Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Reduction-Sensitive and Cysteine Residue-Mediated Streptococcus pneumoniae HrcA Oligomerization In Vitro

  • Received : 2008.06.18
  • Accepted : 2008.11.19
  • Published : 2009.02.28
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Abstract

In both gram-positive and several gram-negative bacteria, the transcription of dnaK and groE operons is negatively regulated by HrcA; however, the mechanism modulating HrcA protein activity upon thermal stress remains elusive. Here, we demonstrate that HrcA is modulated via reduction and oligomerization in vitro. Native-PAGE analysis was used to reveal the oligomeric structure of HrcA. The oligomeric HrcA structure became monomeric following treatment with the reducing agent dithothreitol, and this process was reversed by treatment with hydrogen peroxide. Moreover, the mutant HrcA C118S exhibited reduced binding to CIRCE elements and became less oligomerized, suggesting that cysteine residue 118 is important for CIRCE element binding as well as oligomerization. Conversely, HrcA mutant C280S exhibited increased oligomerization. An HrcA double mutant (C118S, C280S) was monomeric and exhibited a level of oligomerization and CIRCE binding similar to wild type HrcA, suggesting that cysteine residues 118 and 280 may function as checks to one another during oligomer formation. Biochemical fractionation of E. coli cells overexpressing HrcA revealed the presence of HrcA in the membrane fraction. Together, these results suggest that the two HrcA cysteine residues at positions 118 and 280 function as reduction sensors in the membrane and mediate oligomerization upon stress.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation

References

  1. Ahmad, S., Selvapandiyan, A., and Bhantnagar, R.K. (1999). A protein-based phylogenetic tree for gram-positive bacteria derived from hrcA, a unique heat-shock regulatory gene. Int. J. Syst. Bacteriol. 49, 1387-1394 https://doi.org/10.1099/00207713-49-4-1387
  2. Akita, M., Nielsen, E., and Keegstra, K. (1997). Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking. J. Cell Biol. 136, 983-994 https://doi.org/10.1083/jcb.136.5.983
  3. Aruna, B., Ghosh, S., Singh, A.K., Mande, S.C., Srinivas, V., Chauhan, R., and Ehtesham, N.Z. (2003). Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages. Biochemistry 42, 10554-10559 https://doi.org/10.1021/bi034782v
  4. Bukau, B. (1993). Regulation of the Escherichia coli heat shock response. Mol. Microbiol. 9, 671-680 https://doi.org/10.1111/j.1365-2958.1993.tb01727.x
  5. Chastanet, A., Fert, J., and Msadek, T. (2003). Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria. Mol. Microbiol. 47, 1061-1073 https://doi.org/10.1046/j.1365-2958.2003.03355.x
  6. Choi, I.H., Shim, J.H., Kim, S.W., Kim, S.N., Pyo, S.N., and Rhee, D.K. (1999). Limited stress response in Staphylococcus pnenmoniae. Microbiol. Immunol. 43, 807-812 https://doi.org/10.1111/j.1348-0421.1999.tb02474.x
  7. Fehri, L.F., Sirand-Pugnet, P., Gourgues, G., Jan, G., Wroblewski, H., and Blanchard, A. (2005). Resistance to antimicrobial peptides and stress response in Mycoplasma pulmonis. Antimicrob. Agents Chemother. 49, 4154-4165 https://doi.org/10.1128/AAC.49.10.4154-4165.2005
  8. Fukuda, M., Kanno, E., and Mikoshiba, K. (1999). Conserved Nterminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X. J. Biol. Chem. 274, 31421-31427 https://doi.org/10.1074/jbc.274.44.31421
  9. Garnier, C., Barbier, P., Devred, F., Rivas, G., and Peyrot, V. (2002). Hydrodynamic properties and quaternary structure of the 90 kDa heat-shock protein: effects of divalent cations. Biochemistry 41, 11770-11778 https://doi.org/10.1021/bi025650p
  10. Gottesman, S., Wickner, S., and Maurizi, M.R. (1997). Protein quality control: triage by chaperones and proteases. Genes Dev. 11, 815-823 https://doi.org/10.1101/gad.11.7.815
  11. Hecker, M., Schumann, W., and Voker, U. (1996). Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19, 417-428 https://doi.org/10.1046/j.1365-2958.1996.396932.x
  12. Hitomi, M., Nishimura, H., Tsujimoto, Y., Matsui, H., and Watanabe, K. (2003). Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex, indicating a role as a thermosensor. J. Bacteriol. 185, 381-385 https://doi.org/10.1128/JB.185.1.381-385.2003
  13. Horvath, I., Glatz, A., Varvasovszki, V., Torok, Z., Pali, T., Balogh, G., Kovacs, E., Nadasdi, L., Benko, S., Joo, F., et al. (1998). Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a 'fluidity gene'. Proc. Natl. Acad. Sci. USA 95, 3513-3518 https://doi.org/10.1073/pnas.95.7.3513
  14. Hu, Y., Oliver, H.F., Raengpradub, S., Palmer, M.E., Orsi, R.H., Wiedmann, M., and Boor, K.J. (2007). Transcriptomic and phenotypic analyses suggest a network between the transcriptional regulators HrcA and sigmaB in Listeria monocyto-genes. Appl. Environ. Microbiol. 73, 7981-7991 https://doi.org/10.1128/AEM.01281-07
  15. Jha, B.K., Salunke, D.M., and Datta, K. (2002). Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/ p32/gC1qR. Eur. J. Biochem. 269, 298-306 https://doi.org/10.1046/j.0014-2956.2001.02654.x
  16. Kim, S.N., Kim, S.W., Pyo, S.N., and Rhee, D.K. (2001). Molecular cloning and characterization of groESL operon in Streptococcus pneumoniae. Mol. Cells 11, 360-368
  17. Kwon, H.Y., Kim, S.N., Pyo, S.N., and Rhee, D.K. (2005). $Ca^{2+}$- dependent expression of the CIRCE regulon in Streptococcus pneumoniae. Mol. Microbiol. 55, 456-468 https://doi.org/10.1111/j.1365-2958.2004.04416.x
  18. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  19. Lim, J.H., Martin, F., Guiard, B., Pfanner, N., and Voos, W. (2001). The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation. EMBO J. 20, 941-950 https://doi.org/10.1093/emboj/20.5.941
  20. Lin, Z., Schwartz, F.P., and Eisenstein, E. (1995). The hydrophobic nature of GroEL-substrate binding. J. Biol. Chem. 270, 1011-1014 https://doi.org/10.1074/jbc.270.3.1011
  21. Liu, Q., Krzewska, J., Liberek, K., and Craig, E.A. (2001). Mitochondrial Hsp70 Ssc1: role in protein folding. J. Biol. Chem. 276, 6112-6118 https://doi.org/10.1074/jbc.M009519200
  22. Liu, J., Huang, C., Shin, D.H., Yokota, H., Jancarik, J., Kim, J.S., Adams, P.D., Kim, R., and Kim, S.H. (2005). Crystal structure of a heat-inducible transcriptional repressor HrcA from Thermotoga maritima: structural insight into DNA binding and dimerization. J. Mol. Biol. 350, 987-996 https://doi.org/10.1016/j.jmb.2005.04.021
  23. Martirani, L., Raniello, R., Naclerio, G., Ricca, E., and De Felice, M. (2001). Identification of the DNA-binding protein, HrcA, of Streptococcus thermophilus. FEMS Microbiol. Lett. 198, 177-182 https://doi.org/10.1111/j.1574-6968.2001.tb10639.x
  24. McClellan, A.J., Endres, J.B., Vogel, J.P., Palazzi, D., Rose, M.D., and Brodsky, J.L. (1998). Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER. Mol. Biol. Cell 9, 3533-3545 https://doi.org/10.1091/mbc.9.12.3533
  25. Minder, A.C., Fischer, H.M., Hennecke, H., and Narberhaus, F. (2000). Role of HrcA and CIRCE in the heat shock regulatory network of Bradyrhizobium japonicum. J. Bacteriol. 182, 14-22 https://doi.org/10.1128/JB.182.1.14-22.2000
  26. Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F.X., and Schumann, W. (1997). The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J. 16, 4579-4590 https://doi.org/10.1093/emboj/16.15.4579
  27. Mogk, A., Volker, A., Engelmann, S., Hecker, M., Schumann, W., and Volker, U. (1998). Nonnative proteins induce expression of the Bacillus subtilis CIRCE regulon. J. Bacteriol. 180, 2895-2900
  28. Morimoto, R.I., Tissieres, A., and Georgopoulos, C. (1994). The Biology of Heat Shock Proteins and Molecular Chaperones. (Cold Spring Harbor, NY; Cold Spring Harbor Press)
  29. Morrison, D.A., Lacks, S.A., Guild, W.R., and Hageman, J.M. (1983). Isolation and characterization of three new classes of transformation deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination. J. Bacteriol. 156, 281-290
  30. Nagy, E., Balogi, Z., Gombos, I., Akerfelt, M., Bjorkbom, A., Balogh, G., Torok, Z., Maslyanko, A., Fiszer-Kierzkowska, A., Lisowska, K., et al. (2007). Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line. Proc. Natl. Acad. Sci. USA 104, 7945-7950 https://doi.org/10.1073/pnas.0702557104
  31. Narberhaus, F. (1999). Negative regulation of bacterial heat shock genes. Mol. Microbiol. 31, 1-8 https://doi.org/10.1046/j.1365-2958.1999.01166.x
  32. Neidhardt, F.C., and VanBogelen, R.A. (1987). E. coli and Salmonella typhimurium: Cellular and Molecular Biology. F.C., Neidhardt, et al. ed. (Washington, DC: American Society for Microbiology Press), pp. 1334-1345
  33. Nielsen, E., Akita, M., Davila-Aponte, J., and Keegstra, K. (1997). Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone. EMBO J. 16, 935-946 https://doi.org/10.1093/emboj/16.5.935
  34. Oggioni, M.R., Trappetti, C., Kadioglu, A., Cassone, M., Iannelli, F., Ricci, S., Andrew, P.W., and Pozzi, G. (2006). Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol. Microbiol. 61, 1196-1210 https://doi.org/10.1111/j.1365-2958.2006.05310.x.
  35. Okazaki, A., Ikura, T., Nikaido, K., and Kuwajima, K. (1994). The chaperonin GroEL does not recognize apo-alpha-lactalbumin in the molten globule state. Nat. Struct. Biol. 1, 439-446 https://doi.org/10.1038/nsb0794-439
  36. Reischl, S., Wiegert, T., and Schumann, W. (2002). Isolation and analysis of mutant alleles of the Bacillus subtilis HrcA repressor with reduced dependency on GroE function. J. Biol. Chem. 277, 32659-32667 https://doi.org/10.1074/jbc.M201372200
  37. Roncarati, D., Spohn, G., Tango, N., Danielli, A., Delany, I., and Scarlato, V. (2007). Expression, purification and characterization of the membrane-associated HrcA repressor protein of Helicobacter pylori. Protein Expr. Purif. 51, 267-275 https://doi.org/10.1016/j.pep.2006.08.002
  38. Schindler, J., Jung, S., Niedner-Schatteburg, G., Friauf, E., and Nothwang, H.G. (2006). Enrichment of integral membrane proteins from small amounts of brain tissue. J. Neural. Transm. 113, 995-1013 https://doi.org/10.1007/s00702-006-0508-4
  39. Schulz, A., and Schumann, W. (1996). hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J. Bacteriol. 178, 1088-1093 https://doi.org/10.1128/jb.178.4.1088-1093.1996
  40. Servant, P., and Mazodier, P. (2001). Negative regulation of the heat shock response in Streptomyces. Arch. Microbiol. 176, 237-242 https://doi.org/10.1007/s002030100321
  41. Stintzi, A., Marlow, D., Palyada, K., Naikare, H., Panciera, R., Whitworth, L., and Clarke, C. (2005). Use of genome-wide expression profiling and mutagenesis to study the intestinal lifestyle of Campylobacter Infect. Immun. 73, 1797-1810 https://doi.org/10.1128/IAI.73.3.1797-1810.2005
  42. Susin, M.F., Perez, H.R., Baldini, R.L., and Gomes, S.L. (2004). Functional and structural analysis of HrcA repressor protein from Caulobacter crescentus. J. Bacteriol.186, 6759-6767 https://doi.org/10.1128/JB.186.20.6759-6767.2004
  43. Van Dijl, J.M., De Jong, A., Smith, H., Bron, S., and Venema G. (1991). Non-functional expression of Escherichia coli signal peptidase I in Bacillus. J. Gen. Microbiol. 137, 2073-2083 https://doi.org/10.1099/00221287-137-9-2073
  44. Vigh, L., Maresca, B., and Harwood, J. (1998). Does the membrane's physical state control the expression of heat shock and other genes? Trends Biochem. Sci. 23, 369-373 https://doi.org/10.1016/S0968-0004(98)01279-1
  45. Vigh, L., Torok, Z., Balogh, G., Glatz, A., Piotto, S., and Horvath, I. (2007). Molecular Aspects of the Stress Response: Chaperones, Membrances and Networks. P., Csermely, and L., Vigh, eds. (New York: Springer), pp. 114-131
  46. Watanabe, K., Yamamoto, T., and Suzuki, Y. (2001). Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro. J. Bacteriol. 183, 155-161 https://doi.org/10.1128/JB.183.1.155-161.2001
  47. Wiegert, T., and Schumann, W. (2003). Analysis of a DNA-binding motif of the Bacillus subtilis HrcA repressor protein. FEMS Microbiol. Lett. 223, 101-106 https://doi.org/10.1016/S0378-1097(03)00350-1
  48. Wilson, A.C., and Tan, M. (2002). Functional analysis of the heat shock regulator HrcA of Chlamydia trachomatis J. Bacteriol. 184, 6566-6571 https://doi.org/10.1128/JB.184.23.6566-6571.2002
  49. Yuan, G., and Wong, S. (1995). Isolation and charaterization of Bacillus subtilis regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and danK. J. Bacteriol. 177, 6462-6468 https://doi.org/10.1128/jb.177.22.6462-6468.1995
  50. Zhao, Y., Zhang, W., Kho, Y., and Zhao, Y. (2004). Proteomic analysis of integral plasma membrane proteins. Anal. Chem. 76, 1817-1823 https://doi.org/10.1021/ac0354037

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