AiM  Vol.6 No.9 , August 2016
Heterologous Expression of Thermolabile Proteins Enhances Thermotolerance in Escherichia coli
Abstract: Heat shock proteins (HSPs) play important roles in the mechanism of cellular protection against various environmental stresses. It is well known that accumulation of misfolded proteins in a cell triggers the HSPs expression in prokaryotes as well as eukaryotes. In this study, we heterologously expressed two proteins in E. coli, namely, citrate synthase (CpCSY) and malate dehydrogenase (CpMDH) from a psychrophilic bacterium Colwellia psychrerythraea 34H (optimal growth temperature 8°C). Our analyses using circular dichromism along with temperature-dependant enzyme activities measured in purified or direct cell extracts confirmed that the CpCSY and CpMDH are thermolabile and present in misfolded form even at physiological growth temperature. We observed that the cellular levels of HSPs, both GroEL and DnaK cheperonins were increased. Similarly, higher levels were observed for sigma factor s32 which is specific to heat-shock protein expression. These results suggest that the misfolded-thermolabile proteins expressed in E. coli induced the heat shock response. Furthermore, heat treatment (53°C) to wild type E. coli noticeably delayed their growth recovery but cells expressing CpCSY and CpMDH recovered their growth much faster than that of wild type E. coli. This reveals that the HSPs expressed in response to misfolded-thermolabile proteins protected E. coli against heat-induced damage. This novel approach may be a useful tool for investigating stress-tolerance mechanisms of E. coli.
Cite this paper: Ueda, Y. , Yamauchi, S. , Fukata, S. , Okuyama, H. , Morita, E. , Shelake, R. and Hayashi, H. (2016) Heterologous Expression of Thermolabile Proteins Enhances Thermotolerance in Escherichia coli. Advances in Microbiology, 6, 602-612. doi: 10.4236/aim.2016.69060.

[1]   Bukau, B. and Horwich, A.L. (1998) The Hsp70 and Hsp60 Chaperone Machines. Cell, 92, 351-366.

[2]   Hartl, F.U. and Hayer-Hartl, M. (2002) Molecular Chaperones in the Cytosol: From Nascent Chain to Folded Protein. Science, 295, 1852-1858.

[3]   Hengge, R. and Bukau, B. (2003) Proteolysis in Prokaryotes: Protein Quality Control and Regulatory Principles. Molecular Microbiology, 49, 1451-1462.

[4]   Yura, T. and Nakahigashi, K. (1999) Regulation of the Heat-Shock Response. Current Opinion in Microbiology, 2, 153-158.

[5]   Tatsuta, T., Tomoyasu, T., Bukau, B., Kitagawa, M., Mori, H., Karata, K. and Ogura, T. (1998) Heat Shock Regulation in the ftsH Null Mutant of Escherichia coli: Dissection of Stability and Activity Control Mechanisms of σ32 in Vivo. Molecular Microbiology, 30, 583-593.

[6]   Blaszczak, A., Georgopoulos, C. and Liberek, K. (1999) On the Mechanism of FtsH-Dependent Degradation of the σ32 Transcriptional Regulator of Escherichia coli and the Role of the DnaK Chaperone Machine. Molecular Microbiology, 31, 157-166.

[7]   Rodriguez, F., Arsène-Ploetze, F., Rist, W., Rüdiger, S., Schneider-Mergener, J., Mayer, M.P. and Bukau, B. (2008) Molecular Basis for Regulation of the Heat Shock Transcription Factor σ32 by the DnaK and DnaJ Chaperones. Molecular Cell, 32, 347-358.

[8]   Abravaya, K., Myers, M.P., Murphy, S.P. and Morimoto, R.I. (1992) The Human Heat Shock Protein Hsp70 Interacts with HSF, the Transcription Factor That Regulates Heat Shock Gene Expression. Genes & Development, 6, 1153-1164.

[9]   Baler, R., Zou, J. and Voellmy, R. (1996) Evidence for a Role of Hsp70 in the Regulation of the Heat Shock Response in Mammalian Cells. Cell Stress Chaperones, 1, 33-39.<0033:EFAROH>2.3.CO;2

[10]   Kim, B.H. and Schoffl, F. (2002) Interaction between Arabidopsis Heat Shock Transcription Factor 1 and 70 kDa Heat Shock Proteins. Journal of Experimental Botany, 53, 371-375.

[11]   Schulz-Raffelt, M., Lodha, M. and Schroda, M. (2007) Heat Shock Factor 1 Is a Key Regulator of the Stress Response in Chlamydomonas. The Plant Journal, 52, 286-295.

[12]   Kimata, Y., Kimata, Y.I., Shimizu, Y., Abe, H., Farcasanu, I.C., Takeuchi, M., Rose, M.D. and Kohno, K. (2003) Genetic Evidence for a Role of Bip/Kar2 That Regulates Ire1 in Response to Accumulation of Unfolded Proteins. Molecular Biology of the Cell, 14, 2559-2569.

[13]   Morita, R.Y. (1975) Psychrophilic Bacteria. Bacteriological Reviews, 39, 144-167.

[14]   Yamauchi, S., Okuyama, H., Morita, E.H. and Hayashi, H. (2003) Gene Structure and Transcriptional Regulation Specific to the groESL Operon from the Psychrophilic Bacterium Colwellia maris. Archives of Microbiology, 180, 272-278.

[15]   Yamauchi, S., Okuyama, H., Nishiyama, Y. and Hayashi, H. (2004) Gene Structure and Transcriptional Regulation of dnaK and dnaJ Genes from a Psychrophilic Bacterium, Colwellia maris. Extremophiles, 8, 283-290.

[16]   Yamauchi, S., Fukata, S. and Hayashi, H. (2013) Heat Shock Response in Psychrophilic Microorganisms. In: Yumoto, I., Ed., Cold-Adapted Microorganisms, Caister Academic Press, Norfolk, 111-136.

[17]   Watanabe, S., Yamaoka, N., Fukunaga, N. and Takada, Y. (2002) Purification and Characterization of a Cold-Adapted Isocitrate Lyase and Expression Analysis of the Cold-Inducible Isocitrate Lyase Gene from the Psychrophilic Bacterium Colwellia psychrerythraea. Extremophiles, 6, 397-405.

[18]   Gerike, U., Danson, M.J., Russell, N.J. and Hough, D.W. (1997) Sequencing and Expression of the Gene Encoding a Cold-Active Citrate Synthase from an Antarctic Bacterium, Strain DS2-3R. European Journal of Biochemistry, 248, 49-57.

[19]   Oikawa, T., Yamamoto, N., Shimoke, K., Uesato, S., Ikeuchi, T. and Fujioka, T. (2005) Purification, Characterization, and Overexpression of Psychrophilic and Thermolabile Malate Dehydrogenase of a Novel Antarctic Psychrotolerant, Flavobacterium frigidimaris KUC-1. Bioscience, Biotechnology, and Biochemistry, 69, 2146-2154.

[20]   Kitagawa, M., Matsumura, Y. and Tsuchido, T. (2000) Small Heat Shock Proteins, IbpA and IbpB, Are Involved in Resistances to Heat and Superoxide Stresses in Escherichia coli. FEMS Microbiology Letters, 184, 165-171.

[21]   Desmond, C., Fitzgerald, G.F., Stanton, C. and Ross, R.P. (2004) Improved Stress Tolerance of GroESL-Overproducing Lactococcus lactis and Probiotoc Lactobacillus paracasei NFBC 338. Applied and Environmental Microbiology, 70, 5929-5936.

[22]   Jiang, C., Xu, J., Zhang, H., Zhang, X., Shi, J., Li, M. and Ming, F. (2009) A Cytosolic Class I Small Heat Shock Protein, RcHSP17.8, of Rosa chinensis Confers Resistance to a Variety of Stresses to Escherichia coli, Yeast and Arabidopsis thaliana. Plant, Cell & Environment, 32, 1046-1059.

[23]   Liu, D., Lu, Z., Mao, Z. and Liu, S. (2009) Enhanced Thermotolerance of E. coli by Expressed OsHsp90 from Rice (Oryza sativa L.). Current Microbiology, 58, 129-133.

[24]   Patra, M., Roy, S.S., Dasgupta, R. and Basu, T. (2015) GroEL to DnaK Chaperone Network behind the Stability Modulation of σ32 at Physiological Temperature in Escherichia coli. FEBS Letters, 589, 4047-4052.

[25]   Srere, P.A., Brazil, H. and Gonen, L. (1963) The Citrate Condensing Enzyme of Pigeon Breast Muscle and Moth Flight Muscle. Acta Chemica Scandinavica, 17, S129-S134.

[26]   Gill, S.C. and von Hippel, P.H. (1989) Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data. Analytical Biochemistry, 182, 319-326.

[27]   Feller, G. and Gerday, C. (1997) Psychrophilic Enzymes: Molecular Basis of Cold Adaptation. Cellular and Molecular Life Sciences CMLS, 53, 830-841.

[28]   Lonhienne, T., Gerday, C. and Feller, G. (2000) Psychrophilic Enzymes: Revisiting the Thermodynamic Parameters of Activation May Explain Local Flexibility. Biochimica et Biophysica Acta (BBA)—Protein Structure and Molecular Enzymology, 1543, 1-10.

[29]   Aurilia, V., Parracino, A. and D’Auria, S. (2008) Microbial Carbohydrate Esterases in Cold Adapted Environments. Gene, 410, 234-240.

[30]   Tutino, M.L., di Prisco, G., Marino, G. and de Pascale, D. (2009) Cold-Adapted Esterases and Lipases: From Fundamentals to Application. Protein & Peptide Letters, 16, 1172-1180.

[31]   Gerike, U., Danson, M.J. and Hough, D.W. (2001) Cold-Active Citrate Synthase: Mutagenesis of Active-Site Residues. Protein Engineering, Design & Selection, 14, 655-661.

[32]   Tomoyasu, T., Ogura, T., Tatsuta, T. and Bukau, B. (1998) Levels of DnaK and DnaJ Provide Tight Control of Heat Shock Gene Expression and Protein Repair in Escherichia coli. Molecular Microbiology, 30, 567-581.

[33]   Queitsch, C., Hong, S.W., Vierling, E. and Lindquist, S. (2000) Heat Shock Protein 101 Plays a Crucial Role in Thermotolerance in Arabidopsis. The Plant Cell, 12, 479-492.

[34]   Kobayashi, Y., Ohtsu, I., Fujimura, M. and Fukumori, F. (2011) A Mutation in dnaK Causes Stabilization of the Heat Shock Sigma Factor σ32, Accumulation of Heat Shock Proteins and Increase in Toluene-Resistance in Pseudomonas putida. Environmental Microbiology, 13, 2007-2017.

[35]   Voellmy, R. and Boellmann, F. (2007) Chaperone Regulation of the Heat Shock Protein Response. In: Csermely, P. and Vígh, L., Eds., Molecular Aspects of the Stress Response: Chaperones, Membranes and Networks, Springer, New York, 89-99.

[36]   Rasheva, V.I. and Domingos, P.M. (2009) Cellular Responses to Endoplasmic Reticulum Stress and Apoptosis. Apoptosis, 14, 996-1007.