Applied Microbiology and Biotechnology

, Volume 103, Issue 13, pp 5355–5366 | Cite as

The effect of reactive oxygen species (ROS) and ROS-scavenging enzymes, superoxide dismutase and catalase, on the thermotolerant ability of Corynebacterium glutamicum

  • Nawarat NantapongEmail author
  • Ryutarou Murata
  • Sarvitr Trakulnaleamsai
  • Naoya Kataoka
  • Toshiharu Yakushi
  • Kazunobu Matsushita
Applied microbial and cell physiology


The function of two reactive oxygen species (ROS) scavenging enzymes, superoxide dismutase (SOD) and catalase, on the thermotolerant ability of Corynebacterium glutamicum was investigated. In this study, the elevation of the growth temperature was shown to lead an increased intracellular ROS for two strains of Corynebacterium glutamicum, the wild-type (KY9002) and the temperature-sensitive mutant (KY9714). In order to examine the effects of ROS-scavenging enzymes on cell growth, either the SOD or the catalase gene was disrupted or overexpressed in KY9002 and KY9714. In the case of the KY9714 strain, it was shown that the disruption of SOD and catalase disturbs cell growth, while the over-productions of both the enzymes enhances cell growth with a growth temperature of 30 °C and 33 °C. Whereas, in the relatively thermotolerant KY9002 strain, the disruption of both enzymes exhibited growth defects more intensively at higher growth temperatures (37 °C or 39 °C), while the overexpression of at least SOD enhanced the cell growth at higher temperatures. Based on the correlation between the cell growth and ROS level, it was suggested that impairment of cell growth in SOD or catalase-disrupted strains could be a result of an increased ROS level. In contrast, the improvement in cell growth for strains with overexpressed SOD or catalase resulted from a decrease in the ROS level, especially at higher growth temperatures. Thus, SOD and catalase might play a crucial role in the thermotolerant ability of C. glutamicum by reducing ROS-induced temperature stress from higher growth temperatures.


Corynebacterium glutamicum Reactive oxygen species (ROS) Superoxide dismutase (SOD) Catalase Thermotolerant 



The authors wish to thank Sayoko Tanigawa and Seisuke Hatakeyama for their technical support. Part of this work was performed through collaboration with the Core to Core Program, which was supported by the Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT).

Funding information

This work was supported financially by the Advanced Low Carbon Technology Research and Development Program (ALCA: JPMJAL1106) of Japan Science and Technology Agency (JST).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9848_MOESM1_ESM.pdf (1.3 mb)
ESM 1 (PDF 1.28 mb)


  1. Abei H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefGoogle Scholar
  2. Chang R, Lv B, Li B (2017) Quantitative proteomics analysis by iTRAQ revealed underlying changes in thermotolerance of Arthrospira platensis. J Proteome 165:119–131CrossRefGoogle Scholar
  3. Cohen SN, Chang AC, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci 69(8):2110–2114CrossRefGoogle Scholar
  4. Davidson JF, Schiestl RH (2001) Mitochondrial respiratory electron carriers are involved in oxidative stress during heat stress in Saccharomyces cerevisiae. Mol Cell Biol 21(24):8483–8489CrossRefGoogle Scholar
  5. Davidson JF, Whyte B, Bissinger PH, Schiestl RH (1996) Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc Natl Acad Sci 93(10):5116–5121CrossRefGoogle Scholar
  6. Doi H, Hoshino Y, Nakase K, Usuda Y (2014) Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli. Appl Microbiol Biotechnol 98(2):629–639CrossRefGoogle Scholar
  7. Dulley J, Grieve P (1975) A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal Biochem 64(1):136–141CrossRefGoogle Scholar
  8. Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke K-U, Sahm H (1994) Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiol 140(8):1817–1828CrossRefGoogle Scholar
  9. El Shafey HM, Ghanem S (2015) Regulation of expression of sodA and msrA genes of Corynebacterium glutamicum in response to oxidative and radiative stress. Genet Mol Res 14(1):2104–2117CrossRefGoogle Scholar
  10. El Shafey HM, Ghanem S, Merkamm M, Guyonvarch A (2008) Corynebacterium glutamicum superoxide dismutase is a manganese-strict non-cambialistic enzyme in vitro. Microbiol Res 163(1):80–86CrossRefGoogle Scholar
  11. Escuyer V, Haddad N, Frehel C, Berche P (1996) Molecular characterization of a surface-exposed superoxide dismutase of Mycobacterium avium. Microb Pathog 20(1):41–55CrossRefGoogle Scholar
  12. Fedoseeva IV, Pyatrikas DV, Stepanov AV, Fedyaeva AV, Varakina NN, Rusaleva TM, Borovskii GB, Rikhvanov EG (2017) The role of flavin-containing enzymes in mitochondrial membrane hyperpolarization and ROS production in respiring Saccharomyces cerevisiae cells under heat-shock conditions. Sci Rep 7(1):2586CrossRefGoogle Scholar
  13. Flohe L, Ötting F (1984) Superoxide dismutase assays. Methods Enzymol 105:93–104CrossRefGoogle Scholar
  14. Gong H, Li J, Xu A, Tang Y, Ji W, Gao R, Wang S, Yu L, Tian C, Li J, Yen HY, Man Lam S, Shui G, Yang X, Sun Y, Li X, Jia M, Yang C, Jiang B, Lou Z, Robinson CV, Wong LL, Guddat LW, Sun F, Wang Q, Rao Z (2018) An electron transfer path connects subunits of a mycobacterial respiratory supercomplex. Science 362:1020 (eaat8923)CrossRefGoogle Scholar
  15. Gregory E, Fridovich I (1974) Visualization of catalase on acrylamide gels. Anal Biochem 58(1):57–62CrossRefGoogle Scholar
  16. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4):557–580CrossRefGoogle Scholar
  17. Harth G, Horwitz MA (1999) Export of recombinant Mycobacterium tuberculosis superoxide dismutase is dependent upon both information in the protein and mycobacterial export machinery a model for studying export of leaderless proteins by pathogenic mycobacteria. J Biol Chem 274(7):4281–4292CrossRefGoogle Scholar
  18. Hirasawa T, Wachi M, Nagai K (2000) A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and l-glutamate production. J Bacteriol 182(10):2696–2701CrossRefGoogle Scholar
  19. Kao WC, Kleinschroth T, Nitschke W, Baymann F, Neehaul Y, Hellwig P, Richers S, Vonck J, Bott M, Hunte C (2016) The obligate respiratory supercomplex from Actinobacteria. Biochim Biophys Acta 1857(10):1705–1714CrossRefGoogle Scholar
  20. Kim J-S, Holmes RK (2012) Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae. PLoS One 7(3):e31709CrossRefGoogle Scholar
  21. Kim T-H, Park J-S, Kim H-J, Kim Y, Kim P, Lee H-S (2005) The whcE gene of Corynebacterium glutamicum is important for survival following heat and oxidative stress. Biochem Biophys Res Commun 337(3):757–764CrossRefGoogle Scholar
  22. Kinoshita S, Udaka S, Shimono M (1957) Studies on the amino acid fermentation: I. Production of l-glutamic acid by various microorganisms. J Gen Appl Microbiol 503:193–205CrossRefGoogle Scholar
  23. Lanciano P, Khalfaoui-Hassani B, Selamoglu N, Ghelli A, Rugolo M, Daldal F (2013) Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study. Biochim Biophys Acta 1827(11–12):1332–1339CrossRefGoogle Scholar
  24. Man Z, Rao Z, Xu M, Guo J, Yang T, Zhang X, Xu Z (2016) Improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply. Metab Eng 38:310–321CrossRefGoogle Scholar
  25. Matsumoto N, Hattori H, Matsutani M, Matayoshi C, Toyama H, Kataoka N, Yakushi T, Matsushita K (2018) A single-nucleotide insertion in a drug transporter gene induces a thermotolerant phenotype of Gluconobacter frateurii by increasing the NADPH/NADP+ ratio via metabolic change. Appl Environ Microbiol 84(10):e00354–e00318CrossRefGoogle Scholar
  26. Matsushita K, Yamamoto T, Toyama H, Adachi O (1998) NADPH oxidase system as a superoxide-generating cyanide-resistant pathway in the respiratory chain of Corynebacterium glutamicum. Biosci Biotechnol Biochem 62(10):1968–1977CrossRefGoogle Scholar
  27. Matsushita K, Azuma Y, Kosaka T, Yakushi T, Hoshida H, Akada R, Yamada M (2016) Genomic analyses of thermotolerant microorganisms used for high-temperature fermentations. Biosci Biotechnol Biochem 80(4):655–668CrossRefGoogle Scholar
  28. McNamara M, Tzeng S-C, Maier C, Wu M, Bermudez LE (2013) Surface-exposed proteins of pathogenic mycobacteria and the role of cu-zn superoxide dismutase in macrophages and neutrophil survival. Proteome Sci 11(1):45CrossRefGoogle Scholar
  29. Merkamm M, Guyonvarch A (2001) Cloning of the sodA gene from Corynebacterium melassecola and role of superoxide dismutase in cellular viability. J Bacteriol 183(4):1284–1295CrossRefGoogle Scholar
  30. Messner KR, Imlay JA (1999) The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli. J Biol Chem 274(15):10119–10128CrossRefGoogle Scholar
  31. Messner KR, Imlay JA (2002) Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase. J Biol Chem 277(45):42563–42571CrossRefGoogle Scholar
  32. Muffler A, Bettermann S, Haushalter M, Hörlein A, Neveling U, Schramm M, Sorgenfrei O (2002) Genome-wide transcription profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose. J Biotechnol 98(2–3):255–268CrossRefGoogle Scholar
  33. Nantapong N, Otofuji A, Migita TC, Adachi O, Toyama H, Matsushita K (2005) Electron transfer ability from NADH to menaquinone and from NADPH to oxygen of type II NADH dehydrogenase of Corynebacterium glutamicum. Biosci Biotechnol Biochem 69(1):149–159CrossRefGoogle Scholar
  34. Nishio Y, Nakamura Y, Kawarabayasi Y, Usuda Y, Kimura E, Sugimoto S, Matsui K, Yamagishi A, Kikuchi H, Ikeo K (2003) Comparative complete genome sequence analysis of the amino acid replacements responsible for the thermostability of Corynebacterium efficiens. Genome Res 13(7):1572–1579CrossRefGoogle Scholar
  35. Ohnishi J, Hayashi M, Mitsuhashi S, Ikeda M (2003) Efficient 40 °C fermentation of L-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding. Appl Microbiol Biotechnol 62(1):69–75CrossRefGoogle Scholar
  36. Oka A, Sugisaki H, Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147(2):217–226Google Scholar
  37. Pyatrikas DV, Fedoseeva IV, Varakina NN, Rusaleva TM, Stepanov AV, Fedyaeva AV, Borovskii GB, Rikhvanov EG (2015) Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions. FEMS Microbiol Lett 362(12):fnv082CrossRefGoogle Scholar
  38. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Coldspring-Harbour Laboratory Press, United KingdomGoogle Scholar
  39. Schulte C, Arenskötter M, Berekaa MM, Arenskötter Q, Priefert H, Steinbüchel A (2008) Possible involvement of an extracellular superoxide dismutase (SodA) as a radical scavenger in poly (cis-1, 4-isoprene) degradation. Appl Environ Microbiol 74(24):7643–7653CrossRefGoogle Scholar
  40. Tang L, Kwon SY, Kim SH, Kim JS, Choi JS, Cho KY, Sung CK, Kwak SS, Lee HS (2006) Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature. Plant Cell Rep 25(12):1380–1386CrossRefGoogle Scholar
  41. Wang ZL, Zhang LB, Ying SH, Feng MG (2013) Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol 15(2):409–418CrossRefGoogle Scholar
  42. Yokoi H, Ohnishi J, Ochiai K, Yonetani Y, Ozaki A (2000) Novel desensitized aspartokinase. Patent WO 00/63388Google Scholar
  43. Yoshida T, Ayabe Y, Yasunaga M, Usami Y, Habe H, Nojiri H, Omori T (2003) Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp. strain 12S. Microbiol 149(2):431–444CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Preclinical Sciences, Institute of ScienceSuranaree University of TechnologyNakhon RatchasimaThailand
  2. 2.Department of Biological Chemistry, Faculty of AgricultureYamaguchi UniversityYamaguchiJapan
  3. 3.Department of Microbiology, Faculty of ScienceKasetsart UniversityBangkokThailand
  4. 4.Graduate School of Science and Technology for InnovationYamaguchi UniversityYamaguchiJapan
  5. 5.Research Center for Thermotolerant Microbial ResourcesYamaguchi UniversityYamaguchiJapan

Personalised recommendations