Recruitment of the TolA Protein to Cell Constriction Sites in Escherichia coli via Three Separate Mechanisms, and a Critical Role for FtsWI Activity in Recruitment of both TolA and TolQ.

The Tol-Pal system of Gram-negative bacteria helps maintain the integrity of the cell envelope and ensures that invagination of the envelope layers during cell fission occurs in a well-coordinated manner. In Escherichia coli, the five Tol-Pal proteins (TolQ, -R, -A, and -B and Pal) accumulate at cell constriction sites in a manner that normally requires the activity of the cell constriction initiation protein FtsN. While septal recruitment of TolR, TolB, and Pal also requires the presence of TolQ and/or TolA, the latter two can recognize constriction sites independently of the other system proteins. What attracts TolQ or TolA to these sites is unclear. We show that FtsN indirectly attracts both proteins and that PBP1A, PBP1B, and CpoB are dispensable for their septal recruitment. However, the ß-lactam aztreonam readily interferes with the septal accumulation of both TolQ and TolA, indicating that FtsN-stimulated production of septal peptidoglycan by the FtsWI synthase is critical to their recruitment. We also discovered that each of TolA's three domains can separately recognize division sites. Notably, the middle domain (TolAII) is responsible for directing TolA to constriction sites in the absence of other Tol-Pal proteins and CpoB, while recruitment of TolAI requires TolQ and that of TolAIII requires a combination of TolB, Pal, and CpoB. Additionally, we describe the construction and use of functional fluorescent sandwich fusions of the ZipA division protein, which should be more broadly valuable in future studies of the E. coli cell division machinery. IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction will eventually occur. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We studied how two such proteins, TolA and TolQ, which also play a role in maintaining the integrity of the outer membrane, are recruited to the machinery. We find that TolA can be recruited by three separate mechanisms and that both proteins rely on the activity of a well-studied cell division enzyme for their recruitment.

Roles of the DedD Protein in Escherichia coli Cell Constriction.

Two key tasks of the bacterial septal-ring (SR) machinery during cell constriction are the generation of an inward-growing annulus of septal peptidoglycan (sPG) and the concomitant splitting of its outer edge into two layers of polar PG that will be inherited by the two new cell ends. FtsN is an essential SR protein that helps trigger the active constriction phase in Escherichia coli by inducing a self-enhancing cycle of processes that includes both sPG synthesis and splitting and that we refer to as the sPG loop. DedD is an SR protein that resembles FtsN in several ways. Both are bitopic inner membrane proteins with small N-terminal cytoplasmic parts and larger periplasmic parts that terminate with a SPOR domain. Though absence of DedD normally causes a mild cell-chaining phenotype, the protein is essential for division and survival of cells with limited FtsN activity. Here, we find that a small N-terminal portion of DedD (NDedD; DedD1-54) is required and sufficient to suppress ΔdedD-associated division phenotypes, and we identify residues within its transmembrane domain that are particularly critical to DedD function. Further analyses indicate that DedD and FtsN act in parallel to promote sPG synthesis, possibly by engaging different parts of the FtsBLQ subcomplex to induce a conformation that permits and/or stimulates the activity of sPG synthase complexes composed of FtsW, FtsI (PBP3), and associated proteins. We propose that, like FtsN, DedD promotes cell fission by stimulating sPG synthesis, as well as by providing positive feedback to the sPG loop.IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction eventually occurs. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We identify functionally important parts of one of these proteins, DedD, and present evidence supporting a role for DedD in helping to induce and/or sustain a self-enhancing cycle of processes that are executed by fellow septal-ring proteins and that drive the active constriction phase of the cell division cycle.

Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers.

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.

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.

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.

ZipA is required for targeting of DMinC/DicB, but not DMinC/MinD, complexes to septal ring assemblies in Escherichia coli.

The MinC division inhibitor is required for accurate placement of the septal ring at the middle of the Escherichia coli cell. The N-terminal domain of MinC ((Z)MinC) interferes with FtsZ assembly, while the C-terminal domain ((D)MinC) mediates both dimerization and complex formation with either MinD or DicB. Binding to either of these activators greatly enhances the division-inhibitory activity of MinC in the cell. The MinD ATPase plays a crucial role in the rapid pole-to-pole oscillation of MinC that is proposed to force FtsZ ring formation to midcell. DicB is encoded by one of the cryptic prophages on the E. coli chromosome (Qin) and is normally not synthesized. Binding of MinD or DicB to (D)MinC produces complexes that have high affinities for one or more septal ring-associated targets. Here we show that the FtsZ-binding protein ZipA is required for both recruitment of the (D)MinC/DicB complex to FtsZ rings and the DicB-inducible division block normally seen in MinC(+) cells. In contrast, none of the known FtsZ-associated factors, including ZipA, FtsA, and ZapA, appear to be specifically required for targeting of the (D)MinC/MinD complex to rings, implying that the two MinC/activator complexes must recognize distinct features of FtsZ assemblies. MinD-dependent targeting of MinC may occur in two steps of increasing topological specificity: (i) recruitment of MinC from the cytoplasm to the membrane, and (ii) specific targeting of the MinC/MinD complex to nascent septal ring assemblies on the membrane. Using membrane-tethered derivatives of MinC, we obtained evidence that both of these steps contribute to the efficiency of MinC/MinD-mediated division inhibition.

Structural evidence that the P/Q domain of ZipA is an unstructured, flexible tether between the membrane and the C-terminal FtsZ-binding domain.

The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.

ZipA is required for recruitment of FtsK, FtsQ, FtsL, and FtsN to the septal ring in Escherichia coli.

The septal ring in Escherichia coli consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN. Recruitment of FtsK and all downstream components requires the prior localization of FtsA. Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA. The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.