The essentiality of DivIVA_Ef oligomerization for proper cell division in <i>enterococcus faecalis</i> and interaction with a novel cell division protein

Description

DivIVA is a Gram-positive cell division protein involved in chromosome segregation, midcell placement of the cell division machinery, complete septum closure, and polar growth and morphogenesis. Although well conserved across various Gram-positive species, DivIVA is believed to be relatively species specific. One similarity among DivIVA homologues is the ability to oligomerize through coiled-coil interaction into complexes comprising 10-12 monomers. To date, the importance of DivIVA oligomerization and the N-terminal coiled-coil for its proper function in bacterial cell division has not been reported. This study examined the biological significance of DivIVA oligomerization and the N-terminal coiled-coil in bacterial cell division. This research provides evidence that the N-terminal coiled-coil and oligomerization is essential for the proper biological function of DivIVA_Ef in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i>_Ef known to disrupt either the N-terminal coiled-coil or the two central coiled-coils, involved in oligomerization, were found to be lethal unless rescued by <i>in trans</i> expression of wild type DivIVA_Ef. Using this rescue method, the N-terminal <i>divIVA</i>_Ef mutant strain, <i>E. faecalis</i> MWMR5, and the mutant strain with partial disruption of oligomerization, <i>E. faecalis</i> MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of <i>divIVA</i>_Ef mutant strains <i>E. faecalis</i> MWMR5 and MWMR10. Both these strains showed asymmetrical division, loss of normal lancet shape, and irregular chains. Full disruption of oligomerization with point mutations in both central coiled-coils resulted in a dominant lethal phenotype. These results demonstrate the essentiality of the N-terminal coiled-coil and oligomerization of DivIVA_Ef for its proper biological function in <i>E. faecalis</i> cell division.<p>
Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVA_Ef interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVA_Ef and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The <i>in vitro</i> and <i>in vivo</i> results thus confirm interaction between DivIVA_Ef and MLJD1.<p>
Another objective of this study was to determine the localization of DivIVA and MLJD1 in <i>E. faecalis</i>. Localization of DivIVA_Ef in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA_Ef was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA_Ef migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA_Ef in <i>E. faecalis</i>, further implicating MLJD1 as a bacterial cell division protein.<p>
Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVA_Ef and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA_Ef is involved in chromosome replication. This model is similar to the role of RacA in sporulation in <i>B. subtilis</i> where RacA directs the chromosome during sporulation through direct interaction with DivIVA_Bs and Spo0J.<p>
This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.

Tags

<i>Enterococcus faecalis</i>

protein interactions

cell division

DivIVA

oligomerization

immunofluorescence microscopy

Bacterial two-hybrid

Additional Fields

Identifer	oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-04152009-115838
Date	15 April 2009
Creators	Hedlin, Cherise Elizabeth
Contributors	Misra, Vikram, Khachatourians, George G., Howard, S. Peter, Hemmingsen, Sean M., Dillon, Jo-Anne R.
Publisher	University of Saskatchewan
Source Sets	Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada
Language	English
Detected Language	English
Type	text
Format	application/pdf
Source	http://library.usask.ca/theses/available/etd-04152009-115838/
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