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Journal of Bacteriology, September 2008, p. 6048-6059, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00462-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes {triangledown} ,{dagger}

Carine Robichon,1,2 Glenn F. King,3,{ddagger} Nathan W. Goehring,1,§ and Jon Beckwith1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115,1 Institut Pasteur, CNRS URA 2185, Unité de Biochimie des Interactions Macromoléculaires, 25 Rue du Docteur Roux, 75724 Paris Cedex, France,2 Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 060323

Received 4 April 2008/ Accepted 30 June 2008

Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the "bait") to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the "prey") to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 432-1920. Fax: (617) 738-7664. E-mail: jon_beckwith{at}hms.harvard.edu

{triangledown} Published ahead of print on 11 July 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: Division of Chemical and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.

§ Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.

Journal of Bacteriology, September 2008, p. 6048-6059, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00462-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gonzalez, M. D., Beckwith, J. (2009). Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins. J. Bacteriol. 191: 2815-2825 [Abstract] [Full Text]  


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