Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics

Abstract

The peptidoglycan (PG) layers surrounding bacterial cells play an important role in determining cell shape. The machinery controlling when and where new PG is made is not understood, but is proposed to involve interactions between bacterial actin homologs such as Mbl, which forms helical cables within cells, and extracellular multiprotein complexes that include penicillin-binding proteins. It has been suggested that labeled antibiotics that bind to PG precursors may be useful for imaging PG to help determine the genes that control the biosynthesis of this polymer. Here, we compare the staining patterns observed in Bacillus subtilis using fluorescent derivatives of two PG-binding antibiotics, vancomycin and ramoplanin. The staining patterns for both probes exhibit a strong dependence on probe concentration, suggesting antibioticinduced perturbations in PG synthesis. Ramoplanin probes may be better imaging agents than vancomycin probes because they yield clear staining patterns at concentrations well below their minimum inhibitory concentrations. Under some conditions, both ramoplanin and vancomycin probes produce helicoid staining patterns along the cylindrical walls of B. subtilis cells. This sidewall staining is observed in the absence of the cytoskeletal protein Mbl. Although Mbl plays an important role in cell shape determination, our data indicate that other proteins control the spatial localization of the biosynthetic complexes responsible for new PG synthesis along the walls of B. subtilis cells.helix ͉ ramoplanin ͉ vancomycin ͉ Mbl ͉ bacterial cytoskeleton B acterial cells are surrounded by layers of peptidoglycan (PG), a crosslinked carbohydrate polymer that functions as a protective exoskeleton. These PG layers enable bacteria to withstand high internal osmotic pressures and also play an important role in maintaining cell shape (1, 2). Because PG is essential for survival and is a defining feature of bacteria, considerable effort has been focused on understanding its structure and assembly. We know a great deal about how the subunits that comprise PG are synthesized inside the cell (3), but we do not yet understand how these subunits are converted into a complex, 3D polymer on the cell surface (4, 5).Early models of bacterial cell growth held that new PG is made and incorporated at midcell around the time of cell division (6). Subsequent metabolic labeling experiments in Bacillus subtilis and Escherichia coli showed that PG is also made along the cylindrical walls of the cells (7-13). Recent experiments using fluorescent vancomycin, an antibiotic that inhibits PG biosynthesis by binding to PG intermediates (i.e., a substrate-binding antibiotic), suggest that cylindrical wall synthesis in B. subtilis occurs in a helix-like pattern (14). It is currently believed that large biosynthetic machines containing different sets of synthetic and hydrolytic enzymes are involved in wall and septal synthesis during bacterial cell growth and division (15)(16)(17)(18)(19)(20). It has been proposed that the spatial loca...

The Bacterial Actin-Like Cytoskeleton

“…3C) and not in a strain lacking MreB (Fig.mycin and ramoplanin (another PG-binding antibiotic), insertion of nascent PG along the lateral wall of B. subtilis was confirmed to be helical (160). However, in this study, a sidewall helical staining pattern qualitatively similar to that observed in wild-type cells (although less regular) was observed in cells lacking Mbl (160); the authors of that study concluded that Mbl plays an indirect role in directing PG synthesis but that it is not essential for the incorporation of sidewall PG.…”

Bacterial Growth and Cell Division: a Mycobacterial Perspective

“…Van-Fl specifically binds D-Ala-DAla, which is found specifically in newly generated PG, while fluorescently labeled ramoplanin binds initiation sites of PG synthesis and lipid II. These techniques revealed helical patterns of nascent PG similar to those of localized MreB and Mbl (88,406). It is conceivable that PG is not truly inert but that active enzyme complexes are restricted from certain areas and thus there is a lack of turnover in those areas.…”

Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan

“…Among the cellular aspects still poorly addressed, the main one is the mechanism of translocation of lipid II. Perhaps lipid II-antibiotic complexes could be useful tools in these matters, as illustrated by their recent use in the study of the dynamic assembly of peptidoglycan in B. subtilis (49,171).…”