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Transcriptional Analysis Responding to Propanol Stress in Escherichia coli

대장균에서 프로판올 스트레스에 관한 전사분석

  • Park, Hye-Jin (Department of Food Science & Biotechnology, Kyungsung University) ;
  • Lee, Jin-Ho (Department of Food Science & Biotechnology, Kyungsung University)
  • 박혜진 (경성대학교 식품생명공학과) ;
  • 이진호 (경성대학교 식품생명공학과)
  • Received : 2012.02.16
  • Accepted : 2012.03.15
  • Published : 2012.03.30

Abstract

We compared the transcriptome in response to propanol stress in wild-type and propanol-resistant mutant Escherichia coli using the DNA microarray technique. The correlation value of RNA expression between the propanol-treated wild type and the untreated-one was about 0.949, and 50 genes were differentially expressed by more than twofold in both samples. The correlation value of RNA expression between the propanol-treated mutant and the untreated one was about 0.951, and 71 genes in two samples showed differential expression patterns. However, the values between the wild type and mutant, regardless of propanol addition, were 0.974-0.992 and only 1-2 genes were differentially expressed in the two strains. The representative characteristics among differentially expressed genes in W3110 or P19 treated with propanol compared to untreated samples were up-regulation of hest shock response genes and down-regulation of genes relating to ribosome biosynthesis. In addition, many genes were regulated by transcription regulation factors such as ArcA, CRP, FNR, H-NS, GatR, or PurR and overexpressed by sigma factor RpoH. We confirmed that RpoH mediated an important host defense function in propanol stress in E. coli W3110 and P19 by comparison of cell growth rate among the wild type, rpoH disruptant mutant, and rpoH-complemented strain.

대장균 야생주와 프로판올 내성 변이주에서 프로판올 스트레스에 의해 발현이 크게 변화하는 유전자를 DNA microarray 기술을 이용하여 비교 분석하였다. 프로판올 첨가한 야생주와 무첨가한 야생주 사이의 RNA 발현 연관값은 0.949이며, 50개의 유전자 발현이 2배 이상 변화하였다. 프로판올을 첨가한 내성변이주와 무첨가한 변이주 사이의 연관값은 0.952이며, 71개의 유전자 발현이 크게 변화하였다. 그러나, 야생주와 변이주 사이의 연관값은 프로판올 무첨가한 조건과 첨가한 조건에서 각각 0.992 및 0.974로 매우 높았으며, 2배 이상의 발현차이를 나타내는 유전자는 각각 1개 및 2개로, 두 균주는 매우 유사한 발현양상을 보였다. 야생주 또는 변이주에서 프로판올 스트레스에 반응하는 대표적인 유전자들의 특징은 많은 열충격 반응에 관여하는 유전자들의 발현이 크게 증가하였으며, 리보소옴 합성에 필요한 많은 유전자들의 발현이 감소하였다. 또한, 전사조절인자들인 ArcA, CRP, FNR, H-NS, GatR, PurR에 의해 조절받는 유전자들의 발현이 크게 변화하였으며, 시그마인자들 중에서는 RpoH에 의해서 발현되는 유전자들의 발현이 크게 증가하였다. rpoH가 정상적으로 발현되지 못하는 변이주와 야생주를 이용한 프로판올 내성정도를 측정한 결과, RpoH는 대장균에서 프로판올 스트레스에 적응하는데 중요한 기능을 하는 것으로 확인되었다.

Keywords

References

  1. Alexandre, H., Rousseaux, I. and Charpentier, C. 1994. Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata. FEMS Microbiol. Lett. 124, 17-22. https://doi.org/10.1111/j.1574-6968.1994.tb07255.x
  2. Alexeeva, S., Hellingwerf, K. J. and Mattos, T. 2003. Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions. J. Bacteriol. 85, 204-209.
  3. Alsaker, K. V., Paredes, C. and Papoutsakis, E. T. 2010. Metabolite stress and tolerance in the production of biofuels and chemicals: Gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotech. Bioeng. 105, 1131-1147.
  4. Atsumi, S., Cann, A. F., Connor, M. R., Shen, C. R., Smith, K. M., Brynildsen, M. P., Chou, K. J. Y., Hanai, T. and Liao, J. C. 2007. Metabolic engineering of Escherichia coli for 1-butanol production. Metab. Eng. 10, 305-311. https://doi.org/10.1016/j.ymben.2007.08.003
  5. Atsumi, S., Hanai, T. and Liao, J. C. 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86-90. https://doi.org/10.1038/nature06450
  6. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L. and Mori, H. 2006. Construction of E. coli K-12 in-frame, single- gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 1-11.
  7. Baer, S. H., Blaschek, H. P. and Smith, T. L. 1987. Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol tolerant Clostridium acetobutylicum. Appl. Environ. Microbiol. 53, 2854-2861.
  8. Bowles, L. K. and Ellefson, W. L. 1985. Effects of butanol on Clostridium acetobutylicum. Appl. Environ. Microbiol. 50, 1165-1170.
  9. Brynildsen, M. P. and Liao, J. C. 2009. An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol. Syst. Biol. 5, 1-13.
  10. Buttke, T. M. and Ingram, L. O. 1978. Mechanism of ethanol-induced changes in lipid composition of Escherichia coli: Inhibition of saturated fatty acid synthesis in vivo. Biochemistry 17, 637-644. https://doi.org/10.1021/bi00597a012
  11. Buttke, T. M. and Ingram, L. O. 1980. Ethanol-induced changes in lipid composition of Escherichia coli: Inhibition of saturated fatty acid synthesis in vitro. Arch. Biochem. Biophys. 203, 565-571. https://doi.org/10.1016/0003-9861(80)90213-1
  12. Chuang, S. E. and Blattner, F. R. 1993. Characterization of twenty-six new heat shock genes of Escherichia coli. J. Bacteriol. 175, 5242-5252.
  13. Cotter, P. A. and Gunsalus, R. P. 1992. Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli. FEMS Microbiol. Lett. 70, 31-36.
  14. Fic, E., Bonarek, P., Gorecki, A., Kedracka-Krok, S., Mikolajczak, J., Polit, A., Tworzydlo, M., Dziedzicka- Wasylewska, M. and Wasylewski, Z. 2009. cAMP receptor protein from Escherichia coli as a model of signal transduction in proteins. J. Mol. Microbiol. Biotechnol. 17, 1-11. https://doi.org/10.1159/000178014
  15. Gama-Castro, S., Jimenez-Jacinto, V., Peralta-Gil, M., Santos-Zavaleta, A., Penaloza-Spinola, M. I., Contreras- Moreira, B., Segura-Salazar, J., Muniz-Rascado, L., Martinez-Flores, I., Salgado, H., Bonavides-Martinez, C., Abreu-Goodger, C., Rodriguez-Penagos, C., Miranda-Rios, J., Morett, E., Merino, E., Huerta, A. M., Trevino-Quintanilla, L. and Collado-Vides, J. 2008. RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation. Nucleic Acids Res. 36, 120-124. https://doi.org/10.1093/nar/gkn491
  16. Gonzalez, R., Tao, H., Purvis, J. E., York, S. W., Shanmugam, K. T. and Ingram, L. O. 2003. Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: Comparison of KO11 (Parent) to LY01 (resistant mutant). Biotechnol. Prog. 19, 612-623. https://doi.org/10.1021/bp025658q
  17. Kubota, S., Takeo, I., Kume, K., Kanai, M., Shitamukai, A., Mizunuma, M., Miyakawa, T., Shimoi, H., Iefuji, H. and Hirata, D. 2004. Effect of ethanol on cell growth of budding yeast: Genes that are important for cell growth in the presence of ethanol. Biosci. Biotechnol. Biochem. 68, 968-972. https://doi.org/10.1271/bbb.68.968
  18. Lepage, C., Fayolle, F., Hermann, M. and Vandecasteele, J. P. 1987. Changes in membrane-lipid composition of Clostridium acetobutylicum during acetone butanol fermentation- Effects of solvents, growth temperature and pH. J. Gen. Microbiol. 133, 103-110.
  19. Lin, Y. and Tanaka, S. 2006. Ethanol fermentation from biomass resources: current state and prospects. Appl. Microbiol. Biotechnol. 69, 627-642. https://doi.org/10.1007/s00253-005-0229-x
  20. Liu, S. and Qureshi, N. 2009. How microbes tolerate ethanol and butanol. New Biotech. 26, 117-121. https://doi.org/10.1016/j.nbt.2009.06.984
  21. Nobelmann, B. and Lengeler, J. W. 1996. Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism. J. Bacteriol. 178, 6790-6795.
  22. Ogawa, Y., Nitta, A., Uchiyama, H., Imamura, T., Shimoi, H. and Ito, K. 2000. Tolerance mechanism of the ethanol-tolerant mutant of sake yeast. J. Biosci. Bioeng. 90, 313-320. https://doi.org/10.1016/S1389-1723(00)80087-0
  23. Park, S. J., Tseng, C. P. and Gunsalus, R. P. 1995. Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr. Mol. Microbiol. 15, 473-482. https://doi.org/10.1111/j.1365-2958.1995.tb02261.x
  24. Qureshi, N. and Blaschek, H. P. 2001. Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101. J. Ind. Microbiol. Biotech. 27, 287-291. https://doi.org/10.1038/sj.jim.7000114
  25. Repoila, F., Majdalani, N. and Gottesman, S. 2003. Small non-coding RNAs, coordinators of adaptation processes in Escherichia coli: the RpoS paradigm. Mol. Microbiol. 48, 855-861. https://doi.org/10.1046/j.1365-2958.2003.03454.x
  26. Salgueiro, S. P., Sa-Correia, I. and Novais, J. M. 1988. Ethanol-induced leakage in Saccharomyces cerevisiae: Kinetics and relationship to yeast ethanol tolerance and alcohol fermentation productivity. Appl. Environ. Microbiol. 54, 903-909.
  27. Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, G. W. and Gunsalus, R. P. 2003. Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR. J. Biol. Chem. 278, 29837-29855. https://doi.org/10.1074/jbc.M213060200
  28. Sambrook, J. and Russell, D. W. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  29. Shen, C. R. and Liao, J. C. 2008. Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways. Metab. Eng. 10, 312-320. https://doi.org/10.1016/j.ymben.2008.08.001
  30. Terracciano, J. S. and Kashket, E. R. 1986. Intracellular conditions required for initiation of solvent production by Clostridium acetobutylicum. Appl. Environ. Microbiol. 52, 86-91.
  31. Tomas, C. A., Welker, N. E. and Papoutsakis, E. T. 2003. Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and large changes in the cell's transcriptional program. Appl. Environ. Microbiol. 69, 4951-4965. https://doi.org/10.1128/AEM.69.8.4951-4965.2003