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

Nature Chemical Biology volume 12, pages 332–338 (2016)Cite this article

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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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References

  1. Jang, S. & Imlay, J.A. Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes. J. Biol. Chem. 282, 929–937 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Valko, M. et al. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44–84 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Fahey, R.C. Glutathione analogs in prokaryotes. Biochim. Biophys. Acta 1830, 3182–3198 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Cabiscol, E., Tamarit, J. & Ros, J. Oxidative stress in bacteria and protein damage by reactive oxygen species. Int. Microbiol. 3, 3–8 (2000).

    CAS  PubMed  Google Scholar 

  5. Imlay, J.A. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat. Rev. Microbiol. 11, 443–454 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lushchak, V.I. Adaptive response to oxidative stress: bacteria, fungi, plants and animals. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 153, 175–190 (2011).

    Article  PubMed  CAS  Google Scholar 

  7. Wesche, A.M., Gurtler, J.B., Marks, B.P. & Ryser, E.T. Stress, sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens. J. Food Prot. 72, 1121–1138 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Dwyer, D.J. et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proc. Natl. Acad. Sci. USA 111, E2100–E2109 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lamb, C. & Dixon, R.A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 251–275 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Santos, R. et al. Essential role of superoxide dismutase on the pathogenicity of Erwinia chrysanthemi strain 3937. Mol. Plant Microbe Interact. 14, 758–767 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Thi, E.P., Lambertz, U. & Reiner, N.E. Sleeping with the enemy: how intracellular pathogens cope with a macrophage lifestyle. PLoS Pathog. 8, e1002551 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tanaka, A., Christensen, M.J., Takemoto, D., Park, P. & Scott, B. Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. Plant Cell 18, 1052–1066 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Koskimäki, J.J., Pirttilä, A.M., Ihantola, E.L., Halonen, O. & Frank, A.C. The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins. MBio 6, e00039–15 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Pirttilä, A.M., Laukkanen, H., Pospiech, H., Myllylä, R. & Hohtola, A. Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization. Appl. Environ. Microbiol. 66, 3073–3077 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Pirttilä, A.M., Pospiech, H., Laukkanen, H., Myllylä, R. & Hohtola, A. Seasonal variations in location and population structure of endophytes in buds of Scots pine. Tree Physiol. 25, 289–297 (2005).

    Article  PubMed  Google Scholar 

  17. Pohjanen, J. et al. Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro. Tree Physiol. 34, 993–1005 (2014).

    Article  PubMed  Google Scholar 

  18. Pirttilä, A.M., Joensuu, P., Pospiech, H., Jalonen, J. & Hohtola, A. Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures. Physiol. Plant. 121, 305–312 (2004).

    Article  PubMed  Google Scholar 

  19. Pirttilä, A.M., Podolich, O., Koskimäki, J.J., Hohtola, E. & Hohtola, A. Role of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus sylvestris L.). Plant Cell Tissue Organ Cult. 95, 47–55 (2008).

    Article  CAS  Google Scholar 

  20. Fall, R. in Microbial Growth on C1 Compounds, 343–350 (Springer, 1996).

  21. Haces, M.L. et al. Antioxidant capacity contributes to protection of ketone bodies against oxidative damage induced during hypoglycemic conditions. Exp. Neurol. 211, 85–96 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Moore, J., Yin, J.J. & Yu, L.L. Novel fluorometric assay for hydroxyl radical scavenging capacity (HOSC) estimation. J. Agric. Food Chem. 54, 617–626 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Khosravi-Darani, K., Mokhtari, Z.B., Amai, T. & Tanaka, K. Microbial production of poly(hydroxybutyrate) from C1 carbon sources. Appl. Microbiol. Biotechnol. 97, 1407–1424 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Jendrossek, D. & Handrick, R. Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56, 403–432 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Lemoigne, M. Produit de déshydratation et de polymérisation de l'acide β-oxybutyrique. Bull. Soc. Chim. Biol. (Paris) 8, 770–782 (1926).

    CAS  Google Scholar 

  26. Madison, L.L. & Huisman, G.W. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63, 21–53 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lee, S.Y., Lee, Y. & Wang, F. Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals. Biotechnol. Bioeng. 65, 363–368 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Kawata, Y., Kawasaki, K. & Shigeri, Y. Efficient secreted production of (R)-3-hydroxybutyric acid from living Halomonas sp. KM-1 under successive aerobic and microaerobic conditions. Appl. Microbiol. Biotechnol. 96, 913–920 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Doi, Y., Segawa, A., Kawaguchi, Y. & Kunioka, M. Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol. Lett. 55, 165–169 (1990).

    Article  CAS  PubMed  Google Scholar 

  30. Ren, Q. et al. Simultaneous accumulation and degradation of polyhydroxyalkanoates: futile cycle or clever regulation? Biomacromolecules 10, 916–922 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Trautwein, K. et al. Solvent stress response of the denitrifying bacterium “Aromatoleum aromaticum” strain EbN1. Appl. Environ. Microbiol. 74, 2267–2274 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, H., Tomasch, J., Jarek, M. & Wagner-Döbler, I. A dual-species co-cultivation system to study the interactions between Roseobacters and dinoflagellates. Front. Microbiol. 5, 311 (2014).

    PubMed  PubMed Central  Google Scholar 

  33. Krol, E. & Becker, A. Global transcriptional analysis of the phosphate starvation response in Sinorhizobium meliloti strains 1021 and 2011. Mol. Genet. Genomics 272, 1–17 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Balsanelli, E. et al. Molecular adaptations of Herbaspirillum seropedicae during colonization of the maize rhizosphere. Environ. Microbiol. doi:10.1111/1462-2920.12887 (2015).

  35. Wang, Q., Yu, H., Xia, Y., Kang, Z. & Qi, Q. Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application. Microb. Cell Fact. 8, 47 (2009).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  36. Raberg, M., Voigt, B., Hecker, M. & Steinbüchel, A. A closer look on the polyhydroxybutyrate- (PHB-) negative phenotype of Ralstonia eutropha PHB-4. PLoS ONE 9, e95907 (2014).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Aneja, P., Zachertowska, A. & Charles, T.C. Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants. Can. J. Microbiol. 51, 599–604 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Aurass, P. et al. bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection. Appl. Environ. Microbiol. 75, 4506–4515 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kadouri, D., Jurkevitch, E. & Okon, Y. Involvement of the reserve material poly-β-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl. Environ. Microbiol. 69, 3244–3250 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhao, Y.H., Li, H.M., Qin, L.F., Wang, H.H. & Chen, G.Q. Disruption of the polyhydroxyalkanoate synthase gene in Aeromonas hydrophila reduces its survival ability under stress conditions. FEMS Microbiol. Lett. 276, 34–41 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Andersson, S.G. et al. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396, 133–140 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Chien, M. et al. The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305, 1966–1968 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Silva-Gomes, S., Vale-Costa, S., Appelberg, R. & Gomes, M.S. Iron in intracellular infection: to provide or to deprive? Front. Cell. Infect. Microbiol. 3, 96 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Liu, G. et al. Targeted alterations in iron homeostasis underlie plant defense responses. J. Cell Sci. 120, 596–605 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Cao, C. et al. Polyester modification of the mammalian TRPM8 channel protein: implications for structure and function. Cell Reports 4, 302–315 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Klocker, A.A., Phelan, H., Twigg, S.M. & Craig, M.E. Blood β-hydroxybutyrate vs. urine acetoacetate testing for the prevention and management of ketoacidosis in type 1 diabetes: a systematic review. Diabet. Med. 30, 818–824 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Shimazu, T. et al. Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 339, 211–214 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Cheng, B., Lu, H., Bai, B. & Chen, J. D-β-hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress. Neurochem. Int. 62, 620–625 (2013).

    Article  CAS  PubMed  Google Scholar 

  49. Tieu, K. et al. D-β-hydroxybutyrate rescues mitochondrial respiration and mitigates features of Parkinson disease. J. Clin. Invest. 112, 892–901 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang, J. et al. 3-hydroxybutyrate methyl ester as a potential drug against Alzheimer's disease via mitochondria protection mechanism. Biomaterials 34, 7552–7562 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Athlan, A., Braud, C. & Vert, M. Abiotic aging of water-soluble 3-hydroxybutyric acid oligomers as monitored by capillary zone electrophoresis. J. Environ. Polym. Degrad. 5, 243–247 (1997).

    CAS  Google Scholar 

  52. Wider, G. & Dreier, L. Measuring protein concentrations by NMR spectroscopy. J. Am. Chem. Soc. 128, 2571–2576 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Ou, B., Hampsch-Woodill, M. & Prior, R.L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 49, 4619–4626 (2001).

    Article  CAS  PubMed  Google Scholar 

  54. Kehrer, J.P. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 149, 43–50 (2000).

    Article  CAS  PubMed  Google Scholar 

  55. Kim, J.H. et al. Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link. Appl. Microbiol. Biotechnol. 67, 807–815 (2005).

    Article  CAS  PubMed  Google Scholar 

  56. Talaat, A.M., Hunter, P. & Johnston, S.A. Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nat. Biotechnol. 18, 679–682 (2000).

    Article  CAS  PubMed  Google Scholar 

  57. Ostle, A.G. & Holt, J.G. Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate. Appl. Environ. Microbiol. 44, 238–241 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Mucha, J., Guzicka, M., Łakomy, P. & Zadworny, M. Iron and reactive oxygen responses in Pinus sylvestris root cortical cells infected with different species of Heterobasidion annosum sensu lato. Planta 236, 975–988 (2012).

    Article  CAS  PubMed  Google Scholar 

  59. Thordal-Christensen, H., Zhang, Z., Wei, Y. & Collinge, D.B. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J. 11, 1187–1194 (1997).

    Article  CAS  Google Scholar 

  60. Markowitz, V.M. et al. IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115–D122 (2012).

    Article  CAS  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. Genetics and Physiology, University of Oulu, Oulu, Finland

    Janne J Koskimäki, Emmi-Leena Ihantola, Elina Hankala, Marko Suokas, Johanna Pohjanen & Anna Maria Pirttilä

  2. Department of Chemistry, University of Oulu, Oulu, Finland

    Marena Kajula, Ari Turpeinen, Mirva Pääkkönen & Sampo Mattila

  3. Admescope Ltd., Oulu, Finland

    Marena Kajula, Juho Hokkanen & Heidi Hautajärvi

  4. Institute of Clinical Microbiology, University of Eastern Finland, Kuopio, Finland

    Emmi-Leena Ihantola

  5. Department of Agriculture, Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, Albany, California, USA

    Jong H Kim & Bruce C Campbell

  6. Institute of Molecular Biology and Genetics of the National Academy of Sciences Ukraine, Kyiv, Ukraine

    Olga Podolich & Natalia Kozyrovska

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Contributions

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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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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