The essentiality of DivIVAEf oligomerization for proper cell division in enterococcus faecalis and interaction with a novel cell division protein
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 DivIVAEf in Enterococcus faecalis cell division. Introduction of point mutations into chromosomal divIVAEf 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 in trans expression of wild type DivIVAEf. Using this rescue method, the N-terminal divIVAEf mutant strain, E. faecalis MWMR5, and the mutant strain with partial disruption of oligomerization, E. faecalis MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of divIVAEf mutant strains E. faecalis 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 DivIVAEf for its proper biological function in E. faecalis cell division. 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 DivIVAEf 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 DivIVAEf and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The in vitro and in vivo results thus confirm interaction between DivIVAEf and MLJD1. Another objective of this study was to determine the localization of DivIVA and MLJD1 in E. faecalis. Localization of DivIVAEf in E. faecalis was found to be similar to DivIVA localization in Bacillus subtilis and Streptococcus pneumonia. DivIVAEf was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVAEf migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVAEf in E. faecalis, further implicating MLJD1 as a bacterial cell division protein. 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 DivIVAEf and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVAEf is involved in chromosome replication. This model is similar to the role of RacA in sporulation in B. subtilis where RacA directs the chromosome during sporulation through direct interaction with DivIVABs and Spo0J. 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.
Enterococcus faecalis, protein interactions, cell division, DivIVA, oligomerization, immunofluorescence microscopy, Bacterial two-hybrid
Master of Science (M.Sc.)
Microbiology and Immunology
Microbiology and Immunology