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Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals

Abstract

Bacteria rely mainly on enzymes, glutathione and other low–molecular weight thiols to overcome oxidative stress. However, hydroxyl radicals are the most cytotoxic reactive oxygen species, and no known enzymatic system exists for their detoxification. We now show that methyl-esterified dimers and trimers of 3-hydroxybutyrate (ME-3HB), produced by bacteria capable of polyhydroxybutyrate biosynthesis, have 3-fold greater hydroxyl radical–scavenging activity than glutathione and 11-fold higher activity than vitamin C or the monomer 3-hydroxybutyric acid. We found that ME-3HB oligomers protect hypersensitive yeast deletion mutants lacking oxidative stress–response genes from hydroxyl radical stress. Our results show that phaC and phaZ, encoding polymerase and depolymerase, respectively, are activated and polyhydroxybutyrate reserves are degraded for production of ME-3HB oligomers in bacteria infecting plant cells and exposed to hydroxyl radical stress. We found that ME-3HB oligomer production is widespread, especially in bacteria adapted to stressful environments. We discuss how ME-3HB oligomers could provide opportunities for numerous applications in human health.

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Figure 1: Structural analysis of ME-3HB dimers and trimers by LC/MS and NMR from bacterial metabolites.
Figure 2: The ME-3HB dimer and trimer have antioxidant activity toward hydroxyl radical stress.
Figure 3: M. extorquens DSM13060 activates 3-HB–processing genes and mobilizes intracellular PHB storages in response to hydroxyl radical stress.
Figure 4: The promoters for phaC and phaZ are activated in bacteria during early stages of host infection.
Figure 5: M. extorquens mobilizes intracellular PHB storage during early stages of host infection.
Figure 6: Infection by the endophyte M. extorquens induces formation of Fenton reagents H2O2 and Fe2+ in pine seedlings.

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Acknowledgements

The authors thank A. C. Frank, J. Lahdenperä, M. V. Tejesvi, A. Hohtola, E. L. Lagendijk, M. Svenning, P. Ardanov, S. Sutela, O. Halonen, J. Pukki and P. Tegelberg. This work was supported by the Academy of Finland (105586, 118569, 129852, 113607), the University of Oulu, the Niemi Foundation and the Tauno Tönning Foundation.

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A.M.P. performed pine viability assays, J.H. and S.M. fractioned, identified and made structural analysis of antioxidant compounds by LC/MS and NMR. M.K., A.T., E.H. and J.J.K. analyzed biosynthesis and structure of ME-3HB oligomers by 13C-labeled methanol. J.J.K., A.M.P., E.H. and O.P. prepared bacterial cultures for identification of ME-3HB oligomers. O.P. and N.K. prepared and provided cultures of Methylobacterium IMBG290 for analysis of ME-3HB oligomers. M.K., H.H., M.P. and S.M. produced, purified, identified and measured concentration of ME-3HB oligomers for HOSC and yeast bioassays. J.J.K. developed and, with assistance from M.S. and J.P., performed and analyzed HOSC assays. J.H.K. and B.C.C. instructed on performance of yeast deletion mutant assays with antioxidants. J.J.K. developed, and with assistance from E.-L.I., performed yeast deletion mutant assay combined with HOSC. J.J.K. and E.-L.I. generated mCherry-tagged strains of M. extorquens 13061 and tracked colonization of pine seedlings by confocal microscopy. J.J.K. planned and executed, assisted by J.P., RT-qPCR, detection of Fenton reagents and Nile blue A staining experiments. A.M.P. and J.J.K. made phylogenetic analysis. J.P. made statistical analyses. A.M.P., S.M. and B.C.C. supervised the work. A.M.P., B.C.C. and J.J.K. wrote the manuscript.

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Correspondence to Anna Maria Pirttilä.

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The authors declare no competing financial interests.

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Koskimäki, J., Kajula, M., Hokkanen, J. et al. Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals. Nat Chem Biol 12, 332–338 (2016). https://doi.org/10.1038/nchembio.2043

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