Point of
VIEW
Lighting Up the Nascent Cell Wall Wilfred A. van der Donk*
Department of Chemistry, University of Illinois at Urbana–Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801
T
he manner by which eubacteria assemble their cell walls has been intensely investigated for more than half a century, primarily because this process is targeted by many important antibiotics, including the -lactams, vancomycin, fosfomycin, nisin, and bacitracin (1). The eubacterial cell wall consists of layers of peptidoglycan (PG) polymer (also termed murein) made up of alternating disaccharides composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) (Figure 1) that are cross-linked through short peptides attached to the lactyl group of each MurNAc (2). This cross-linking by penicillin-binding proteins (PBPs) generates a netlike, 3D structure that provides the cell with the strength to withstand intracellular pressures of several atmospheres. Whereas the cytoplasmic biosynthesis of lipid II, the precursor for the polymerization of PG, is well understood (3), the mechanism by which this monomer is inserted into existing PG in a growing or dividing cell is still largely unknown. For the details of PG biosynthesis to be understood, knowledge about the subcellular localization of the biosynthetic machinery and its substrates is essential. In a recent issue of Proceedings of the National Academy of Sciences, U.S.A., Walker and co-workers (4) provide a powerful and general tool for investigating the spatial distribution of sites of nascent PG biosynthesis. Although the molecular details are not known, ramoplanin binds in the region of MurNAc pyrophosphate found both in lipid II and in the reducing end of the growing glycan polymer (5), where new lipid II molwww.acschemicalbiology.org
ecules are inserted through a transglycosylation reaction catalyzed by the highmolecular-weight PBPs (Figure 1). Walker et al. (4) used microscopy and a series of fluorescently labeled ramoplanin analogues to visualize the sites of new PG biosynthesis. The staining patterns observed showed fluorescent patches at the hemispherical cell poles and at new cell-division septa at midcell of the rod-shaped bacterium Bacillus subtilis. In addition, helical staining patterns were observed along the cylindrical side wall of the cell (Figure 2). A similar helicoid pattern along the longitudinal cell axis had been reported previously by Daniel and Errington (6), who used fluorescently labeled vancomycin. This molecule binds to the D-Ala-D-Ala segment present in lipid II as well as along PG chains that have not been fully cross-linked by transpeptidases nor hydrolyzed by D,D-carboxypeptidases (Figure 1) (2). On the basis of the respective recognition sites for ramoplanin and vancomycin, one might have expected that the former would be more specific for staining initiation sites of nascent PG biosynthesis; however, similar (although not identical) patterns were in fact observed when either probe was used (4). One advantage of ramoplanin staining over vancomycin staining is that presumably because of its higher affinity for its target, lower concentrations of reagents could be used to visualize the coiled patterns. Because both molecules are antibiotics, the use of lower concentrations could be important to ensure that the reagents themselves do not influence the distribution of sites of nascent PG biosynthesis. The observation that cell poles are
A B S T R A C T Many antibiotics target the assembly of the cell wall of eubacteria, a netlike 3D structure composed of layers of peptidoglycan (PG). Very little is known about how the lipid precursor of PG, lipid II, is inserted into the existing cell wall in a growing and dividing cell. A new study provides a powerful tool for investigating this insertion process and opens the door to understanding the mechanism of eubacterial cell wall biogenesis.
*Corresponding author,
[email protected].
Published online August 18, 2006 10.1021/cb600308w CCC: $33.50 © 2006 by American Chemical Society
VOL.1 NO.7 • ACS CHEMICAL BIOLOGY
425
Figure 1. Schematic representation of new PG biosynthesis. The structure of lipid II is shown in blue. It is inserted into existing PG via a transglycosylation reaction. After incorporation, the sidechain amine of a lysine (or in certain cases, a diaminopimelic acid) of one nascent chain is cross-linked with another PG chain by a transpeptidation reaction in which the terminal D-Ala of one of the MurNAc-linked pentapeptides is displaced. The binding sites of vancomycin, which primarily inhibits transpeptidation, and ramoplanin, which inhibits transglycosylation, are indicated. Inset: Nascent PG after one transpeptidation cross-link. M ⴝ MurNAc, G ⴝ GlcNAc, blue spheres are the first three amino acids of pentapeptide, and green spheres represent D-Ala-D-Ala. In mature PG, the terminal D-Ala can be removed as a result of transpeptidation or alternatively by proteolysis by a D,D-carboxypeptidase.
stained by ramoplanin, with its selectivity for binding to initiation sites, suggests that the poles are not inert and that PG biosynthesis persists. The detection of helical localization of the biosynthetic substrates for PG biosynthesis is very intriguing, especially because it had been long thought that PG biosynthesis occurred in a dispersed fashion along the cylindrical cell of rod-shaped bacteria. It raises the million-dollar question: what positions the substrate lipid II and/or the biosynthetic machinery along spirals wrapping the cylindrical cell membrane? Interestingly, the helical distribution of sites of new PG biosynthesis comes at a time when more and more studies report similar sublocalizations of proteins in bacteria. Recent years have seen the identification of bacterial homologues of the major eukaryotic cytoskeletal proteins. MreB is the bacterial homologue of actin, FtsZ is the homologue of tubulin, and Crescentin is the homologue of intermediate filament (7). All three are distributed at certain times during bacterial cell cycles in helicoid patterns. Because the 426
VOL.1 NO.7 • 425–428 • 2006
shape of the PG layer is a critical determinant of bacterial morphology (2), many mutants identified in various nonspherical bacteria on the basis of a change of shape (rod mutants) have provided candidate proteins that may determine the spatial organization of the machinery for new PG biosynthesis. Among these mutants are genes in the mre gene cluster (murein cluster e). Like actin, MreB assembles into filaments that form large fibrous spirals in the cytoplasm, just under the cell membrane of the rodshaped bacteria B. subtilis (8, 9) and Escherichia coli (10), as well as at the start of the cell cycle of the crescent-shaped organism Caulobacter crescentus (11, 12). Visualization of these structures by immunofluorescence microscopy or GFP-fusion imaging demonstrates that in B. subtilis and C. crescentus the spiral consists of three or four turns along the length of the cell, whereas in E. coli the helix forms one or two turns. Interestingly, Pbp2, whose gene is in the same operon as mreB, is also organized along helical bands encircling the cell in C. crescentus. This spiral arrangement of Pbp2 is VAN DER DONK
dependent on MreB, and Pbp2 coimmunoprecipitated with several other PBPs; this suggests it is part of a multienzyme PG biosynthetic complex (11). Bacterial cell-wall biosynthesis in rod-shaped bacteria is thought to take place in two stages, one involving cell division at the septum and a subsequent stage in which elongation of the side wall takes place (2). Pbp2 is essential for the latter process and is a membrane protein with a cytoplasmic and periplasmic domain that catalyzes cross-linking of PG by transpeptidation. Collectively, these data suggest a model in which the intracellular MreB helix might position the Pbp2 spiral, thus serving as a spatial and temporal cytoskeletal scaffold directing PG biosynthesis during cell elongation. However, several other studies suggest a more complex picture. The helical positioning of fluorescently labeled vancomycin reporting on the location of nascent PG biosynthesis was not dependent on MreB (6). Furthermore, during the cell cycle of C. crescentus, the coiled distribution of MreB dynamically changed to a midcell localization at the onset of cell diviwww.acschemicalbiology.org
Point of
VIEW Figure 2. Schematic representation of the sites of nascent PG biosynthesis as revealed by vancomycin and ramoplanin staining patterns. When 3D deconvolution microscopy was used, bands were observed at the cell poles and the division septa and patterns consistent with helices were also seen. These spirals run along the cylindrical part of the rod-shaped cell. This figure is not representative of the intensities of the staining or the details of the patterns, which vary according to conditions. For the actual fluorescent microscopy images, see refs 4 and 6.
sion, whereas Pbp2 remained organized in spirals (11, 13). If the biosynthetic complex for PG biosynthesis does not appear to track along MreB filaments, how does it then become organized along helices during cell elongation? The membrane proteins MreC and MreD are essential for determining a rodlike shape and also form helical cables around the longitudinal axes of various bacteria (13–15). Moreover, MreC is required for the spiral distribution of PG precursors in B. subtilis (15). Interestingly, simultaneous visualization of cytosolic MreB and periplasmic MreC in C. crescentus showed that their helices do not colocalize and also that MreC helix formation is independent of MreB (13). Thus, transfer of the information about localization from the cytoplasm to the periplasm does not appear to be achieved through a direct physical interaction between these two protein helices. On the other hand, the mere fact that the two coils do not overlay suggests that some form of communication must occur. The Pbp2 helix did partially overlap with the MreC spiral, and in the absence of functional MreC or MreB, newly synthesized Pbp2 mislocalized to the diviwww.acschemicalbiology.org
sion plane. This mislocalization was shown to be dependent on the tubulin homologue FtsZ (13), which forms a ring at midcell in the earliest known event of cell division and then recruits all other proteins associated with cytokinesis (16). In cells containing chemically inactivated MreB and that were depleted of FtsZ, Pbp2 once again was localized along helical patterns. To explain these observations, Theriot et al. (13) recently hypothesized that MreB prevents Pbp2 accumulation at the divisional site and that MreC actively promotes helical localization along the cylindrical side wall. When either is absent or inactivated, mislocalization of Pbp2 occurs. The reason that FtsZ depletion counteracts MreB inactivation in this model is that in the absence of FtsZ no PG precursors accumulate at midcell that can recruit Pbp2. Unlike C. crescentus, many bacteria have ⬎1 MreB homologue. B. subtilis contains three such proteins: MreB, Mbl (MreB-like), and MreBH. Like MreB, Mbl is important in determining cell shape and forms spiral patterns along the cylindrical part of the cell (8). The importance of these Mbl fibers for positioning new PG biosynthesis during cell elongation is currently under debate. Daniel and Errington (6) reported that the helical staining pattern with fluorescein-labeled vancomycin was abolished in mbl null mutants. On the other hand, Walker and co-workers (4) used either labeled vancomycin or ramoplanin to show a qualitatively similar pattern of helicoid staining of wildtype and mbl– cells. The reasons for this discrepancy are not known at present. To date most studies have looked at localization of Pbp2 by fluorescence imaging and at transpeptidation and transglycosylation sites using fluorescently labeled vancomycin and ramoplanin. However, PBPs themselves need not necessarily track along cytoskeletal filaments; in principle only one component of the multienzyme PG biosynthetic complex must be properly positioned for the entire complex to be correctly local-
ized. In the spherical bacterium Staphylococcus aureus, Pbp2 appears to be recruited to sites of new PG synthesis (division site) by the presence of its substrate (17). Thus, other means for proper spatial and/or temporal control over PG biosynthesis in rod-shaped bacteria could involve coordinated localization of lipid II in the outer leaflet of the cytoplasmic membrane. At present, the translocase that exports intracellularly synthesized lipid II to the outside of the membrane remains unidentified; hence, its subcellular localization is unknown. Another possibility suggested by Walker and co-workers (4) is based on the observed helical bands of proteins of the general secretory (Sec) machinery (18). The PBPs are exported by the Sec pathway, and hence the observed helical localization of SecA and SecY may play a role in determining the timing and place of PBP membrane insertion and therefore new PG biosynthesis. In closing, the long-held view of bacteria as bags of relatively uniformly distributed biomolecules enclosed by cell walls and membranes was abandoned some time ago with the discovery of bacterial cell cycles and polarity. Now the mechanisms by which subcellular localization is governed are starting to emerge. In one example discussed here, recent years have seen major developments in our understanding of how the spatial and temporal coordination of PG biosynthesis is achieved. Although many questions still remain, given the rapid pace of progress in the area, the advances in the spatial and temporal resolution of fluorescence imaging, and the development of small molecules as additional tools, the near future is likely to see major new discoveries. REFERENCES 1. Walsh, C. T. (2003) Antibiotics: Actions, Origins, Resistance pp 1–49, ASM Press, Washington DC. 2. Höltje, J. V. (1998) Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli, Microbiol. Mol. Biol. Rev. 62, 181–203. VOL.1 NO.7 • 425–428 • 2006
427
3. van Heijenoort, J. (2001) Recent advances in the formation of the bacterial peptidoglycan monomer unit, Nat. Prod. Rep. 18, 503–519. 4. Tiyanont, K., Doan, T., Lazarus, M. B., Fang, X., Rudner, D. Z., and Walker, S. (2006) Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics, Proc. Natl. Acad. Sci. U.S.A. 103, 11033–11038. 5. Walker, S., Chen, L., Hu, Y., Rew, Y., Shin, D., and Boger, D. L. (2005) Chemistry and biology of ramoplanin: a lipoglycodepsipeptide with potent antibiotic activity, Chem. Rev. 105, 449–476. 6. Daniel, R. A., and Errington, J. (2003) Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell, Cell 113, 767–776. 7. Møller-Jensen, J., and Löwe, J. (2005) Increasing complexity of the bacterial cytoskeleton, Curr. Opin. Cell Biol. 17, 75–81. 8. Jones, L. J., Carballido-Lopez, R., and Errington, J. (2001) Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis, Cell 104, 913–922. 9. van den Ent, F., Amos, L. A., and Löwe, J. (2001) Prokaryotic origin of the actin cytoskeleton, Nature 413, 39–44. 10. Shih, Y. L., Le, T., and Rothfield, L. (2003) Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles, Proc. Natl. Acad. Sci. U.S.A. 100, 7865–7870. 11. Figge, R. M., Divakaruni, A. V., and Gober, J. W. (2004) MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus, Mol. Microbiol. 51, 1321–1332.
428
VOL.1 NO.7 • 425–428 • 2006
12. Gitai, Z., Dye, N., and Shapiro, L. (2004) An actinlike gene can determine cell polarity in bacteria, Proc. Natl. Acad. Sci. U.S.A. 101, 8643–8648. 13. Dye, N. A., Pincus, Z., Theriot, J. A., Shapiro, L., and Gitai, Z. (2005) Two independent spiral structures control cell shape in Caulobacter, Proc. Natl. Acad. Sci. U.S.A. 102, 18608–18613. 14. Divakaruni, A. V., Loo, R. R., Xie, Y., Loo, J. A., and Gober, J. W. (2005) The cell-shape protein MreC interacts with extracytoplasmic proteins including cell wall assembly complexes in Caulobacter crescentus, Proc. Natl. Acad. Sci. U.S.A. 102, 18602–18607. 15. Leaver, M., and Errington, J. (2005) Roles for MreC and MreD proteins in helical growth of the cylindrical cell wall in Bacillus subtilis, Mol. Microbiol. 57, 1196–1209. 16. Errington, J., Daniel, R. A., and Scheffers, D. J. (2003) Cytokinesis in bacteria, Microbiol. Mol. Biol. Rev. 67, 52–65 17. Pinho, M. G., and Errington, J. (2005) Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates, Mol. Microbiol. 55, 799–807. 18. Campo, N., Tjalsma, H., Buist, G., Stepniak, D., Meijer, M., Veenhuis, M., Westermann, M., Müller, J. P., Bron, S., Kok, J., Kuipers, O. P., and Jongbloed, J. D. (2004) Subcellular sites for bacterial protein export, Mol. Microbiol. 53, 1583–1599.
VAN DER DONK
www.acschemicalbiology.org
