Murein (Peptidoglycan) Binding Property of the Essential Cell Division Protein FtsN from Escherichia coli

Abstract

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.Division of the rod-shaped bacterium Escherichia coli includes the formation of two new polar caps of the daughter cells. Division is facilitated by the so-called divisome, a ring structure at the tip of the inward-growing septum (12, 44). About twelve known essential cell division proteins (Fts proteins) were shown to localize at this site (47). Probably the best characterized Fts protein is FtsZ, a homolog of eukaryotic tubulin, which is the first known protein that localizes at the division site and which forms a ring-like polymeric structure (5,16,39,40,52). The localization of all other cell division proteins depends on the presence of FtsZ. It is assumed that FtsZ may provide not only the platform for the assembly of the other components of the divisome but also the force for constriction by its ability to utilize energy from GTP hydrolysis. The FtsZ ring is stabilized by and maybe connected to the membrane via ZipA (21, 29, 30, 38) and FtsA, which has an actin-like fold (2,42,43,49,55,59). The assembly of the divisome then continues with the sequential localization of the predicted ABC transporter FtsEX (54), followed by the membrane proteins FtsK (3), FtsQ (7, 9), FtsL (15,23,27), YgbQ (now termed FtsB) (8), and FtsW (6, 46) at the FtsZ-FtsAZipA ring. After FtsW, the monofunctional murein transpeptidase penicillin-binding protein 3 (PBP3; also named FtsI) localizes at the site of division (48, 61, 62), foll...

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

“…Purified SPOR domains bind to purified PG sacculi in a cosedimentation assay, but whether this binding reflects the specific association of the SPOR domains with septal PG is not known (15,17,20,21). To address this question, we purified hexahistidine (His 6 )-tagged fusions of GFP to the SPOR domains from four E. coli cell-division proteins: DamX, DedD, FtsN, and RlpA.…”

“…In support of this hypothesis, in a cosedimentation assay the periplasmic domain of FtsN (which includes the SPOR domain) binds much better to wild-type E. coli sacculi than to sacculi from a triple-amidase mutant (20). Moreover, the purified periplasmic domain of FtsN binds to long glycan strands (≥25 disaccharides) released by amidase digestion of PG (20). The length requirement suggests binding is cooperative or involves the formation of a higher-order structure of the SPOR domain with PG.…”

“…SPOR domains are thought to direct proteins to the division site by binding to denuded glycans created by the cell-wall amidases that mediate daughter-cell separation. In support of this hypothesis, in a cosedimentation assay the periplasmic domain of FtsN (which includes the SPOR domain) binds much better to wild-type E. coli sacculi than to sacculi from a triple-amidase mutant (20). Moreover, the purified periplasmic domain of FtsN binds to long glycan strands (≥25 disaccharides) released by amidase digestion of PG (20).…”

Diversity of Penicillin-binding Proteins

“…Binding of FmtA ⌬27 to Peptidoglycan-These experiments were carried out following a published protocol with minor modifications (43). Briefly, peptidoglycan from S. aureus RN6390 was isolated as described previously (44).…”

The bacterial divisome: ready for its close-up

“…This was at first surprising, as FtsA is an early recruit to the Z ring, while FtsN is mainly a late recruit [10]. However, it was found that a small amount of FtsN molecules are recruited early by FtsA [35], which initiates a positive feedback loop that involves septal wall synthesis [36], consistent with FtsN's ability to contact peptidoglycan directly [37]. The result is a robust localization of FtsN to the divisome at late stages.…”