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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Jang, S. & Imlay, J.A. Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes. J. Biol. Chem. 282, 929–937 (2007).
Valko, M. et al. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44–84 (2007).
Fahey, R.C. Glutathione analogs in prokaryotes. Biochim. Biophys. Acta 1830, 3182–3198 (2013).
Cabiscol, E., Tamarit, J. & Ros, J. Oxidative stress in bacteria and protein damage by reactive oxygen species. Int. Microbiol. 3, 3–8 (2000).
Imlay, J.A. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat. Rev. Microbiol. 11, 443–454 (2013).
Lushchak, V.I. Adaptive response to oxidative stress: bacteria, fungi, plants and animals. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 153, 175–190 (2011).
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).
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).
Lamb, C. & Dixon, R.A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 251–275 (1997).
Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Fall, R. in Microbial Growth on C1 Compounds, 343–350 (Springer, 1996).
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).
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).
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).
Jendrossek, D. & Handrick, R. Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56, 403–432 (2002).
Lemoigne, M. Produit de déshydratation et de polymérisation de l'acide β-oxybutyrique. Bull. Soc. Chim. Biol. (Paris) 8, 770–782 (1926).
Madison, L.L. & Huisman, G.W. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63, 21–53 (1999).
Lee, S.Y., Lee, Y. & Wang, F. Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals. Biotechnol. Bioeng. 65, 363–368 (1999).
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).
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).
Ren, Q. et al. Simultaneous accumulation and degradation of polyhydroxyalkanoates: futile cycle or clever regulation? Biomacromolecules 10, 916–922 (2009).
Trautwein, K. et al. Solvent stress response of the denitrifying bacterium “Aromatoleum aromaticum” strain EbN1. Appl. Environ. Microbiol. 74, 2267–2274 (2008).
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).
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).
Balsanelli, E. et al. Molecular adaptations of Herbaspirillum seropedicae during colonization of the maize rhizosphere. Environ. Microbiol. doi:10.1111/1462-2920.12887 (2015).
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).
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).
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).
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).
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).
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).
Andersson, S.G. et al. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396, 133–140 (1998).
Chien, M. et al. The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305, 1966–1968 (2004).
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).
Liu, G. et al. Targeted alterations in iron homeostasis underlie plant defense responses. J. Cell Sci. 120, 596–605 (2007).
Cao, C. et al. Polyester modification of the mammalian TRPM8 channel protein: implications for structure and function. Cell Reports 4, 302–315 (2013).
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).
Shimazu, T. et al. Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 339, 211–214 (2013).
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).
Tieu, K. et al. D-β-hydroxybutyrate rescues mitochondrial respiration and mitigates features of Parkinson disease. J. Clin. Invest. 112, 892–901 (2003).
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).
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).
Wider, G. & Dreier, L. Measuring protein concentrations by NMR spectroscopy. J. Am. Chem. Soc. 128, 2571–2576 (2006).
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).
Kehrer, J.P. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 149, 43–50 (2000).
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).
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).
Ostle, A.G. & Holt, J.G. Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate. Appl. Environ. Microbiol. 44, 238–241 (1982).
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).
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).
Markowitz, V.M. et al. IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115–D122 (2012).
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.
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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