Piet A. J. de Boer

Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E. coli.

FtsZ is a soluble, tubulin-like GTPase that forms a membrane-associated ring at the division site of bacterial cells. While this ring is thought to drive cell constriction, it is not well understood how it is assembled or how it affects cell wall invagination. Here we report that FtsZ binds directly to a novel integral inner membrane protein in E. coli that we call ZipA. We present genetic and morphological evidence indicating that this interaction is required for cell division, and show that a fluorescent ZipA-Gfp fusion protein is located in a ring structure at the division site, both before and during cell wall invagination. ZipA is an essential component of the division machinery, and, by binding to both FtsZ and the cytoplasmic membrane, is likely to be directly involved in the assembly and/or function of the FtsZ ring.

Pattern formation in Escherichia coli: a model for the pole-to-pole oscillations of Min proteins and the localization of the division site.

Proper cell division requires an accurate definition of the division plane. In bacteria, this plane is determined by a polymeric ring of the FtsZ protein. The site of Z ring assembly in turn is controlled by the Min system, which suppresses FtsZ polymerization at noncentral membrane sites. The Min proteins in Escherichia coli undergo a highly dynamic localization cycle, during which they oscillate between the membrane of both cell halves. By using computer simulations we show that Min protein dynamics can be described accurately by using the following assumptions: (i) the MinD ATPase self-assembles on the membrane and recruits both MinC, an inhibitor of Z ring formation, and MinE, a protein required for MinC/MinD oscillation, (ii) a local accumulation of MinE is generated by a pattern formation reaction that is based on local self-enhancement and a long range antagonistic effect, and (iii) it displaces MinD from the membrane causing its own local destabilization and shift toward higher MinD concentrations. This local destabilization results in a wave of high MinE concentration traveling from the cell center to a pole, where it disappears. MinD reassembles on the membrane of the other cell half and attracts a new accumulation of MinE, causing a wave-like disassembly of MinD again. The result is a pole-to-pole oscillation of MinC/D. On time average, MinC concentration is highest at the poles, forcing FtsZ assembly to the center. The mechanism is self-organizing and does not require any other hypothetical topological determinant.

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.

Dynamic localization cycle of the cell division regulator MinE in Escherichia coli

The MinC protein directs placement of the division septum to the middle of Escherichia coli cells by blocking assembly of the division apparatus at other sites. MinD and MinE regulate MinC activity by modulating its cellular location in a unique fashion. MinD recruits MinC to the membrane, and MinE induces MinC/MinD to oscillate rapidly between the membrane of opposite cell halves. Using fixed cells, we previously found that a MinE–green fluorescent protein fusion accumulated in an annular structure at or near the midcell, as well as along the membrane on only one side of the ring. Here we show that in living cells, MinE undergoes a rapid localization cycle that appears coupled to MinD oscillation. The results show that MinE is not a fixed marker for septal ring assembly. Rather, they support a model in which MinE stimulates the removal of MinD from the membrane in a wave‐like fashion. These waves run from a midcell position towards the poles in an alternating sequence such that the time‐averaged concentration of division inhibitor is lowest at midcell.

The trans‐envelope Tol–Pal complex is part of the cell division machinery and required for proper outer‐membrane invagination during cell constriction in E. coli

Fission of bacterial cells involves the co‐ordinated invagination of the envelope layers. Invagination of the cytoplasmic membrane (IM) and peptidoglycan (PG) layer is likely driven by the septal ring organelle. Invagination of the outer membrane (OM) in Gram‐negative species is thought to occur passively via its tethering to the underlying PG layer with generally distributed PG‐binding OM (lipo)proteins. The Tol–Pal system is energized by proton motive force and is well conserved in Gram‐negative bacteria. It consists of five proteins that can connect the OM to both the PG and IM layers via protein–PG and protein–protein interactions. Although the system is needed to maintain full OM integrity, and for class A colicins and filamentous phages to enter cells, its precise role has remained unclear. We show that all five components accumulate at constriction sites in Escherichia coli and that mutants lacking an intact system suffer delayed OM invagination and contain large OM blebs at constriction sites and cell poles. We propose that Tol–Pal constitutes a dynamic subcomplex of the division apparatus in Gram‐negative bacteria that consumes energy to establish transient trans‐envelope connections at/near the septal ring to draw the OM onto the invaginating PG and IM layers during constriction.

The MinE Ring: An FtsZ-Independent Cell Structure Required for Selection of the Correct Division Site in E. coli

E. coli cell division is mediated by the FtsZ ring and associated factors. Selection of the correct division site requires the combined action of an inhibitor of FtsZ ring formation (MinCD) and of a topological specificity factor that somehow prevents MinCD action at the middle of the cell (MinE). Here we show that a biologically active MinE-Gfp fusion accumulates in an annular structure near the middle of young cells. Formation of the MinE ring required MinD but was independent of MinC and continued in nondividing cells in which FtsZ function was inhibited. The results indicate that the MinE ring represents a novel cell structure, which allows FtsZ ring formation at midcell by suppressing MinCD activity at this site.

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.

Advances in understanding E. coli cell fission.

Much of what we know about cytokinesis in bacteria has come from studies with Escherichia coli, and efforts to comprehensively understand this fundamental process in this organism continue to intensify. Major recent advances include in vitro assembly of a membrane-tethered version of FtsZ into contractile rings in lipid tubules, in vitro dynamic patterning of the Min proteins and a deeper understanding of how they direct assembly of the FtsZ-ring to midcell, the elucidation of structures, biochemical activities and interactions of other key components of the cell fission machinery, and the uncovering of additional components of this machinery with often redundant but important roles in invagination of the three cell envelope layers.

ZipA-Induced Bundling of FtsZ Polymers Mediated by an Interaction between C-Terminal Domains

FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.

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.