кандидат биологических наук, доцент.

пр. Независимости, 4, 220030, Минск.

Katsiaryna G. Verameyenka - Ph. D. (Biol.), Associate Professor, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

veremeenkoKatya@yandex.ruШапироМ. А.ShapiraM. A.

Шапиро Михаил Анатольевич - научный сотрудник.

ул. Академика В.Ф. Купревича, 5/2, 220141, Минск.

Michail A. Shapira – Researcher, Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus.

5/2, Academician V.F. Kuprevich Str., 220141, Minsk.

shapiromihailanatolevich@gmail.comНаумовскаяО. А.NaumouskayaV. A.

Наумовская Ольга Алексеевна - магистрант.

Agencja Rozwoju Aglomeracji Wroclawskiej SA pl. Solny 14, 50-062 Wroclaw.

Volga A. Naumouskaya – Undergraduate, Wroclaw University of Natural Sciences.

Agencja Rozwoju Aglomeracji Wroclawskiej SA pl. Solny 14, 50-062 Wroclaw.

nnn060399@tut.byАшманкевичД. Д.AshmankevichD. D.

Ашманкевич Денис Дмитриевич - специалист.

Товарищеский пер., 2а, 220037, Минск.

Denis D. Ashmankevich – specialist, Center for Examinations and Tests in Health Service Republican Unitary Enterprise.

2a, Tovarishcheski Lane, 220037, Minsk.

denis.ashmankevich@gmail.comМаксимоваН. П.MaximovaN. P.

Максимова Наталья Павловна - доктор биологических наук, профессор, заведующий кафедрой.

пр. Независимости, 4, 220030, Минск.

Natalia P. Maximova - D. Sc. (Biol.), Professor, Head of the Department, Belarusian State University.

4, Nezavisimosti Ave., 220030, Minsk.

nataliamaximova@yahoo.comБелорусский государственный университетBelarusian State UniversityИнститут биоорганической химии НАН БеларусиInstitute of Bioorganic Chemistry of the National Academy of Sciences of BelarusВроцлавский университет естественных наукWroclaw University of Natural SciencesЦентр экспертиз и испытаний в здравоохраненииCenter for Examinations and Tests in Health Service Republican Unitary Enterprise20220202202267191104Copyright © Веремеенко Е.Г., Шапиро М.А., Наумовская О.А., Ашманкевич Д.Д., Максимова Н.П., 20222022Веремеенко Е.Г., Шапиро М.А., Наумовская О.А., Ашманкевич Д.Д., Максимова Н.П.Verameyenka K.G., Shapira M.A., Naumouskaya V.A., Ashmankevich D.D., 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/790

Протеомный анализ - высокоэффективный метод идентификации бактерий и определения содержания белка в прокариотических клетках при различных условиях роста бактериальной культуры. Однако данный подход практически не используется для характеристики продуцентов биологически активных веществ. Использование методов протеомного профилирования при изучении бактерий позволяет получить данные о метаболических процессах, протекающих в клетках прокариот, что дает возможность оптимизировать подходы к созданию продуцентов биологически значимых соединений.

Целью данной работы было проведение протеомного профилирования мутантных штаммов бактерий P. chlororaphis subsp. aurantiaca, способных к сверхпродукции феназиновых антибиотиков, с помощью микробиологических и биохимических методов.

B ходе проведения протеомного анализа штаммов-продуцентов феназиновых антибиотиков бактерий P. chlororaphis subsp. aurantiaca впервые было продемонстрировано достаточно раннее (в log-фазе) начало экспрессии отдельных генов phz-оперона, кодирующего ферменты синтеза феназинов. Наиболее высокое содержание белка PhzO, ген которого находится за пределами phz-оперона, было зарегистрировано для штамма дикого типа. Не обнаружено корреляции между содержанием данного белка и концентрацией продуктов реакции, которую он катализирует. Кроме того, выявлена общая тенденция штаммов-продуцентов к накоплению ферментов и белков, входящих в комплекс системы антиоксидантной защиты. У штаммов-продуцентов также отмечено существенное увеличение содержания белков, принимающих участие в репарации ДНК, и шаперонов, способствующих восстановлению нативной конформации белков.

Proteomic analysis is a highly effective method for bacteria identification and the elucidation of protein's content in prokaryotic cells at different growth conditions. To our knowledge this approach is hardly ever used for characterization of producers of biologically active substances. The understanding of the changes in protein profile in mutant strains capable of biologically active substances overproduction helps to recognize the biochemical and molecular basis of metabolic changes which lead to overproduction. So that, proteomic analysis could be especially useful for optimization the producer's creation techniques.

The purpose of current research was to carry out proteomic profiling of bacteria P. chlororaphis subsp. aurantiaca mutant strains capable of overproduction of phenazine antibiotics. Microbiological and biochemical methods were used for these aims.

In current research a proteomic analysis of strains of P. chlororaphis subsp. aurantiaca producing phenazines was carried out. An early (during log-phase) onset of expression of individual genes of phz-operon which codes enzymes for phenazines synthesis was demonstrated. It was also found that the wild type strain has the highest level of PhzO protein. The gene encoding this protein is located outside the phz-operon. We weren't able to establish the correlation among PhzO protein content and concentration of the derivatives for which appearance PhzO is responsible. A general tendency of producer strains towards the accumulation of enzymes and proteins of the antioxidant defense system was revealed. Producer strains also demonstrate a significant increase in the concentration of proteins involved in DNA repair as well as chaperones involved in the native protein conformation maintenance.

феназиныпротеомphz-оперонантиоксидантные ферментыпероксид водородарепаративные процессысверхпродукциягенphenazinesproteomephz -operonantioxidant enzymeshydrogen peroxidereparative processessuperproductiongeneReferencesВозможности применения протеомного анализа в инфектологии / Н. С. Страшникова [и др.] // Бюл. Сибир. медицины. - 2019. - T. 18, № 2. - С. 248-261.Strashnikova N. S., Martynova G. P., Salmina A. B., Olovyannikova R. Ya., Kutyakov V. A., Tokhidpur A. Possibilities of using proteomic analysis in infectious diseases. Byulleten' Sibirskoi meditsiny [Bulletin of Siberian medicine], 2019, vol. 18, no. 2, pp. 248-261 (in Russian).Беспятых, Ю. А. Протеомные подходы в изучении микобактерий / Ю. А. Беспятых, Е. А. Шитиков, Е. Н. Ильина // Acta Naturae. - 2017. - Т. 9, № 1. - С. 16-26.Bespyatykh Yu. A., Shitikov E. A., Il'ina E. N. Proteomic approaches in the study of mycobacteria. Acta Naturae, 2017, vol. 9, no. 1, pp. 16-26 (in Russian).A proteomics strategy for the analysis of bacterial biodegradation pathways / S. I. L. Kim [et al.] // OMICS: J. Integr. Biol. - 2007. - Vol. 11, N 3. - P. 280-294. https://doi.org/10.1089/omi.2007.0019Kim S. I. L., Choi J. S., Kahng H. Y. A Proteomics strategy for the analysis of bacterial biodegradation pathways. OMICS: Journal of Integrative Biology, 2007, vol. 11, no. 3, pp. 280-294. https://doi.org/10.1089/omi.2007.0019Proteomic analysis of bacterial response to a 4-hydroxybenzylidene indolinone compound, which re-sensitizes bacteria to traditional antibiotics / C. Opoku-Temeng [et al.] // J. Proteomics. - 2019. - Vol. 202. - Art. 103368. https://doi.org/10.1016/j.jprot.2019.04.018Opoku-Temeng C., Onyedibeb K. I., Aryalc U. K., Sintimb H. O. Proteomic analysis of bacterial response to a 4-hydro-xybenzylidene indolinone compound, which re-sensitizes bacteria to traditional antibiotics. Journal of Proteomics, 2019, vol. 202, art. 103368. https://doi.org/10.1016/j.jprot.2019.04.018Synthesis of dibenzo[a,j]phenazine compounds using hemicucurbit[6]uril-catalyzed oxidative dimerization of 2-aryl-amines / Heng Li [et al.] // ChemCatChem. - 2020. - Vol. 12, N 22. - P. 5727-5732. https://doi.org/10.1002/cctc.202000956Heng Li, Juan Yu, Qingmei Ge, Mao Liu, Zhu Tao, Hang Cong. Synthesis of dibenzo[a,j]phenazine compounds using hemicucurbit[6]uril-catalyzed oxidative dimerization of 2-arylamines. ChemCatChem, 2020, vol. 12, no. 22, pp. 5727-5732. https://doi.org/10.1002/cctc.202000956Veremeenko, E. G. Creation and characterization of regulatory mutants of Pseudomonas aurantiaca B-162 resistant to toxic analogues of aromatic amino acids / E. G. Veremeenko // Biodiversity. Ecology. Adaptation. Evolution: materials of III Int. conf. of young scientists. - 2007. - P. 231-232.Veremeenko E. G. Creation and characterization of regulatory mutants of Pseudomonas aurantiaca B-162 resistant to toxic analogues of aromatic amino acids. Biodiversity. Ecology. Adaptation. Evolution: materials of III International conference of young scientists, 2007, pp. 231-232.Veremeenko, E. G. Increase of phenazine antibiotic production in bacteria Pseudomonas aurantiaca by cloning the cluster of PhzIR-genes / E. G. Veremeenko, N. P. Maksimova // Adv. Med. Biol. - 2012. - Vol. 50. - P. 195-206.Veremeenko E. G., Maksimova N. P. Increase of phenazine antibiotic production in bacteria Pseudomonas aurantiaca by cloning the cluster of PhzIR-genes. Advances in Medicine and Biology, 2012, vol. 50, pp. 195-206.Statistical approach to protein quantification / S. Gerster [et al.] // Mol. Cell Proteomics. - 2014. - Vol. 13, N 2. -P. 666-677. https://doi.org/10.1074/mcp.M112.025445Gerster S., Kwon T., Ludwig C., Matondo M., Vogel C., Marcotte E. M., Aebersold R., Buhlmann P Statistical approach to protein quantification. Molecular and Cellular Proteomics, 2014, vol. 13, no. 2, pp. 666-677. https://doi.org/10.1074/mcp.M112.025445.Protein measurement with the folin phenol reagent / O. H. Lowry [et al.] // J. Biol. Chem. - 1951. - Vol. 193, N 1. -P. 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 1951, vol. 193, no. 1, pp. 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa / B. Alqarni [et al.] // J. Microbiol. Meth. - 2016. - Vol. 127. - P. 182-187. https://doi.org/10.1016/j.mimet.2016.06.008Alqarni B., Colley B., Klebensberger Ja., McDougald D., Rice S. A. Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa. Journal of Microbiological Methods, 2016, vol. 127, pp. 182-187. https://doi.org/10.1016/j.mimet.2016.06.008Levitch, M. E. A study of the biosynthesis of phenazine-1-carboxylic acid. / M. E. Levitch, E. R. Stadtman // Arch. Biochem. Biophys. - 1964. - Vol. 106. - P. 194-199. https://doi.org/10.1107/S1744309111012346Levitch M. E., Stadtman E. R. A study of the biosynthesis of phenazine-1-carboxylic acid. Archive Biochemica et Biophysica, 1964, vol. 106, pp. 194-199. https://doi.org/10.1107/S1744309111012346Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation / D. V. Mavrodi [et al.] // Ann. Rev. Phytopathol. - 2006. - Vol. 44. - P. 417-445. https://doi.org/10.1146/annurev.phyto.44.013106.145710Mavrodi D. V., Blankenfeldt W., Thomashow L. S., Mentel M. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annual Review of Phytopathology, 2006, vol. 44, pp. 417-445. https://doi.org/10.1146/annurev.phyto.44.013106.145710Price-Whelan, A. Rethinking “secondary” metabolism: physiological roles for phenazine antibiotics / A. Price-Whelan, L. E. P. Dietrich, D. K. Newman // Nat. Chem. Biol. - 2006. - Vol. 2, N 2. - P. 71-78. https://doi.org/10.1038/nchembio764Price-Whelan A., Dietrich L. E. P., Newman D. K. Rethinking “secondary” metabolism: physiological roles for phenazine antibiotics. Natural Chemical Biology, 2006, vol. 2, no. 2, pp. 71-78. https://doi.org/10.1038/nchembio764Of two make one: the biosynthesis of phenazines / M. Mentel [et al.] // ChemBioChem. - 2009. - Vol. 10, N 14. -P. 2295-2304. https://doi.org/10.1002/cbic.200900323Mentel M., Ahuja E. G., Mavrodi D. V., Breinbauer R., Thomashow L. S., Blankenfeldt W. Of two make one: the biosynthesis of phenazines. ChemBioChem, 2009, vol. 10, no. 14, pp. 2295-2304. https://doi.org/10.1002/cbic.200900323Veremeenko, E. G. Activation of the antioxidant complex in Pseudomonas aurantiaca - producer of phenazine antibiotics / E. G. Veremeenko, N. P. Maksimova // Microbiology. - 2010. - Vol. 79, N 4. - P. 439-444. https://doi.org/10.1134/S0026261710040041Veremeenko E. G., Maksimova N. P. Activation of the antioxidant complex in Pseudomonas aurantiaca - producer of phenazine antibiotics. Microbiology, 2010, vol. 79, no. 4, pp. 439-444. https://doi.org/10.1134/S0026261710040041A comparison of thiol peroxidase mechanisms / L. Flohe [et al.] // Antioxid. Redox Signal. - 2011. - Vol. 15, N 3. -P. 763-780. https://doi.org/10.1089/ars.2010.3397Flohe L., Toppo S., Cozza G., Ursini F. A comparison of thiol peroxidase mechanisms. Antioxidants & Redox Signaling, 2011, vol. 15, no. 3, pp. 763-780. https://doi.org/10.1089/ars.2010.3397Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens / T. Leeper [et al.] // Acta Crystallographica. Sec. F, Struct. boil. crystal. comm. - 2011. - Vol. 67, N 9. - P. 1141-1147. https://doi.org/10.1107/S1744309111012346Leeper T., Zhang S., Van Voorhis W. C., Myler P. J., Varani G. Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens. Acta Crystallographica, Section F, Structural biology and crystallization communications, 2011, vol. 67, no. 9, pp. 1141-1147. https://doi.org/10.1107/S1744309111012346Dubbs, J. M. Peroxiredoxins in bacterial antioxidant defense / J. M. Dubbs, S. Mongkolsuk // Peroxiredoxin Systems. -2007. - Vol. 44. - P. 143-193. https://doi.org/10.1007/978-1-4020-6051-9_7Dubbs J. M., Mongkolsuk S. Peroxiredoxins in bacterial antioxidant defense. Peroxiredoxin Systems, 2007, vol. 44, pp. 143-193. https://doi.org/10.1007/978-1-4020-6051-9_7Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress / T. Shen [et al.] // Mol. BioSyst. - 2013. - Vol. 9, N 1. - P. 121132. https://doi.org/10.1039/c2mb25285fShen T., Rui B., Zhou H., Zhang X., Yi Y., Wen H., Shi Y. Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress. Molecular BioSystems, 2013, vol. 9, no. 1, pp. 121-132. https://doi.org/10.1039/c2mb25285fMullarky, E. Diverting glycolysis to combat oxidative stress / E. Mullarky, L. C. Cantley // Innovative Medicine: Basic Research and Development / eds. : K. Nakao, N. Minato, S. Uemoto. - Tokyo, 2015. - P. 3-23.Mullarky E., Cantley L. C. Diverting glycolysis to combat oxidative stress. Innovative Medicine: Basic Research and Development. Tokyo, 2015, pp. 3-23.Characterization of two signal transduction systems involved in intracellular macrophage survival and environmental stress response in Enterococcus faecalis / C. Muller [et al.] // J. Mol. Microbiol. Biotechnol. - 2008. - Vol. 14. - P. 59-66. https://doi.org/10.1159/000106083Muller C., Sanguinetti M., Riboulet E., Hubert L., Posteraro B., Fadda G., Auffray Y., Rinc A. Characterization of two signal transduction systems involved in intracellular macrophage survival and environmental stress response in Enterococcus faecalis. Journal of Molecular Microbiological Biotechnology, 2008, vol. 14, pp. 59-66. https://doi.org/10.1159/000106083Alkhateeb, A. A. Nuclear ferritin: a new role for ferritin in cell biology / A. A. Alkhateeb, J. R. Connor // Biochim. Biophys. Acta. Gen. Sub. - 2010. - Vol. 1080, N 8. - P. 793-797. https://doi.org/10.1016/j.bbagen.2010.03.017Alkhateeb A. A., Connor J. R. Nuclear ferritin: a new role for ferritin in cell biology. Biochimica et Biophysica Acta (BBA) - General Subjects, 2010, vol. 1080, no. 8, pp. 793-797. https://doi.org/10.1016/j.bbagen.2010.03.017Lund, P. A. Microbial molecular chaperones / P. A. Lund // Adv. Microb. Physiol. - 2001. - Vol. 44. - P. 93-140. https://doi.org/10.1016/s0065-2911(01)44012-4Lund P. A. Microbial molecular chaperones. Advances in Microbiological Physiology, 2001, vol. 44, pp. 93-140. https://doi.org/10.1016/s0065-2911(01)44012-4The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation / C. Chang [et al.] // Biochemistry. -2012. - Vol. 51, N 24. - P. 4822-4834. https://doi.org/10.1021/bi300412yChang C., Myhre R. O., McCallum K. C., Maynard J. A. Entzminger K. C. The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation. Biochemestry, 2012, vol. 51, no. 24, pp. 4822-4834. https://doi.org/10.1021/bi300412yUnal, C. M. Microbial peptidyl-prolyl cis/trans isomerases (ppiases): virulence factors and potential alternative drug targets / C. M. Unal, M. Steinert // Microbiol. Mol. Biol. Rev. - 2014. - Vol. 78, N 3. - P. 544-571. https://doi.org/10.1128/MMBR.00015-14Unal C. M., Steinert M. Microbial peptidyl-prolyl cis/trans isomerases (ppiases): virulence factors and potential alternative drug targets. Microbiology Molecular Biology Review, 2014, vol. 78, no. 3, pp. 544-571. https://doi.org/10.1128/MMBR.00015-14Antony, E. Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes / E. Antony, T. M. Lohman // Sem. Cell Develop. Biol. - 2019. - Vol. 86. - P. 102-111. https://doi.org/10.1016/j.semcdb.2018.03.017Antony E., Lohman T. M. Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes. Seminars of Cell Development Biology, 2019, vol. 86, pp. 102-111. https://doi.org/10.1016/j.semcdb.2018.03.017Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase / R. Zhang [et al.] // Biochemistry. - 1999. - Vol. 38, N 15. - P. 4691-4700. https://doi.org/10.1021/bi982858vZhang R., Evans G., Rotella F. J., Westbrook E. M., Beno D., Huberman E., Chimiak A. J., Collart F. R. Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase. Biochemistry, 1999, vol. 38, no. 15, pp. 46914700. https://doi.org/10.1021/bi982858vRapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa / R. Velasco-Gartfa [et al.] // J. Bacteriol. - 1999. - Vol. 181, N 4. - P. 1292-1300. https://doi.org/10.1128/JB.181.4.1292-1300.1999Velasco-Garda R., Mujica-Jimenez C., Mendoza-Hernandez G., Munoz-Clares R. A. Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa. Journal of Bacteriology, 1999, vol. 181, no. 4, pp. 12921300. https://doi.org/10.1128/JB.181.4.1292-1300.1999Полевой, С. А. Анализ активности ферментов антиоксидантного комплекса у бактерий Pseudomonas chloro-raphis subsp. aurantiaca, устойчивых к пероксиду водорода / С. А. Полевой, Е. Г. Веремеенко // 75-я науч. конф. студентов и аспирантов Белорус. гос. ун-та : материалы конф., Минск, 14-23 мая 2018 г. : в 3 ч. / Белорус. гос. ун-т ; редкол. : В. Г. Сафонов (пред.) [и др.]. - Минск, 2018. - Ч. 2. - С. 330-333.Polevoi S. A., Veremeenko E. G. Analysis of the activity of enzymes of the antioxidant complex in the bacteria Pseudomonas chlororaphis subsp. aurantiaca resistant to hydrogen peroxide. 75-ya nauchnaya konferentsiya studentov i aspirantov Belorusskogo gosudarstvennogo universiteta: materialy konferentsii (Minsk, 14-23 maya 2018 g.). Chast' 2 [The 75th scientific conference of students and postgraduates of the Belarusian State University: conference proceedings (Minsk, May 14-23, 2018). Part 2]. Minsk, 2018, pp. 330-333 (in Russian).

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