Thomas G. Bernhardt

The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway

The N‐acetylmuramoyl‐l‐alanine amidases of Escherichia coli (AmiA, B and C) are periplasmic enzymes that remove murein cross‐links by cleaving the peptide moiety from N‐acetylmuramic acid. Ami– cells form chains, indicating that the amidases help to split the septal murein. Interestingly, cells defective in the twin‐arginine protein transport (Tat) pathway show a similar division defect. We find that both AmiA and AmiC are routed to the periplasm via Tat, providing an explanation for the Tat– division phenotype. Taking advantage of the ability of Tat to export prefolded (fluorescent) green fluorescent protein (GFP) to the periplasm, we sublocalized AmiA and AmiC in live cells using functional fusions to GFP. Interestingly, the periplasmic localization of the fusions differed markedly. AmiA–GFP appeared to be dispersed throughout the periplasm in all cells. AmiC–GFP similarly appeared throughout the periplasm in small cells, but was concentrated almost exclusively at the septal ring in constricting cells. Recruitment of AmiC to the ring was mediated by an N‐terminal non‐amidase targeting domain and required the septal ring component FtsN. AmiC therefore replaces FtsN as the latest known recruit to the septal ring and is the first entirely periplasmic component to be localized.

RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli

The bacterial MreB actin cytoskeleton is required for cell shape maintenance in most non‐spherical organisms. In rod‐shaped cells such as Escherichia coli, it typically assembles along the long axis in a spiral‐like configuration just underneath the cytoplasmic membrane. How this configuration is controlled and how it helps dictate cell shape is unclear. In a new genetic screen for cell shape mutants, we identified RodZ (YfgA) as an important transmembrane component of the cytoskeleton. Loss of RodZ leads to misassembly of MreB into non‐spiral structures, and a consequent loss of cell shape. A juxta‐membrane domain of RodZ is essential to maintain rod shape, whereas other domains on either side of the membrane have critical, but partially redundant, functions. Though one of these domains resembles a DNA‐binding motif, our evidence indicates that it is primarily responsible for association of RodZ with the cytoskeleton.

Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis

During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC− defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.

Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity

Bacterial cytokinesis is driven by the septal ring apparatus, the assembly of which in Escherichia coli is directed to mid‐cell by the Min system. Despite suffering aberrant divisions at the poles, cells lacking the minCDE operon (Min–) have an almost normal growth rate. We developed a generally applicable screening method for synthetic lethality in E. coli, and used it to select for transposon mutations (slm) that are synthetically lethal (or sick) in combination with ΔminCDE. One of the slm insertions mapped to envC (yibP), proposed to encode a lysostaphin‐like, metallo‐endopeptidase that is exported to the periplasm by the general secretory (Sec) pathway. Min– EnvC– cells showed a severe division defect, supporting a role for EnvC in septal ring function. Accordingly, we show that an EnvC–green fluorescent protein fusion, when directed to the periplasm via the twin‐arginine export system, is both functional and part of the septal ring apparatus. Using an in‐gel assay, we also present evidence that EnvC possesses murein hydrolytic activity. Our results suggest that EnvC plays a direct role in septal murein cleavage to allow outer membrane constriction and daughter cell separation. By uncovering genetic interactions, the synthetic lethal screen described here provides an attractive new tool for studying gene function in E. coli.

Nucleoid occlusion factor SlmA is a DNA-activated FtsZ polymerization antagonist

The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.

Beta-Lactam Antibiotics Induce a Lethal Malfunctioning of the Bacterial Cell Wall Synthesis Machinery

Penicillin and related beta-lactams comprise one of our oldest and most widely used antibiotic therapies. These drugs have long been known to target enzymes called penicillin-binding proteins (PBPs) that build the bacterial cell wall. Investigating the downstream consequences of target inhibition and how they contribute to the lethal action of these important drugs, we demonstrate that beta-lactams do more than just inhibit the PBPs as is commonly believed. Rather, they induce a toxic malfunctioning of their target biosynthetic machinery involving a futile cycle of cell wall synthesis and degradation, thereby depleting cellular resources and bolstering their killing activity. Characterization of this mode of action additionally revealed a quality control function for enzymes that cleave bonds in the cell wall matrix. The results thus provide insight into the mechanism of cell wall assembly and suggest how best to interfere with the process for future antibiotic development.

LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli

Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of beta-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM(-) cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.

An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring

ATP-binding cassette transporters are ubiquitous membrane protein complexes that move substrates across membranes. They do so using ATP-induced conformational changes in their nucleotide-binding domains to alter the conformation of the transport cavity formed by their transmembrane domains. In Escherichia coli, an ATP-binding cassette transporter-like complex composed of FtsE (nucleotide-binding domain) and FtsX (transmembrane domain) has long been known to be important for cytokinesis, but its role in the process has remained mysterious. Here we identify FtsEX as a regulator of cell-wall hydrolysis at the division site. Cell-wall material synthesized by the division machinery is shared initially by daughter cells and must be split by hydrolytic enzymes called “amidases” to drive daughter-cell separation. We recently showed that the amidases require activation at the cytokinetic ring by proteins with LytM domains, of which EnvC is the most critical. In this report, we demonstrate that FtsEX directly recruits EnvC to the septum via an interaction between EnvC and a periplasmic loop of FtsX. Importantly, we also show that FtsEX variants predicted to be ATPase defective still recruit EnvC to the septum but fail to promote cell separation. Our results thus suggest that amidase activation via EnvC in the periplasm is regulated by conformational changes in the FtsEX complex mediated by ATP hydrolysis in the cytoplasm. Since FtsE has been reported to interact with the tubulin-like FtsZ protein, our model provides a potential mechanism for coupling amidase activity with the contraction of the FtsZ cytoskeletal ring.

Self-enhanced accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR domain (DamX, DedD, and RlpA) in Escherichia coli cell constriction.

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an alpha-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain ((E)FtsN) of at most 35 residues that is centered about helix H2 in the periplasm. (E)FtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain ((S)FtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of (S)FtsN depended on the ability of cells to constrict and, thus, on the activity of (E)FtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.

MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis

Building the cell wall is flipping difficult The cell wall of bacteria is constructed from a polysaccharide called peptidoglycan (PG). It forms a matrix that surrounds cells and is essential for the integrity of the cytoplasmic membrane. Many of our most successful antibiotics target PG synthesis. The synthetic pathway involves the assembly of sugar building blocks on a lipid carrier at the inner face of the cytoplasmic membrane. The reactions that produce this so-called lipid II precursor and the enzymes that catalyze them have been known for decades. However, the identity of the flippase enzyme that “flips” lipid II in the membrane to expose the sugar building blocks on the cell surface for polymerization has remained highly controversial. Sham et al. now show that the essential protein MurJ is the long sought-after flippase responsible for the translocation of lipid-linked cell wall precursors across the bacterial cytoplasmic membrane (see the Perspective by Young). The work completes the cell wall biogenesis pathway and defines the function of an attractive target for new antibiotics. Science, this issue p. 220; see also p. 139 The identity of the final essential component of the bacterial peptidoglycan biogenesis pathway is elucidated. [Also see Perspective by Young] Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic strategy to rapidly and specifically block flippase function. We combined these approaches to demonstrate that MurJ is the lipid II flippase in Escherichia coli.