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Classic Spotlight: Cellular Sites of Peptidoglycan Synthesis Revealed Piet A. J. de Boer Department of Molecular Biology & Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA
T
he peptidoglycan (PG) sacculus of bacteria is a giant cellshaped molecule that forms a fine meshed enclosure around the cytoplasmic membrane (CM). It consists of glycan strands that are cross-linked by peptide bridges. The latter contain amino acids not found anywhere else in the cell, such as meso-diaminopimelic acid (DAP) and D-stereoisomers of alanine and glutamic acid. The sacculus helps maintain cell shape, and it is critical to cell viability because even small breaches in the PG meshwork can result in turgor-driven extrusion of the CM and cell lysis. The molecule is also dynamic in that it is extended during cell growth and then separated into two intact sacculi during cell fission. Growth and fission of the sacculus involve a large number of synthetic and degradative enzymes whose activities are tightly coordinated and regulated at the molecular level. How this translates to patterns of growth at the cellular level is the topic of a classic Journal of Bacteriology paper by Miguel de Pedro and colleagues (1). In principle, the sacculus of a rod-shaped cell could elongate by insertion of new PG in specific cell zones (e.g., midcell or cell pole) or in a more dispersed fashion. Early attempts to determine saccular growth modes relied on autoradiographic methods (2). This involved metabolic incorporation of radiolabeled DAP, overlay of cells or purified sacculi with a photographic emulsion, and after weeks of exposure and development, examination of the positions of silver grains relative to cells or sacculi by electron microscopy. The first application of this method on sacculi of Escherichia coli suggested the presence of a single growth zone at midcell (2). However, subsequent studies indicated that this midcell zone primarily reflected the synthesis of septal PG (sPG) and that insertion of new cylindrical PG (cPG) likely occurred in a more diffuse manner (3, 4). The autoradiographical method was time-consuming and required specialized skills and equipment, and the spatial resolution was inherently limited by the size of silver grains (5). The development of a superior method by de Pedro and colleagues (1), therefore, provided an important impetus to the field. Their label-and-chase method took smart advantage of the old discovery that externally added D-amino acids that are normally absent from PG, including D-cysteine, can become covalently incorporated into the sacculus of bacterial cells (6, 7). Thus, cells were first grown at length in the presence of D-Cys, ensuring uniform incorporation of the “label” in the sacculus, and then “chased” by subsequent growth for various periods in fresh medium lacking the D-amino acid. Their sacculi were then purified, and incorporated D-Cys was specifically biotinylated with sulfhydryl-reactive reagents, allowing pre- and postchase PG to be clearly distinguished by electron or fluorescence microscopy after
July 2016 Volume 198 Number 13
an appropriate immunolabeling step (1). The new method allowed the authors to obtain unambiguous support for older suggestions that, in E. coli, incorporation of most new cPG is diffuse rather than zonal, that sPG is newly synthesized, and that polar PG is very stable (1). In addition, it confirmed the synthesis of a narrow band of cPG at midcell immediately prior to the start of sPG synthesis and cell constriction (8). Curiously, moreover, formation of this preseptal cPG at midcell was shown to require FtsZ but not FtsA, FtsQ, or the sPG-specific transpeptidase PBP3 (1, 8). Importantly, the paper also inspired the more recent creation of fluorescent versions of nonnatural D-amino acids. These provide even simpler means of detecting cellular sites of active PG synthesis, helping to reveal an unexpected diversity in PG growth modes among the Bacteria (9). REFERENCES 1. de Pedro MA, Quintela JC, Höltje J-V, Schwarz H. 1997. Murein segregation in Escherichia coli. J Bacteriol 179:2823–2834. 2. Ryter A, Hirota Y, Schwarz U. 1973. Process of cellular division in Escherichia coli. Growth pattern of E.coli murein. J Mol Biol 78:185–195. 3. Verwer RWH, Nanninga N. 1980. Pattern of meso-DL-2,6-diaminopimelic acid incorporation during the division cycle of Escherichia coli. J Bacteriol 144:327–336. 4. Burman LG, Raichler J, Park JT. 1983. Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus. J Bacteriol 155: 983–988. 5. Caro LG. 1962. High-resolution autoradiogaphy. II. The problem of resolution. J Cell Biol 15:189 –199. 6. Lark C, Lark KG. 1961. Studies on the mechanism by which D-amino acids block cell wall synthesis. Biochim Biophys Acta 49:308 –322. 7. Tsuruoka T, Tamura A, Miyata A, Takei T, Iwamatsu K, Inouye S, Matsuhashi M. 1984. Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli. J Bacteriol 160:889 – 894. 8. Wientjes FB, Nanninga N. 1989. Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge. J Bacteriol 171:3412–3419. 9. Kuru E, Hughes HV, Brown PJ, Hall E, Tekkam S, Cava F, de Pedro MA, Brun YV, VanNieuwenhze MS. 2012. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew Chem Int Ed Engl 51:12519 –12523.
Citation de Boer PAJ. 2016. Classic spotlight: cellular sites of peptidoglycan synthesis revealed. J Bacteriol 198:1795. doi:10.1128/JB.00310-16. Address correspondence to [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved. The views expressed in this Editorial do not necessarily reflect the views of the journal or of ASM.
Journal of Bacteriology
jb.asm.org
1795
Classic Spotlight: Cellular Sites of Peptidoglycan Synthesis Revealed Piet A. J. de Boer Department of Molecular Biology & Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA
T
he peptidoglycan (PG) sacculus of bacteria is a giant cellshaped molecule that forms a fine meshed enclosure around the cytoplasmic membrane (CM). It consists of glycan strands that are cross-linked by peptide bridges. The latter contain amino acids not found anywhere else in the cell, such as meso-diaminopimelic acid (DAP) and D-stereoisomers of alanine and glutamic acid. The sacculus helps maintain cell shape, and it is critical to cell viability because even small breaches in the PG meshwork can result in turgor-driven extrusion of the CM and cell lysis. The molecule is also dynamic in that it is extended during cell growth and then separated into two intact sacculi during cell fission. Growth and fission of the sacculus involve a large number of synthetic and degradative enzymes whose activities are tightly coordinated and regulated at the molecular level. How this translates to patterns of growth at the cellular level is the topic of a classic Journal of Bacteriology paper by Miguel de Pedro and colleagues (1). In principle, the sacculus of a rod-shaped cell could elongate by insertion of new PG in specific cell zones (e.g., midcell or cell pole) or in a more dispersed fashion. Early attempts to determine saccular growth modes relied on autoradiographic methods (2). This involved metabolic incorporation of radiolabeled DAP, overlay of cells or purified sacculi with a photographic emulsion, and after weeks of exposure and development, examination of the positions of silver grains relative to cells or sacculi by electron microscopy. The first application of this method on sacculi of Escherichia coli suggested the presence of a single growth zone at midcell (2). However, subsequent studies indicated that this midcell zone primarily reflected the synthesis of septal PG (sPG) and that insertion of new cylindrical PG (cPG) likely occurred in a more diffuse manner (3, 4). The autoradiographical method was time-consuming and required specialized skills and equipment, and the spatial resolution was inherently limited by the size of silver grains (5). The development of a superior method by de Pedro and colleagues (1), therefore, provided an important impetus to the field. Their label-and-chase method took smart advantage of the old discovery that externally added D-amino acids that are normally absent from PG, including D-cysteine, can become covalently incorporated into the sacculus of bacterial cells (6, 7). Thus, cells were first grown at length in the presence of D-Cys, ensuring uniform incorporation of the “label” in the sacculus, and then “chased” by subsequent growth for various periods in fresh medium lacking the D-amino acid. Their sacculi were then purified, and incorporated D-Cys was specifically biotinylated with sulfhydryl-reactive reagents, allowing pre- and postchase PG to be clearly distinguished by electron or fluorescence microscopy after
July 2016 Volume 198 Number 13
an appropriate immunolabeling step (1). The new method allowed the authors to obtain unambiguous support for older suggestions that, in E. coli, incorporation of most new cPG is diffuse rather than zonal, that sPG is newly synthesized, and that polar PG is very stable (1). In addition, it confirmed the synthesis of a narrow band of cPG at midcell immediately prior to the start of sPG synthesis and cell constriction (8). Curiously, moreover, formation of this preseptal cPG at midcell was shown to require FtsZ but not FtsA, FtsQ, or the sPG-specific transpeptidase PBP3 (1, 8). Importantly, the paper also inspired the more recent creation of fluorescent versions of nonnatural D-amino acids. These provide even simpler means of detecting cellular sites of active PG synthesis, helping to reveal an unexpected diversity in PG growth modes among the Bacteria (9). REFERENCES 1. de Pedro MA, Quintela JC, Höltje J-V, Schwarz H. 1997. Murein segregation in Escherichia coli. J Bacteriol 179:2823–2834. 2. Ryter A, Hirota Y, Schwarz U. 1973. Process of cellular division in Escherichia coli. Growth pattern of E.coli murein. J Mol Biol 78:185–195. 3. Verwer RWH, Nanninga N. 1980. Pattern of meso-DL-2,6-diaminopimelic acid incorporation during the division cycle of Escherichia coli. J Bacteriol 144:327–336. 4. Burman LG, Raichler J, Park JT. 1983. Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus. J Bacteriol 155: 983–988. 5. Caro LG. 1962. High-resolution autoradiogaphy. II. The problem of resolution. J Cell Biol 15:189 –199. 6. Lark C, Lark KG. 1961. Studies on the mechanism by which D-amino acids block cell wall synthesis. Biochim Biophys Acta 49:308 –322. 7. Tsuruoka T, Tamura A, Miyata A, Takei T, Iwamatsu K, Inouye S, Matsuhashi M. 1984. Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli. J Bacteriol 160:889 – 894. 8. Wientjes FB, Nanninga N. 1989. Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge. J Bacteriol 171:3412–3419. 9. Kuru E, Hughes HV, Brown PJ, Hall E, Tekkam S, Cava F, de Pedro MA, Brun YV, VanNieuwenhze MS. 2012. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew Chem Int Ed Engl 51:12519 –12523.
Citation de Boer PAJ. 2016. Classic spotlight: cellular sites of peptidoglycan synthesis revealed. J Bacteriol 198:1795. doi:10.1128/JB.00310-16. Address correspondence to [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved. The views expressed in this Editorial do not necessarily reflect the views of the journal or of ASM.
Journal of Bacteriology
jb.asm.org
1795
