Protein-Protein Interaction Domains of Bacillus subtilis DivIVA

Abstract

e DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent proteintagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.

Genetic Dissection of DivIVA Functions in Listeria monocytogenes

“…subtilis DivIVA is not functional in L. monocytogenes. Previous work has shown that DivIVA from L. monocytogenes (DivIVA Lm ) is not functional in B. subtilis, even though the two proteins share 65% sequence identity (18) and are almost of the same length (175 versus 164 amino acids, respectively) (Fig. 1A).…”

“…Hydrophobic amino acids are exposed to the solvent at the apical end of this structure, and DivIVA interacts with membranes through the insertion of these side chains into the phospholipid bilayer (16,17). In addition to membrane binding, the lipid binding domain also mediates the interaction of DivIVA with binding partners, such as MinJ (18). In Bacillus subtilis, MinJ serves as a molecular bridge between DivIVA and MinD to ensure septal and polar recruitment of the division site selection proteins MinCD (10,11).…”

“…In Bacillus subtilis, MinJ serves as a molecular bridge between DivIVA and MinD to ensure septal and polar recruitment of the division site selection proteins MinCD (10,11). MinJ is a polytopic transmembrane protein and interacts with that domain of DivIVA, which already is in closest proximity to the membrane, the lipid binding domain (18). The C-terminal domain of DivIVA, which is oriented toward the cytoplasm, also associates with binding partners, such as the centromere-binding protein RacA (18), contributing to chromosome segregation in B. subtilis cells (19,20).…”

“…MinJ is a polytopic transmembrane protein and interacts with that domain of DivIVA, which already is in closest proximity to the membrane, the lipid binding domain (18). The C-terminal domain of DivIVA, which is oriented toward the cytoplasm, also associates with binding partners, such as the centromere-binding protein RacA (18), contributing to chromosome segregation in B. subtilis cells (19,20). Considering that RacA is a soluble cytoplasmic protein, it therefore seems that DivIVA interacts with transmembrane proteins via its lipid binding domain and with cytoplasmic proteins through its C-terminal domain (18).…”

Hortense: Horizontal gene transfer detection directly from proteomic MS/MS data

“…The HGT organism in this data set is Bacillus subtilis BSN238, a transgenic organism that is a chimera of B. subtilis 168 where the DivIVA protein has been replaced with the DivIVA from Listeria monocytogenes strain EGD-e (van Baarle et al, 2012). The Listeria DivIVA protein is located on the complement strand at positions 2'100'224-2'100'751 (NC 003210.1).…”