Characterization of a “hypothetical protein”, EF1025, from Enterococcus faecalis: role in cell length and shape

Abstract

DivIVA plays multifaceted roles in Gram-positive organisms by associating with various cell division and non-cell division proteins. While the interaction of DivIVA with other proteins has been studied in many Gram-positive bacteria, no information is available about DivIVA- associating proteins in E. faecalis. This research reports a novel DivIVAEf interacting protein named EF1025 (encoded by EF1025) (confirmed using Bacterial Two-Hybrid, Glutathione S-Transferase pull-down, and co-immunoprecipitation assays) that affects cell length and morphology in E. faecalis. EF1025 is predominantly conserved in Gram-positive bacteria and contains a conserved N-terminal DNA binding Helix-turn-Helix (HTH) domain and two Cystathionine β-Synthase (CBS) domains located centrally and at the C-terminus. The protein, EF1025, oligomerizes to form a higher-order oligomer and the two CBS domains are responsible for its self-interaction. Viable cells were recovered after insertional inactivation or deletion of EF1025 only through complementation of EF1025 in trans. These cells were longer than the average length of E. faecalis cells and had distorted shapes. Overexpression of EF1025 also resulted in cell elongation but had no effect on cell shape. Immuno-staining revealed comparable localization patterns of EF1025 and DivIVAEf in the later stages of division in E. faecalis cells. The EF1025 homologue in Bacillus subtilis, CcpN, is a transcriptional repressor in Bacillus subtilis. In the presence of glucose, CcpN binds to the promoter region of gapB and pckA and downregulates their expression. CcpN interacted with DivIVA of B. subtilis in B2H and GST-pull down assays. A heterologous interaction between EF1025 and DivIVABs was also identified in a GST-pull down assay. Insertional inactivation of ccpN leads to cell elongation and growth of cells in straight chains. These findings suggest an additional function of CcpN in B. subtilis, therefore, CcpN is a dual function performing protein involved in both gluconeogenesis and cell elongation. E. faecalis contains homologues of divisome proteins FtsZ, FtsA, FtsK, FtsQ, FtsL, FtsI and FtsB, however, the cell division interactome of E. faecalis, by contrast, is not presently known. This thesis also presents the unique interactome of E. faecalis divisome proteins (i.e. FtsZEf, FtsAEf, FtsQEf, FtsLEf, FtsIEf, FtsWEf, DivIVAEf, and FtsBEf), established using Bacterial-two hybrid system. The interaction of FtsA with FtsI, FtsL, and FtsZ, is common among E. faecalis, S. pneumoniae and S. aureus cell division interactomes. One unique interaction i.e. FtsZEf-FtsIEf was identified in E. faecalis cell division interactome. While studying the divisome interactome of E. faecalis, it was observed that EF1025 is not a part of the divisome machinery in E. faecalis as it did not interact with any divisome protein except DivIVAEf.