Analysis of Protease Effect on Biofilm Structure of Azospirillum Brasilense Strain Sp245 and Its Flagellation-Defective mmsB1 and fabG1 Mutants
Azospirillum bacteria are engaged in associative interactions with a wide range of plants. In this type of interaction, there formed no specialized structures like nodules, which are characteristic of the legume-Rhizobium symbiosis. The formation of biofilms by Azospirillum on the plant root surface can be important for the successful functioning of plant-microbe associations. Scarce data exist on the role of cell surface protein structures in the formation and stabilization of Azospirillum biofilms. It is known that as compared to A. brasilense Sp245, its flagellation-defective mmsB1 and fabG1 mutants form biofilms on a hydrophobic surface (polystyrene) worse and only mutant mmsB forms thinner biofilms on a hydrophilic surface (glass). The aim of this work was to examine the role of protease-sensitive nonflagellar protein structures in the stabilization of biomass of Azospirillum biofilms. The presence of contacts between cells and the thickness/biomass of biofilms before and after protease treatment were determined by direct microscopic examination and by crystal violet staining of biofilms. The quantitative measure of biofilm biomass thickness was the А590 of a solution of desorbed dye. The hemagglutinating activity of suspension of biofilms washed off the glass surface was determined by using trypsinized rabbit erythrocytes. In this study, it was shown that the protein components of the Azospirillum cell surface which are different from flagella and are sensitive to pronase and trypsin are necessary for strong connection of bacteria in biofilms on glass and polystyrene. These proteins contribute to the attachment of biofilms to polystyrene under a rich liquid medium and probably are hemagglutinins of Azospirillum. Our previous data show that the protein structures that stabilize biofilms on abiotic surfaces can function similarly with biofilms formed by azospirilla on the roots of plants.
1. Fibach-Paldi S., Burdman S., Okon Y. Key physiological properties contributing to rhizosphere adaptation and plant growth promoting abilities of Azospirillum brasilense // FEMS Microbiol. Lett. 2012. Vol. 326, № 2. P. 99–108.
2. Pedrosa F. O. Physiology, biochemistry and genetics of Azospirillum and other root-associated nitrogen-fi xing bacteria // Crit. Rev. Plant Sci. 1988. Vol. 6, № 4. P. 345–384.
3. ПетроваЛ. П., Шелудько А. В., КацыЕ. И. Плазмидные перестройки и изменения в формировании биопленок Azospirillum brasilense // Микробиология. 2010. Т. 79, № 1. С. 129–132.
4. Шелудько А. В., Широков А. А., Соколова М. К., Соколов О. И., Петрова Л. П., Матора Л. Ю., Кацы Е. И. Колонизация корней пшеницыбактериями Azospirillum brasilense с различной подвижностью // Микробио- логия. 2010. Т. 79, № 5. С. 696–704.
5. Ramey B. E., Koutsoudis M., Bodman S. B. von, Fuqua C. Biofi lm formation in plant–microbe associations // Curr. Opin. Microbiol. 2004. Vol. 7, № 6. P. 602–609.
6. Lopez D., Vlamakis H., Kolter R. Biofi lms // Cold Spring Harb. Perspect. Biol. 2010. Vol. 2, № 7. P. a000398.
7. Flemming H.-C., Wingender J. The biofi lm matrix // Nature Rev. Microbiology. Vol. 8. P. 623–633.
8. Шелудько А. В., Кулибякина О. В., Широков А. А., Петрова Л. П., Матора Л. Ю., Кацы Е. И. Влияние мутаций в синтезе липополисахаридов и полисахаридов, связывающих калькофлуор, на формирование биопленок Azospirillum brasilense // Микробиология. 2008. Т. 77, № 3. C. 358–363.
9. Wisniewski-Dye F., Borziak K., Khalsa-Moyers G., Alexandre G., Sukharnikov L. O., Wuichet K., Hurst G. B., McDonald W. H., Robertson J. S., Barbe V., Calteau A., Rouy Z., Mangenot S., Prigent-Combaret C., Normand P., Boyer M., Siguier P., Dessaux Y., Elmerich C., Condemine G., Krishnen G., Kennedy I., Paterson A. H., Gonzalez V., Mavingui P., Zhulin I. B. Azospirillum genomes reveal transition of bacteria from aquatic to terrestrial environments // PLoS Genetics. 2011. Vol. 7, № 12. P. e1002430.
10. Шелудько А. В., Филипьечева Ю. А., Шумилова Е. М., Хлебцов Б. Н., Буров А. М., Л. П. Петрова Л. П., Кацы Е. И. Изменения в формировании биопленок у fl hB1 мутанта бактерии Azospirillum brasilense Sp245, лишенного жгутиков // Микробиология. 2015. Т. 84, № 2. C. 175–183.
11. Шумилова Е. М., Шелудько А. В., Филипьечева Ю. А., Евстигнеева С. С., Пономарева Е. Г., Петрова Л. П., Кацы Е. И. Изменение свойств клеточной поверхности и эффективности формирования биопленок у мутантов бактерии Azospirillum brasilense Sp245 по предполага- емым генам липидного метаболизма mmsB1 и fabG1 // Микробиология. 2016. Т. 85, № 2. С. 162–170.
12. Ковтунов Е. А., Шелудько А. В., Чернышова М. П., Петрова Л. П., Кацы Е. И. Мутанты бактерии Azospirillum brasilense Sp245 со вставкой омегона в генах липидного метаболизма mmsB или fabG дефектны по подвижности и жгутикованию // Генетика. 2013. Т. 49, № 11. С. 1270–1275.
13. Baldani V. L. D., Baldani J. I., Dobereiner J. Effects of Azospirillum inoculation on root infection and nitrogen incorporation in wheat // Can. J. Microbiol. 1983. Vol. 29, № 8. P. 924–929.
14. Dobereiner J., Day J. M. Associative symbiosis in tropical grass: Characterization of microorganisms and dinitrogen fi xing sites // Symposium on Nitrogen Fixation / eds. W. E. Newton, C. J. Nijmans. Pullman : Washington State University Press, 1976. P. 518–538.
15. Sambrook J., Fritsch E. F., Maniatis T. Molecular Cloning : a Laboratory Manual. 2nd ed. Cold Spring Harbor : Cold Spring Harbor Laboratory Press, 1989.
16. Никитина В. Е., Пономарева Е. Г., АленькинаС. А., Кон- нова С. А. Участие бактериальных лектинов клеточной поверхности в агрегации азоспирилл // Микробиоло- гия. 2001. Т. 70, № 4. С. 471–476.
17. Шелудько А. В., Пономарева Е. Г., Варшаломидзе О. Э., ВетчинкинаЕ. П., КацыЕ. И., НикитинаВ. Е. Гемагглютинирующая активность и подвижность бактерий Azospirillum brasilense в присутствии разных источников азота // Микробиология. 2009. Т. 78, № 6. С. 749–756.