In the last year, we have focused on three specific events that occur during the life cycle of the model organism Bacillus subtilis in order to study how proteins localize within a cell and how they subsequently assemble into larger structures. First, we have examined the localization and assembly of the protein DivIVA during cell division. Second, we have studied how the peptide SpoVM orchestrates the timing of assembly of two different supramolecular structures during the developmental program of spore formation. Third, we have studied how the protein SpoIVA utilizes ATP to form stable filaments that envelope the maturing spore. During bacterial cell division, the tubulin homolog FtsZ assembles into a ring at mid-cell and constricts the plasma membrane during cytokinesis. In B. subtilis, this constriction is inhibited by a complex of three proteins whose subcellular localization depends on DivIVA. In the last year, using deconvolution fluorescence microscopy, we have seen that DivIVA assembles into rings at mid-cell and patches at the poles. By examining time-lapse images of actively growing cells, we have determined the kinetics of DivIVA ring assembly and the stability of these rings once they have formed. However, DivIVA also forms patches elsewhere in the cell. How does one protein form two distinct structures? Again, by using time-lapse techniques, we observed that patches form when DivIVA rings collapse during cell separation;that is, when the architecture of the cell changes. Finally, in collaboration with Joseph Pogliano at UCSD, by employing a super resolution technique called structured illumination microscopy, we observed that DivIVA actually forms two adjacent rings on either side of nascent division septa. Thus, DivIVA represents an unusual example of a protein that assembles into a higher order structure, whose shape is determined by a pre-existing template: in this case, the curvature of the plasma membrane as defined by the shape of the cell. Taken together with our previous finding that DivIVA localizes by detecting negative membrane curvature, we have proposed that DivIVA localizes quickly to invaginating membranes (nascent cell division sites) and recruits FtsZ polymerization inhibitors in order to prevent aberrant cell division immediately adjacent to recent cell division sites. We have submitted a manuscript for publication that describes these results. When B. subtilis is faced with starvation conditions, it initiates a developmental program called sporulation, wherein the rod-shaped cell forms a spherical internal organelle called the forespore. The forespore is eventually encased in two concentric shells: a proteinaceous outer shell called the coat, and an inner shell made of peptidoglycan called the cortex. During sporulation, initiation of cortex assembly is dependent on the proper initiation of the coat, suggesting that a developmental checkpoint exists which links the morphogenesis of both structures. This checkpoint is mediated by a small protein called SpoVM. We identified two amino acids in SpoVM whose substitution specifically disrupts cortex assembly, but not coat assembly, thereby separating the coat assembly function of SpoVM from its cortex assembly function. By employing classical genetic techniques we discovered a novel gene, which we have named cmpA (cortex morphogenetic protein), that encodes a short protein that participates in this pathway with SpoVM. In the last year, we have shown that CmpA is synthesized under the same transcriptional control as SpoVM and that it co-localizes with SpoVM during sporulation. Furthermore, we have demonstrated that CmpA is a repressor of cortex assembly that delays the initiation of cortex assembly until coat assembly has initiated. Current efforts are aimed towards identifying what downstream target is inhibited by CmpA and understanding how the inhibition of CmpA is relieved. Proper localization of SpoVM to the surface of the forespore marks this surface as the site for the future localization of proteins that form the coat. The first coat protein that is recruited by SpoVM is SpoIVA which, as mentioned above, forms the basement layer of the coat. Our previous studies had shown that SpoIVA binds ATP via a Walker A motif and subsequently hydrolyzes the bound ATP, and we have shown that both binding and hydrolysis are required for the polymerization of the protein in vitro into long filaments. Our hypothesis is that ATP hydrolysis results in a conformational change in SpoIVA that promotes and stabilizes filament assembly, and that these filaments eventually encircle the forespore and provide a platform on top of which the rest of the coat forms. In order to test this model, we wondered if we could isolate variants of SpoIVA that are able to bind, but not hydrolyze ATP. Recently, we have identified several conserved residues in SpoIVA, including ones that comprise a Walker B motif, that are specifically required for ATP hydrolysis, but not ATP binding. Disruption of these motifs resulted in drastic sporulation defects in cells producing these proteins, and purified SpoIVA harboring a disrupted Walker B motif displayed reduced ATP hydrolysis activity. Finally, variants of SpoIVA that are disrupted for ATP hydrolysis were unable to polymerize in vitro. Taken together, we conclude that SpoIVA is a novel morphogenetic protein that utilizes ATP hydrolysis to drive assembly.

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Decker, Amanda R; Ramamurthi, Kumaran S (2017) Cell Death Pathway That Monitors Spore Morphogenesis. Trends Microbiol 25:637-647
Updegrove, Taylor B; Ramamurthi, Kumaran S (2017) Geometric protein localization cues in bacterial cells. Curr Opin Microbiol 36:7-13
Eswara, Prahathees J; Ramamurthi, Kumaran S (2017) Bacterial Cell Division: Nonmodels Poised to Take the Spotlight. Annu Rev Microbiol :
Wu, Yicong; Chandris, Panagiotis; Winter, Peter W et al. (2016) Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy. Optica 3:897-910
Ramamurthi, Kumaran S (2016) Editorial overview: Growth and development: prokaryotes. Curr Opin Microbiol 34:vii-viii
Fu, Riqiang; Gill Jr, Richard L; Kim, Edward Y et al. (2015) Spherical nanoparticle supported lipid bilayers for the structural study of membrane geometry-sensitive molecules. J Am Chem Soc 137:14031-14034
Wu, I-Lin; Narayan, Kedar; Castaing, Jean-Philippe et al. (2015) A versatile nano display platform from bacterial spore coat proteins. Nat Commun 6:6777
Tan, Irene S; Weiss, Cordelia A; Popham, David L et al. (2015) A Quality-Control Mechanism Removes Unfit Cells from a Population of Sporulating Bacteria. Dev Cell 34:682-93
Gill Jr, Richard L; Castaing, Jean-Philippe; Hsin, Jen et al. (2015) Structural basis for the geometry-driven localization of a small protein. Proc Natl Acad Sci U S A 112:E1908-15
Castaing, Jean-Philippe; Lee, Scarlett; Anantharaman, Vivek et al. (2014) An autoinhibitory conformation of the Bacillus subtilis spore coat protein SpoIVA prevents its premature ATP-independent aggregation. FEMS Microbiol Lett 358:145-53

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