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Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum

  • Ning Xu
  • Hongfang Lv
  • Liang Wei
  • Yuan Liang
  • Jiansong Ju
  • Jun Liu
  • Yanhe Ma
Applied microbial and cell physiology
  • 86 Downloads

Abstract

The industrial organism Corynebacterium glutamicum is often subjected to acid stress during large-scale fermentation for the production of bio-based chemicals. The capacity of the cells to thrive in acidic environments is a prerequisite for achieving high product yields. In this study, we obtained an acid-adapted strain using an adaptive laboratory evolution strategy. Physiological characterizations revealed that the adapted strain achieved improved cell viability after acid-stress challenge, with a higher cytoplasmic pHin level, a lower intracellular reactive oxygen species (ROS), and an enhanced morphological integrity of the cells, when compared to those of the original control strain. Transcriptome analysis indicated that several important cellular processes were altered in the adapted strain, including sulfur metabolism, iron transport, and central metabolic pathways. Further research displayed that KatA and Dps cooperatively mediated intracellular ROS scavenging, which was required for resistance to low-pH stress in C. glutamicum. Furthermore, the repression of sulfur assimilation by the McbR regulator also contributed to the improvement of acid-stress tolerance. Moreover, two copper chaperone genes cg1328 and cg3292 were found to be involved in promoting cell survival under acid-stress conditions. Finally, a new recombinant C. glutamicum strain with enhanced acid tolerance was generated by the combined overexpression of katA, dps, mcbR, and cg1328, showing 18.4 ± 2.5% higher biomass yields than the wild-type strain under acid-stress conditions. These findings will provide new insights into the understanding and genetic improvement of acid tolerance in C. glutamicum.

Keywords

C. glutamicum Adaptive laboratory evolution Acid resistance Oxidative stress Sulfur assimilation 

Notes

Acknowledgements

We are grateful to Prof. Masayuki Inui (Research Institute of Innovative Technology for the Earth, Japan) for generously providing plasmids.

Funding

This study was supported by the National Natural Science Foundation of China (No. 31500044), the Natural Science Foundation of Tianjin (No. 17JCQNJC09600, No. 17JCYBJC24000), the Tianjin Science and Technology Project (15PTCYSY00020), the Foundation of Hebei Educational Committee (ZD2017047) and the “Hundred Talents Program” of the Chinese Academy of Sciences.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_9585_MOESM1_ESM.pdf (430 kb)
ESM 1 (PDF 429 kb)

References

  1. Andrews SC, Robinson AK, Rodríguez-Quiñones F (2003) Bacterial iron homeostasis. FEMS Microbiol Rev 27(2–3):215–237.  https://doi.org/10.1016/S0168-6445(03)00055-X CrossRefGoogle Scholar
  2. Beales N (2004) Adaptation of microorganisms to cold temperatures, weak acid preservatives, low pH, and osmotic stress: a review. Compr Rev Food Sci F 3(1):1–20.  https://doi.org/10.1111/j.1541-4337.2004.tb00057.x CrossRefGoogle Scholar
  3. Bellapadrona G, Ardini M, Ceci P, Stefanini S, Chiancone E (2010) Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell. Free Radic Biol Med 48(2):292–297.  https://doi.org/10.1016/j.freeradbiomed.2009.10.053 CrossRefGoogle Scholar
  4. Brune I, Werner H, Huser AT, Kalinowski J, Puhler A, Tauch A (2006) The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum. BMC Genomics 7(21):21.  https://doi.org/10.1186/1471-2164-7-21 CrossRefGoogle Scholar
  5. Cecchini G (2003) Function and structure of complex II of the respiratory chain. Annu Rev Biochem 72:77–109.  https://doi.org/10.1146/annurev.biochem.72.121801.161700 CrossRefGoogle Scholar
  6. Chung HJ, Bang W, Drake MA (2006) Stress response of Escherichia coli. Compr Rev Food Sci Food Saf 5(3):52–64.  https://doi.org/10.1111/j.1541-4337.2006.00002.x CrossRefGoogle Scholar
  7. Djoko KY, Phan MD, Peters KM, Walker MJ, Schembri MA, McEwan AG (2017) Interplay between tolerance mechanisms to copper and acid stress in Escherichia coli. Proc Natl Acad Sci U S A 114(26):6818–6823.  https://doi.org/10.1073/pnas.1620232114 Google Scholar
  8. Follmann M, Ochrombel I, Krämer R, Trötschel C, Poetsch A, Rückert C, Hüser A, Persicke M, Seiferling D, Kalinowski J, Marin K (2009) Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis. BMC Genomics 10:621.  https://doi.org/10.1186/1471-2164-10-621 CrossRefGoogle Scholar
  9. Foster JW (2004) Escherichia coli acid resistance: tales of an amateur acidophile. Nat Rev Microbiol 2(11):898–907.  https://doi.org/10.1038/nrmicro1021 CrossRefGoogle Scholar
  10. Genevaux P, Georgopoulos C, Kelley WL (2007) The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions. Mol Microbiol 66(4):840–857.  https://doi.org/10.1111/j.1365-2958.2007.05961.x CrossRefGoogle Scholar
  11. Giuffre A, Borisov VB, Arese M, Sarti P, Forte E (2014) Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress. Biochim Biophys Acta 1837(7):1178–1187.  https://doi.org/10.1016/j.bbabio.2014.01.016 CrossRefGoogle Scholar
  12. Harrison MD, Jones CE, Dameron CT (1999) Copper chaperones: function, structure and copper-binding properties. J Biol Inorg Chem 4(2):145–153.  https://doi.org/10.1007/s007750050297 CrossRefGoogle Scholar
  13. Heydari H, Siow CC, Tan MF, Jakubovics NS, Wee WY, Mutha NV, Wong GJ, Ang MY, Yazdi AH, Choo SW (2014) CoryneBase: Corynebacterium genomic resources and analysis tools at your fingertips. PLoS One 9(1):e86318.  https://doi.org/10.1371/journal.pone.0086318 CrossRefGoogle Scholar
  14. Jakoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Tech 13(6):437–441.  https://doi.org/10.1023/A:1008968419217 CrossRefGoogle Scholar
  15. Kakinuma Y (1998) Inorganic cation transport and energy transduction in Enterococcus hirae and other streptococci. Microbiol Mol Biol Rev 62(4):1021–1045Google Scholar
  16. Kanjee U, Houry WA (2013) Mechanisms of acid resistance in Escherichia coli. Annu Rev Microbiol 67:65–81.  https://doi.org/10.1146/annurev-micro-092412-155708 CrossRefGoogle Scholar
  17. Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175(17):5595–5603.  https://doi.org/10.1128/jb.175.17.5595-5603.1993 CrossRefGoogle Scholar
  18. Kobayashi H, Saito H, Kakegawa T (2000) Bacterial strategies to inhabit acidic environments. J Gen Appl Microbiol 46(5):235–243.  https://doi.org/10.2323/Jgam.46.235 CrossRefGoogle Scholar
  19. Krulwich TA, Sachs G, Padan E (2011) Molecular aspects of bacterial pH sensing and homeostasis. Nat Rev Microbiol 9(5):330–343.  https://doi.org/10.1038/nrmicro2549 CrossRefGoogle Scholar
  20. Lee JY, Na YA, Kim E, Lee HS, Kim P (2016) The actinobacterium Corynebacterium glutamicum, an industrial workhorse. J Microbiol Biotechnol 26(5):807–822.  https://doi.org/10.4014/jmb.1601.01053 CrossRefGoogle Scholar
  21. Lee JY, Seo J, Kim ES, Lee HS, Kim P (2013) Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling. Biotechnol Lett 35(5):709–717.  https://doi.org/10.1007/s10529-012-1135-9 CrossRefGoogle Scholar
  22. Letek M, Fiuza M, Ordonez E, Villadangos AF, Ramos A, Mateos LM, Gil JA (2008) Cell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicum. Antonie Van Leeuwenhoek 94(1):99–109.  https://doi.org/10.1007/s10482-008-9224-4 CrossRefGoogle Scholar
  23. Liu Y, Yang X, Yin Y, Lin J, Chen C, Pan J, Si M, Shen X (2016) Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE. J Gen Appl Microbiol 62(3):144–153.  https://doi.org/10.2323/jgam.2016.02.001 CrossRefGoogle Scholar
  24. Liu YP, Tang HZ, Lin ZL, Xu P (2015) Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation. Biotechnol Adv 33(7):1484–1492.  https://doi.org/10.1016/j.biotechadv.2015.06.001 CrossRefGoogle Scholar
  25. Lund P, Tramonti A, De Biase D (2014) Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiol Rev 38(6):1091–1125.  https://doi.org/10.1111/1574-6976.12076 CrossRefGoogle Scholar
  26. Marles-Wright J, Lewis RJ (2007) Stress responses of bacteria. Curr Opin Struct Biol 17(6):755–760.  https://doi.org/10.1016/j.sbi.2007.08.004 CrossRefGoogle Scholar
  27. Milse J, Petri K, Ruckert C, Kalinowski J (2014) Transcriptional response of Corynebacterium glutamicum ATCC 13032 to hydrogen peroxide stress and characterization of the OxyR regulon. J Biotechnol 190:40–54.  https://doi.org/10.1016/j.jbiotec.2014.07.452 CrossRefGoogle Scholar
  28. Mols M, van Kranenburg R, van Melis CC, Moezelaar R, Abee T (2010) Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation. Environ Microbiol 12(4):873–885.  https://doi.org/10.1111/j.1462-2920.2009.02132.x CrossRefGoogle Scholar
  29. Okibe N, Suzuki N, Inui M, Yukawa H (2011) Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid. J Microbiol Meth 85(2):155–163.  https://doi.org/10.1016/j.mimet.2011.02.012 CrossRefGoogle Scholar
  30. Palumaa P (2013) Copper chaperones. The concept of conformational control in the metabolism of copper. FEBS Lett 587(13):1902–1910.  https://doi.org/10.1016/j.febslet.2013.05.019 CrossRefGoogle Scholar
  31. Papadimitriou K, Alegria A, Bron PA, de Angelis M, Gobbetti M, Kleerebezem M, Lemos JA, Linares DM, Ross P, Stanton C, Turroni F, van Sinderen D, Varmanen P, Ventura M, Zuniga M, Tsakalidou E, Kok J (2016) Stress physiology of lactic acid bacteria. Microbiol Mol Biol Rev 80(3):837–890.  https://doi.org/10.1128/MMBR.00076-15 CrossRefGoogle Scholar
  32. Park S, Imlay JA (2003) High levels of intracellular cysteine promote oxidative DNA damage by driving the Fenton reaction. J Bacteriol 185(6):1942–1950.  https://doi.org/10.1128/jb.185.6.1942-1950.2003 CrossRefGoogle Scholar
  33. Radmacher E, Alderwick LJ, Besra GS, Brown AK, Gibson KJ, Sahm H, Eggeling L (2005) Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum. Microbiology 151(Pt 7):2421–2427.  https://doi.org/10.1099/mic.0.28012-0 CrossRefGoogle Scholar
  34. Rausch T, Wachter A (2005) Sulfur metabolism: a versatile platform for launching defence operations. Trends Plant Sci 10(10):503–509.  https://doi.org/10.1016/j.tplants.2005.08.006 CrossRefGoogle Scholar
  35. Rey DA, Pühler A, Kalinowski J (2003) The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum. J Biotechnol 103(1):51–65.  https://doi.org/10.1016/S0168-1656(03)00073-7 CrossRefGoogle Scholar
  36. Rivera M (2017) Bacterioferritin: structure, dynamics, and protein-protein interactions at play in iron storage and mobilization. Acc Chem Res 50(2):331–340.  https://doi.org/10.1021/acs.accounts.6b00514 CrossRefGoogle Scholar
  37. Ruckert C, Milse J, Albersmeier A, Koch DJ, Puhler A, Kalinowski J (2008) The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules. BMC Genomics 9:483.  https://doi.org/10.1186/1471-2164-9-483 CrossRefGoogle Scholar
  38. Si M, Zhao C, Burkinshaw B, Zhang B, Wei D, Wang Y, Dong TG, Shen X (2017) Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis. Proc Natl Acad Sci U S A 114(11):E2233–E2242.  https://doi.org/10.1073/pnas.1614902114 CrossRefGoogle Scholar
  39. Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2(5):a000414.  https://doi.org/10.1101/cshperspect.a000414 CrossRefGoogle Scholar
  40. Teramoto H, Inui M, Yukawa H (2013) OxyR acts as a transcriptional repressor of hydrogen peroxide-inducible antioxidant genes in Corynebacterium glutamicum R. FEBS J 280(14):3298–3312.  https://doi.org/10.1111/febs.12312 CrossRefGoogle Scholar
  41. Wang T, Gao F, Kang Y, Zhao C, Su T, Li M, Si M, Shen X (2016) Mycothiol peroxidase MPx protects Corynebacterium glutamicum against acid stress by scavenging ROS. Biotechnol Lett 38(7):1221–1228.  https://doi.org/10.1007/s10529-016-2099-y CrossRefGoogle Scholar
  42. Wennerhold J, Krug A, Bott M (2005) The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR. J Biol Chem 280(49):40500–40508.  https://doi.org/10.1074/jbc.M508693200 CrossRefGoogle Scholar
  43. Wieschalka S, Blombach B, Bott M, Eikmanns BJ (2013) Bio-based production of organic acids with Corynebacterium glutamicum. Microb Biotechnol 6(2):87–102.  https://doi.org/10.1111/1751-7915.12013 CrossRefGoogle Scholar
  44. Winterbourn CC (1995) Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxico Lett 82-83:969–974.  https://doi.org/10.1016/0378-4274(95)03532-X CrossRefGoogle Scholar
  45. Wu C, He G, Zhang J (2014) Physiological and proteomic analysis of Lactobacillus casei in response to acid adaptation. J Ind Microbiol Biotechnol 41(10):1533–1540.  https://doi.org/10.1007/s10295-014-1487-3 CrossRefGoogle Scholar
  46. Wu C, Zhang J, Wang M, Du G, Chen J (2012) Lactobacillus casei combats acid stress by maintaining cell membrane functionality. J Ind Microbiol Biotechnol 39(7):1031–1039.  https://doi.org/10.1007/s10295-012-1104-2 CrossRefGoogle Scholar
  47. Xu N, Wei L, Liu J (2017) Biotechnological advances and perspectives of γ-aminobutyric acid production. World J Microbiol Biot 33(3):64.  https://doi.org/10.1007/s11274-017-2234-5 CrossRefGoogle Scholar
  48. Xu N, Zheng YY, Wang XC, Krulwich TA, Ma YH, Liu J (2018) The lysine 299 residue endows the multisubunit Mrp1 antiporter with dominant roles in Na+ resistance and pH homeostasis in Corynebacterium glutamicum. Appl Environ Microbiol 84(10):e00110–e00118.  https://doi.org/10.1128/AEM.00110-18 CrossRefGoogle Scholar
  49. Zhang J, Fu RY, Hugenholtz J, Li Y, Chen J (2007) Glutathione protects Lactococcus lactis against acid stress. Appl Environ Microbiol 73(16):5268–5275.  https://doi.org/10.1128/AEM.02787-06 CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Ning Xu
    • 1
  • Hongfang Lv
    • 2
  • Liang Wei
    • 1
  • Yuan Liang
    • 1
  • Jiansong Ju
    • 2
  • Jun Liu
    • 1
    • 3
  • Yanhe Ma
    • 1
  1. 1.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin Airport Economic AreaPeople’s Republic of China
  2. 2.College of Life SciencesHebei Normal UniversityShijiazhuangPeople’s Republic of China
  3. 3.Key Laboratory of Systems Microbial BiotechnologyChinese Academy of SciencesTianjinPeople’s Republic of China

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