Journal of Biochemistry Advance Access originally published online on April 3, 2008
Journal of Biochemistry 2008 144(1):63-76; doi:10.1093/jb/mvn044
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 The Japanese Biochemical Society
Identification of the Coiled-coil Domains of Enterococcus faecalis DivIVA that Mediate Oligomerization and their Importance for Biological Function
,
1Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5; 2Department of Microbiology and Immunology, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5; 3Vaccine and Infectious Disease Organization, 120 Veterinary Road, Saskatoon, Saskatchewan S7N 5E3; and 4Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, Saskatchewan, S7N 5E2, Canada
To whom correspondence should be addressed. Tel: 1-306-966-4232, Fax: 1-306-966-8839, E-mail: j.dillon{at}usask.ca
Received October 24, 2007; Accepted March 13, 2008
 |
Abstract |
|---|
Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10–12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVAEf self-interacts and forms a 10–12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVAEf self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVAEf did not affect DivIVAEf oligomerization. The introduction of DivIVAEf mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVAEf in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVAEf are indispensable for proper biological function. Overexpression of wild-type DivIVAEf in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVAEf mutations failed to induce such alterations.
Key Words: cell division, DivIVA, Enterococcus faecalis, mutagenesis, protein interactions
Abbreviations: Bs, Bacillus subtilis; DivIVAEf, E. faecalis DivIVA; Ef, Enterococcus faecalis; NGE, Native Gel Electrophoresis; SDM, site-directed mutagenesis; SEC, size exclusion chromatography; Y2H, yeast two-hybrid assays
*Present addresses: Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, Ontario, Canada K1G 4J5.
College of Arts and Science, University of Saskatchewan, Room 226, Arts Building, 9 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A5.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Hempel, S.-b. Wang, M. Letek, J. A. Gil, and K. Flardh Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth in Streptomyces coelicolor J. Bacteriol., November 15, 2008; 190(22): 7579 - 7583. [Abstract] [Full Text] [PDF]
|
|