This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Feilmeier, B. J.
Right arrow Articles by Phillips, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feilmeier, B. J.
Right arrow Articles by Phillips, G. J.

 Previous Article  |  Next Article 

Journal of Bacteriology, July 2000, p. 4068-4076, Vol. 182, No. 14
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli†

Bradley J. Feilmeier, Ginger Iseminger, Diane Schroeder, Hannali Webber, and Gregory J. Phillips*

Department of Microbiology, Iowa State University, Ames, Iowa 50011

Received 18 December 1998/Accepted 19 January 2000

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta -galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

* Corresponding author. Mailing address: Department of Microbiology, Science I Bldg., Rm. 207, Iowa State University, Ames, IA 50011. Phone: (515) 294-1525. Fax: (515) 294-6019. E-mail: gregory{at}iastate.edu.

dagger Journal Paper no. J-18214 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. IOW3220.

Journal of Bacteriology, July 2000, p. 4068-4076, Vol. 182, No. 14
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Gorla, P., Pandey, J. P., Parthasarathy, S., Merrick, M., Siddavattam, D. (2009). Organophosphate Hydrolase in Brevundimonas diminuta Is Targeted to the Periplasmic Face of the Inner Membrane by the Twin Arginine Translocation Pathway. J. Bacteriol. 191: 6292-6299 [Abstract] [Full Text]  
  • Uehara, T., Dinh, T., Bernhardt, T. G. (2009). LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli. J. Bacteriol. 191: 5094-5107 [Abstract] [Full Text]  
  • Arends, S. J. R., Kustusch, R. J., Weiss, D. S. (2009). ATP-Binding Site Lesions in FtsE Impair Cell Division. J. Bacteriol. 191: 3772-3784 [Abstract] [Full Text]  
  • McKenzie, N. L., Nodwell, J. R. (2009). Transmembrane topology of the AbsA1 sensor kinase of Streptomyces coelicolor. Microbiology 155: 1812-1818 [Abstract] [Full Text]  
  • Pradel, N., Delmas, J., Wu, L. F., Santini, C. L., Bonnet, R. (2009). Sec- and Tat-Dependent Translocation of {beta}-Lactamases across the Escherichia coli Inner Membrane. Antimicrob. Agents Chemother. 53: 242-248 [Abstract] [Full Text]  
  • Kolkman, M. A. B., van der Ploeg, R., Bertels, M., van Dijk, M., van der Laan, J., van Dijl, J. M., Ferrari, E. (2008). The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway. Appl. Environ. Microbiol. 74: 7507-7513 [Abstract] [Full Text]  
  • Kaserer, W. A., Jiang, X., Xiao, Q., Scott, D. C., Bauler, M., Copeland, D., Newton, S. M. C., Klebba, P. E. (2008). Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane. J. Bacteriol. 190: 4001-4016 [Abstract] [Full Text]  
  • Real, G., Fay, A., Eldar, A., Pinto, S. M., Henriques, A. O., Dworkin, J. (2008). Determinants for the Subcellular Localization and Function of a Nonessential SEDS Protein. J. Bacteriol. 190: 363-376 [Abstract] [Full Text]  
  • Olichon, A., Surrey, T. (2007). Selection of Genetically Encoded Fluorescent Single Domain Antibodies Engineered for Efficient Expression in Escherichia coli. J. Biol. Chem. 282: 36314-36320 [Abstract] [Full Text]  
  • Tatar, L. D., Marolda, C. L., Polischuk, A. N., van Leeuwen, D., Valvano, M. A. (2007). An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling. Microbiology 153: 2518-2529 [Abstract] [Full Text]  
  • Wilks, J. C., Slonczewski, J. L. (2007). pH of the Cytoplasm and Periplasm of Escherichia coli: Rapid Measurement by Green Fluorescent Protein Fluorimetry. J. Bacteriol. 189: 5601-5607 [Abstract] [Full Text]  
  • Lehrer, J., Vigeant, K. A., Tatar, L. D., Valvano, M. A. (2007). Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide. J. Bacteriol. 189: 2618-2628 [Abstract] [Full Text]  
  • Watt, R. M., Wang, J., Leong, M., Kung, H.-f., Cheah, K. S.E., Liu, D., Danchin, A., Huang, J.-D. (2007). Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes. Nucleic Acids Res 35: e37-e37 [Abstract] [Full Text]  
  • Duffy, E. B., Barquera, B. (2006). Membrane Topology Mapping of the Na+-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis. J. Bacteriol. 188: 8343-8351 [Abstract] [Full Text]  
  • Kikuchi, Y., Date, M., Itaya, H., Matsui, K., Wu, L.-F. (2006). Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869. Appl. Environ. Microbiol. 72: 7183-7192 [Abstract] [Full Text]  
  • Tomich, M., Fine, D. H., Figurski, D. H. (2006). The TadV Protein of Actinobacillus actinomycetemcomitans Is a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins.. J. Bacteriol. 188: 6899-6914 [Abstract] [Full Text]  
  • Scheffers, D.-J., Pinho, M. G. (2005). Bacterial Cell Wall Synthesis: New Insights from Localization Studies. Microbiol. Mol. Biol. Rev. 69: 585-607 [Abstract] [Full Text]  
  • McDonough, J. A., Hacker, K. E., Flores, A. R., Pavelka, M. S. Jr, Braunstein, M. (2005). The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial {beta}-Lactamases. J. Bacteriol. 187: 7667-7679 [Abstract] [Full Text]  
  • Daley, D. O., Rapp, M., Granseth, E., Melen, K., Drew, D., von Heijne, G. (2005). Global Topology Analysis of the Escherichia coli Inner Membrane Proteome. Science 308: 1321-1323 [Abstract] [Full Text]  
  • Paris, S., Sesboue, R. (2004). Metastasis models: the green fluorescent revolution?. Carcinogenesis 25: 2285-2292 [Abstract] [Full Text]  
  • Handrick, R., Reinhardt, S., Kimmig, P., Jendrossek, D. (2004). The "Intracellular" Poly(3-Hydroxybutyrate) (PHB) Depolymerase of Rhodospirillum rubrum Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases. J. Bacteriol. 186: 7243-7253 [Abstract] [Full Text]  
  • Marques, J. P., Schattat, M. H., Hause, G., Dudeck, I., Klosgen, R. B. (2004). In vivo transport of folded EGFP by the {Delta}pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana. J Exp Bot 55: 1697-1706 [Abstract] [Full Text]  
  • Kesty, N. C., Kuehn, M. J. (2004). Incorporation of Heterologous Outer Membrane and Periplasmic Proteins into Escherichia coli Outer Membrane Vesicles. J. Biol. Chem. 279: 2069-2076 [Abstract] [Full Text]  
  • Ding, Z., Christie, P. J. (2003). Agrobacterium tumefaciens Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion. J. Bacteriol. 185: 760-771 [Abstract] [Full Text]  
  • Abanes-De Mello, A., Sun, Y.-L., Aung, S., Pogliano, K. (2002). A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore. Genes Dev. 16: 3253-3264 [Abstract] [Full Text]  
  • Mintz, K. P., Moskovitz, J., Wu, H., Fives-Taylor, P. M. (2002). Peptide methionine sulfoxide reductase (MsrA) is not a major virulence determinant for the oral pathogen Actinobacillus actinomycetemcomitans. Microbiology 148: 3695-3703 [Abstract] [Full Text]  
  • DeLisa, M. P., Samuelson, P., Palmer, T., Georgiou, G. (2002). Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria. J. Biol. Chem. 277: 29825-29831 [Abstract] [Full Text]  
  • Park, S.-K., Jiang, F., Dalbey, R. E., Phillips, G. J. (2002). Functional Analysis of the Signal Recognition Particle in Escherichia coli by Characterization of a Temperature-Sensitive ffh Mutant. J. Bacteriol. 184: 2642-2653 [Abstract] [Full Text]  
  • Drew, D., Sjostrand, D., Nilsson, J., Urbig, T., Chin, C.-n., de Gier, J.-W., von Heijne, G. (2002). Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis. Proc. Natl. Acad. Sci. USA 10.1073/pnas.052018199v1 [Abstract] [Full Text]  
  • Tanudji, M., Hevi, S., Chuck, S. L. (2002). Improperly folded green fluorescent protein is secreted via a non-classical pathway. J. Cell Sci. 115: 3849-3857 [Abstract] [Full Text]  
  • Hanson, M. R., Kohler, R. H. (2001). GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 52: 529-539 [Abstract] [Full Text]  
  • Santini, C.-L., Bernadac, A., Zhang, M., Chanal, A., Ize, B., Blanco, C., Wu, L.-F. (2001). Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock. J. Biol. Chem. 276: 8159-8164 [Abstract] [Full Text]  
  • Drew, D., Sjostrand, D., Nilsson, J., Urbig, T., Chin, C.-n., de Gier, J.-W., von Heijne, G. (2002). Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis. Proc. Natl. Acad. Sci. USA 99: 2690-2695 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»