MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2005, p. 585–607 Vol. 69, No. 41092-2172/05/$08.00⫹0 doi:10.1128/MMBR.69.4.585–607.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.Bacterial Cell Wall Synthesis: New Insights from Localization StudiesDirk-Jan Scheffers1* and Mariana G. Pinho2*Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, Amsterdam, The Netherlands,1and Microbial Pathogenesis and Cell Biology Laboratory, Instituto de Tecnologia Quı´mica e Biolo´gica, Oeiras, Portugal2INTRODUCTION .......................................................................................................................................................585PEPTIDOGLYCAN STRUCTURE AND COMPOSITION...................................................................................585BIOCHEMICAL REACTIONS FOR PEPTIDOGLYCAN SYNTHESIS ............................................................587ENZYMES INVOLVED IN THE LAST STAGES OF PEPTIDOGLYCAN SYNTHESIS................................587GROWTH OF THE PEPTIDOGLYCAN: HYPOTHETICAL MULTIENZYME COMPLEXES FORMEDBY PENICILLIN-BINDING PROTEINS ........................................................................................................589LOCALIZATION OF PEPTIDOGLYCAN SYNTHESIS ......................................................................................591Incorporation of Labeled Peptidoglycan Precursors .........................................................................................591Localization of PBPs ..............................................................................................................................................594PBP localization in B. subtilis ...........................................................................................................................594PBP localization in E. coli .................................................................................................................................595PBP localization in S. aureus ............................................................................................................................595PBP localization in S. pneumoniae....................................................................................................................597PBP localization in other bacteria ...................................................................................................................597Localization of Peptidoglycan-Degrading Enzymes ...........................................................................................597Localization of Other Proteins Involved in Bacterial Cell Wall Synthesis ....................................................598ROLE OF THE BACTERIAL CYTOSKELETON IN DIRECTING CELL WALL SYNTHESIS ...................599Actin Homologues in Bacteria and Their Role in Cell Wall Synthesis ..........................................................599A Reappraisal of the Role of the Tubulin Homologue FtsZ in Cell Wall Synthesis ....................................600A MODEL FOR PBP LOCALIZATION .................................................................................................................600FUTURE DIRECTIONS ............................................................................................................................................603ACKNOWLEDGMENTS ...........................................................................................................................................603REFERENCES ............................................................................................................................................................603INTRODUCTIONThe cell wall is the principal stress-bearing and shape-main-taining element in bacteria, and its integrity is of critical im-portance to cell viability. In both gram-positive and gram-negative bacteria, the scaffold of the cell wall consists of thecross-linked polymer peptidoglycan (PG). Many studies haveaddressed the relationship between PG synthesis and bacterialgrowth and cell shape by looking at changes in cell shape inmutants that lack one or several enzymes involved in the syn-thesis of PG or other cell wall components or by looking at theincorporation of labeled PG precursors into the cell wall (see41, 57, 80, 159). Recent developments have prompted a re-newed effort to understand cell wall growth and shape deter-mination. First, the application of fluorescence microscopy tobacteria has made it possible to study the localization of en-zymes involved in PG synthesis in growing cells, as well as tolook at localization of newly incorporated PG in live cells.Second, the discovery of an actin-like cytoskeleton involved inbacterial cell shape determination has raised the question ofhow structural information from inside the cell is translated tothe cell wall. In this review, we discuss the recent data onlocalization of PG-synthesizing enzymes in the light of what isknown about PG synthesis from previous studies, and we dis-cuss the role of bacterial cytoskeletal proteins in organizing thecell wall synthesis process.We focus this review not only on the usual model organisms,the rod-shaped bacteria Bacillus subtilis and Escherichia coli,but also on two cocci, namely, Staphylococcus aureus and Strep-tococcus pneumoniae, both of which are clinically relevantpathogens. Rod-shaped bacteria always divide through thesame medial plane and are thought to have two modes of cellwall synthesis: one responsible for the elongation of the celland one responsible for the formation of the division septum(Fig. 1). The two modes of synthesis appear to be catalyzed bydifferent protein complexes. Coccoid bacteria like S. aureusdivide using three different perpendicular planes in three con-secutive cycles of cell division and seem to have only one modeof cell wall synthesis at the septum. S. pneumoniae cells are not“true” cocci, as their shape is not totally round, but insteadhave the shape of a rugby ball and synthesize cell wall not only atthe septum but also at the so called “equatorial rings” (Fig. 1).These differences in the mode of division and sites for cell wallsynthesis reflect some of the diversity existing in bacteria, afundamental aspect of bacterial cell biology.PEPTIDOGLYCAN STRUCTURE AND COMPOSITIONPeptidoglycan, also called murein, is a polymer that consistsof long glycan chains that are