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<sub>Ef</sub> in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i><sub>Ef</sub> 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<sub>Ef</sub>. Using this rescue method, the N-terminal <i>divIVA</i><sub>Ef</sub> 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><sub>Ef</sub> 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<sub>Ef</sub> 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<sub>Ef</sub> 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<sub>Ef</sub> 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<sub>Ef</sub> 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<sub>Ef</sub> in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA<sub>Ef</sub> was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA<sub>Ef</sub> migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA<sub>Ef</sub> 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<sub>Ef</sub> and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA<sub>Ef</sub> 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<sub>Bs</sub> 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.
Identifer | oai:union.ndltd.org:USASK/oai:usask.ca: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 | University of Saskatchewan Library |
Language | English |
Detected Language | English |
Type | text |
Format | application/pdf |
Source | http://library.usask.ca/theses/available/etd-04152009-115838/ |
Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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