Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Abstract

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to "denuded" glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division.A defining feature of bacteria is the peptidoglycan (PG) cell wall or "sacculus" that confers cell shape, protects the cell against lysis caused by turgor pressure, and is the ultimate target of many clinically relevant antibiotics, including β-lactams and vancomycin (1-4). The structure of PG is characterized by a network of glycan strands connected at regular intervals by oligopeptide cross-links (Fig. 1). The glycans are built from a repeating disaccharide of β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), from which branch the oligopeptides that mediate cross-linking. During cell division, a new PG wall is assembled between the incipient daughter cells (5, 6), and subsequent cell separation depends on the activity of a collection of PG hydrolases that cleave various bonds in the PG mesh (7).In the model Gram-negative bacterium Escherichia coli, daughter-cell separation is driven primarily by three cell-wall amidases (8-10). These enzymes cleave the amide bond that joins the lactyl group of the MurNAc to the α-amino group of the first amino acid (L-Ala) of the stem peptide, releasing as products free oligopeptides and "denuded" glycan strands (Fig. 1). Lytic transglycosylases (LTs) that cleave the MurNAc-GlcNAc glycosidic bond and endopeptidases that cleave stem peptides make minor but still significant contributions to daughter-cell separation (9, 11). In Bacillus subtilis, a model Gram-positive species, the enzymology of daughter-cell separation is not as well characterized. The...

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division

“…What's more, this was shown to be true in both E. coli and B. subtilis (15), organisms whose evolutionary paths diverged over 1 billion years ago. More recently, we extended this paradigm to S. aureus, an organism that does not even have SPOR domain proteins (Fig.…”

“…These issues were laid to rest by using fluorescence microscopy to visualize purified GFP-SPOR fusion proteins bound to isolated PG sacculi (15). This assay revealed GFP-SPOR proteins derived from DamX, DedD, FtsN, and RlpA localized to division sites (Fig.…”

“…This minireview focuses on one important type of PG binding domain used to target proteins to the division site, namely, the "sporulation-related repeat" or "SPOR" domain (Pfam 05036) (10)(11)(12)(13). SPOR domains serve as septal targeting domains on account of their affinity for denuded glycans, regions where the PG lacks peptide sidechains (11,14,15) (Fig. 1A).…”

FtsEX acts on FtsA to regulate divisome assembly and activity

“…The third step involves activation of the divisome complex by the arrival of FtsN to start peptidoglycan (PG) synthesis (12,13). The onset of septal PG synthesis results in the recruitment of many additional proteins involved in cell wall remodeling and invagination of the envelope, including amidases (14), the denuded PG strand binding proteins (containing SPOR domains) (12,15,16), the transenvelope Tol-Pal complex, and a number of other proteins with unknown function (2,17). Together, these proteins ensure that the constriction process proceeds smoothly and that daughter cells are separated without lysis.…”

Bacterial Division: FtsZ Treadmills to Build a Beautiful Wall

“…This more dominant role for FtsZ in driving local peptidoglycan synthesis compared with MreB makes sense, because sidewall synthesis continues an existing template, whereas septum synthesis must initiate wall assembly in a new orthogonal direction. Moreover, septal peptidoglycan is chemically distinct from sidewall peptidoglycan [15].…”