NEWS AND VIEWS A metabolic assembly line in bacteria Matthew T. Cabeen and Christine Jacobs-Wagner The bacterial cytoplasm is rich in filament-forming proteins, from homologues of eukaryotic cytoskeletal elements to other scaffolding and segregation proteins. We now learn that even the metabolic enzyme CTP synthase forms cytoplasmic filaments that affect bacterial cell shape. Bacteria keep surprising us. It was not so long in mediating cell curvature in Caulobacter cres- and analysing their function later. Using high- ago that they were thought to be mere bags of centus9; subsequent characterization revealed resolution electron cryotomography (ECT), an chemicals, possessing only the cell wall as a sort its intermediate filament-like properties9. But unbiased method which uses no labels, Jensen of exoskeleton to hold everything together. As what about proteins with functions that would and colleagues uncovered several filament-like it turns out, bacterial cells have a sophisticated never suggest any polymerizing property? structures in the cytoplasm of C. crescentus internal organization. They possess counter- Recent work has approached the discovery of that could not be identified by disrupting or parts of tubulin, actin and intermediate fila- subcellular structures from the opposite direc- eliminating known cytoskeletal structures10. ment proteins, suggesting that a cytoskeleton tion by searching for filamentous structures first Meanwhile, in another unbiased approach, first evolved in bacteria. Moreover, in recent years the known bacterial filament-forming proteins have expanded beyond the traditional cytoskeleton to include DNA segregators, structural scaffolds and proteins, the function of which are still unknown. On page 739 of this TubZ issue, Ingerson-Mahar et al. now show that even a protein doing an everyday biochemical task — making the nucleotide CTP — is organ- Alp7A ized into a filamentous structure1. AlfA At first, filament-forming proteins in bacteria were discovered purely on the basis of their cel- Cell wall MreB lular functions. For example, the tubulin homo- logue, FtsZ, was initially found for its role in Crescentin MamK CTP cell division2, and the actin homologue, MreB, synthase 3 for its function in determination of rod-shape . Bactofilin Their weak sequence similarity to the eukaryo- FtsZ FilP tic proteins left their cytoskeletal properties ParM unsuspected for years. Only later did micro- 1900–1990 scopy and crystallography allow us to appre- 1990–2000 ciate their striking structural properties and 2001–2005 similarity to eukaryotic cytoskeletal elements4–7 (Fig. 1). Even crescentin, the founding member in a growing family of bacterial intermediate fil- 8 Since ament-like proteins , was uncovered for its role 2006 Matthew T. Cabeen is at Yale University, Department of Molecular, Cellular & Developmental Biology, New Haven, CT 06511, USA. Christine Jacobs Wagner is an HHMI Investigator at Yale University, Department of Molecular, Cellular & Developmental Biology and at Yale University School of Medicine, Microbial Pathogenesis Section, New Haven, CT 06511, USA. Figure 1 The recent increase in knowledge about filamentous structures in the cytoplasm of walled e-mail: [email protected] bacteria. Examples of various protein families are shown. NATURE CELL BIOLOGY VOLUME 12 | NUMBER 8 | AUGUST 2010 731 © 2010 Macmillan Publishers Limited. All rights reserved NEWS AND VIEWS a CTP synthase But why would a CTP synthase assemble into a filamentous structure? In C. crescentus, Inhibition of cell curvature CtpS appears to have an inhibitory effect on crescentin-mediated cell curvature1 (Fig. 2a). Overproduction of CtpS causes the crescentin structure to collapse into a single spot, lead- ing to loss of cell curvature. Conversely, CtpS Crescentin structure depletion results in hypercurvature. Analysis of CtpS point mutants that are unable to cata- b Active Inactive state state lyse the synthesis of CTP and either retain or lose the ability to polymerize shows that CtpS assembly, but not enzymatic activity, is required for its inhibitory effect on the cres- centin structure. Interestingly, this inhibition does not seem to be a C. crescentus-specific Stabilization of active state adaptation. C. crescentus cells producing E. coli CtpS rather than the native enzyme are viable, c Activation domain display filamentous localization of CtpS, and have wild-type curvature. Thus, the E. coli enzyme possesses all the enzymatic, polym- erization and curvature-regulation properties of the native protein. Activation site A key question raised by this work is why d a housekeeping enzyme like CtpS would have the unexpected property of polymerizing into a filamentous structure. The observation that Bipartite E. coli CtpS — which has no cell curvature active site to regulate — also polymerizes1, suggests that Reconstituted this property may be an integral part of its active site enzymatic function rather than a secondary adaptation. Filament formation by metabolic Figure 2 Possible purposes of CTP synthase polymerization. (a) CTP synthase polymers interact with enzymes is not without precedent, as ani- the crescentin structure and inhibit its cell curvature function through an unknown mechanism. (b) Polymerization results in stabilization of an active state. (c) Polymerization causes each CTP synthase mal acetyl coenzyme A carboxylase (ACC) subunit to be activated by binding to the adjacent subunit. (d) Each complete active site is formed from polymerizes in vitro into an active form when two adjacent subunits in the polymer. induced by citrate, an allosteric activator12. Recent work shows that an accessory protein Gitai and colleagues undertook a genome-wide C. crescentus cells by light microscopy corre- (MIG12) induces ACC polymerization, and project with the goal of fluorescently tagging spond to inner curvature-localized filaments hence activity, in vitro and in vivo, arguing that as many C. crescentus proteins as possible and visible by ECT. Importantly, a CtpS-specific polymerization is physiologically relevant13. In cataloguing their localization patterns by fluo- inhibitory drug causes diffuse cytoplasmic the case of ACC, allosteric activation precedes rescence microscopy11. From this screen, they distribution of fluorescently tagged CtpS as polymerization slightly, and the polymers may discovered three proteins that had a filament- well as disappearance of the ECT filaments, therefore stabilize the individual enzymes like localization. Unsurprisingly, the inter- suggesting that these filaments are indeed in their active state14. It is possible that the mediate filament-like protein crescentin was composed of CtpS. The CtpS filaments polymerization of CtpS fulfills a similar sta- one of these proteins, but the other two were appear to be anchored by the membrane- bilizing function (Fig. 2b). Alternatively, CtpS highly unexpected: UDP-N-acetylmuramate- associated crescentin structure at the inner polymerization itself may promote enzymatic alanine ligase, a member of the cell wall bio- cell curvature, as the CtpS filaments become activity. This is analogous to the idea that stim- synthesis pathway, and CTP synthase (CtpS), cytoplasmic in crescentin-null C. crescentus ulation of GTPase activity by polymerization an essential enzyme that aminates uridine tri- cells. Affinity between crescentin and CtpS of tubulin or FtsZ is accomplished by inter- phosphate to make the nucleotide, cytidine is supported by their co-localization into subunit interactions, with each subunit being triphosphate. filamentous structures when both proteins activated by its neighbour15 (Fig. 2c) or with In this issue, the Gitai and Jensen groups are exogenously produced in Escherichia coli. adjacent subunits in the polymer combining work together to confirm and character- Yet, CtpS filaments assemble independently to form an active site16 (Fig. 2d). Whether this ize this unusual property of CtpS1. They of crescentin in crescentin-null C. crescentus is the case awaits more work on the structure first show that fluorescently labelled CtpS and in E. coli and fission yeast, which lack and dynamics of CtpS subunits and polymers structures seen at the inner curvature of crescentin, as well as in vitro. in vitro. Apart from CtpS, three other enzymes 732 NATURE CELL BIOLOGY VOLUME 12 | NUMBER 8 | AUGUST 2010 © 2010 Macmillan Publishers Limited. All rights reserved NEWS AND VIEWS of the pyrimidine biosynthesis pathway were COMPETING FINANCIAL INTERESTS 9. Ausmees, N., Kuhn, J. R. & Jacobs-Wagner, C. Cell 115, 705–713 (2003). The authors declare no competing financial interests. tagged in the high-throughput C. crescentus 10. Briegel, A. et al. Mol. Microbiol. 62, 5–14 screen; all three showed diffuse cytoplasmic 1. Ingerson-Mahar, M., Briegel, A., Werner, J. N., Jensen, (2006). localization11, suggesting that filament assem- G. J. & Gitai, Z. Nat. Cell Biol. 12, 739–746 (2010). 11. Werner, J. N. et al. Proc. Natl Acad. Sci. USA 106, 2. Lutkenhaus, J. F., Wolf-Watz, H. & Donachie, W. D. 7858–7863 (2009). bly is not a general property of enzymes in this J. Bacteriol. 142, 615–620 (1980). 12. Kleinschmidt, A. K., Moss, J. & Lane, D. M. Science pathway. Are there other metabolic enzymes 3. Doi, M. et al. J. Bacteriol. 170, 4619–4624 (1988). 166, 1276–1278 (1969). 4. Bi, E. F. & Lutkenhaus, J. Nature 354, 161–164 13. Kim, C. W. et al. Proc. Natl Acad. Sci. USA 107, with polymerizing properties in bacteria? The (1991). 9626–9631 (2010). filament-like