William Margolin

FtsZ and the division of prokaryotic cells and organelles

Binary fission of many prokaryotes as well as some eukaryotic organelles depends on the FtsZ protein, which self-assembles into a membrane-associated ring structure early in the division process. FtsZ is homologous to tubulin, the building block of the microtubule cytoskeleton in eukaryotes. Recent advances in genomics and cell-imaging techniques have paved the way for the remarkable progress in our understanding of fission in bacteria and organelles.

Ca2+‐mediated GTP‐dependent dynamic assembly of bacterial cell division protein FtsZ into asters and polymer networks in vitro

FtsZ, a tubulin‐like GTPase that forms a dynamic ring marking the division plane of prokaryotic cells, is essential for cytokinesis. It is not known what triggers FtsZ ring assembly. In this work, we use a FtsZ–green fluorescent protein (Gfp) chimera to assay FtsZ assembly over time by using fluorescence microscopy. We show that FtsZ polymers can assemble dynamically in solution in a GTP‐dependent manner. Initially, FtsZ nucleation centers grow into aster‐like structures that dramatically resemble microtubule organizing centers. As assembly proceeds further, protofilament bundles emanating from different asters interconnect, mimicking the closure of the FtsZ ring in vivo. Surprisingly, millimolar levels of Ca2+ promote FtsZ dynamic assembly. FtsZ can undergo repeated GTP‐dependent assembly and disassembly in solution by sequential addition and removal of Ca2+. In addition, GTP binding and hydrolysis by FtsZ are regulated by Ca2+ concentration. Although the concentration of Ca2+ required for FtsZ assembly in vitro is high, its clear and specific effect on FtsZ dynamics suggests the possibility that Ca2+ may have a role in regulating FtsZ ring assembly in the cell.

FtsZ ring clusters in min and partition mutants: role of both the Min system and the nucleoid in regulating FtsZ ring localization

To understand further the role of the nucleoid and the min system in selection of the cell division site, we examined FtsZ localization in Escherichia coli cells lacking MinCDE and in parC mutants defective in chromosome segregation. More than one FtsZ ring was sometimes found in the gaps between nucleoids in min mutant filaments. These multiple FtsZ rings were more apparent in longer cells; double or triple rings were often found in the nucleoid‐free gaps in ftsI min and ftsA min double mutant filaments. Introducing a parC mutation into the ftsA min double mutant allowed the nucleoid‐free gaps to become significantly longer. These gaps often contained dramatic clusters of FtsZ rings. In contrast, filaments of the ftsA parC double mutant, which contained active MinCDE, assembled only one or two rings in most of the large nucleoid‐free gaps. These results suggest that all positions along the cell length are competent for FtsZ ring assembly, not just sites at mid‐cell or at the poles. Consistent with previous results, unsegregated nucleoids also correlated with a lack of FtsZ localization. A model is proposed in which both the inhibitory effect of the nucleoid and the regulation by MinCDE ensure that cells divide precisely at the midpoint.

FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli.

Prokaryotes contain cytoskeletal proteins such as the tubulin-like FtsZ, which forms the Z ring at the cell center for cytokinesis, and the actin-like MreB, which forms a helix along the long axis of the cell and is required for shape maintenance. Using time-lapse analysis of Escherichia coli cells expressing FtsZ-GFP, we found that FtsZ outside of the Z ring also localized in a helix-like pattern and moved very rapidly within this pattern. The movement occurred independently of the presence of Z rings and was most easily detectable in cells lacking Z rings. Moreover, we observed oscillation waves of FtsZ-GFP in the helix-like pattern, particularly in elongated cells, and the period of this oscillation was similar to that of the Min proteins. The MreB helix was not required for the rapid movement of FtsZ or the oscillation of MinD. The results suggest that FtsZ not only forms the Z ring but also is part of a highly dynamic, potentially helical cytoskeleton in bacterial cells.

Sculpting the bacterial cell.

Prokaryotes come in a wide variety of shapes, determined largely by natural selection, physical constraints, and patterns of cell growth and division. Because of their relative simplicity, bacterial cells are excellent models for how genes and proteins can directly determine morphology. Recent advances in cytological methods for bacteria have shown that distinct cytoskeletal filaments composed of actin and tubulin homologs are important for guiding growth patterns of the cell wall in bacteria, and that the glycan strands that constitute the wall are generally perpendicular to the direction of growth. This cytoskeleton-directed cell wall patterning is strikingly reminiscent of how plant cell wall growth is regulated by microtubules. In rod-shaped bacilli, helical cables of actin-like MreB protein stretch along the cell length and orchestrate elongation of the cell wall, whereas the tubulin-like FtsZ protein directs formation of the division septum and the resulting cell poles. The overlap and interplay between these two systems and the peptidoglycan-synthesizing enzymes they recruit are the major driving forces of cylindrical shapes. Round cocci, on the other hand, have lost their MreB cables and instead must grow mainly via their division septum, giving them their characteristic round or ovoid shapes. Other bacteria that lack MreB homologs or even cell walls use distinct cytoskeletal systems to maintain their distinct shapes. Here I review what is known about the mechanisms that determine the shape of prokaryotic cells.

A gain-of-function mutation in ftsA bypasses the requirement for the essential cell division gene zipA in Escherichia coli

ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of Escherichia coli. The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins. Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*). Cells with ftsA* in single copy in place of WT ftsA or with ftsA* alone on a multicopy plasmid divide mostly normally, whether they are zipA+ or zipA−. Experiments with ftsQAZ and ftsQA*Z on multicopy plasmids indicate that ftsQAZ/zipA+ and ftsQA*Z/zipA− cells divide fairly normally, whereas ftsQAZ/zipA− cells divide poorly and ftsQA*Z/zipA+ cells display a phenotype that suggests their septa are unusually stable. In support of the idea that ftsA* stabilizes Z rings, single-copy ftsA* confers resistance to excess MinC, which destabilizes Z rings. The inhibitory effect of excess ZipA on division is also suppressed by ftsA*. These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA. This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.

The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling During Infection

Phage Invasion Bacteriophages are responsible for much of bacterial evolution, both by imposing selection for resistance to infection and by horizontal gene transfer of host genes to new bacteria. However, we know surprisingly little about the initiation of phage infection. Hu et al. (p. 576, published online 10 January) used high-throughput cryo-electron tomography and sub-volume analysis to examine Escherichia coli minicells infected with both wild-type and mutant T7 bacteriophages. High-resolution views of phage structures at different stages of infection reveal the de novo formation of an extended tail by the ejection of internal head proteins, in order to form the channel for DNA transport into the cytoplasm. Cryo–electron tomography captures T7 bacteriophage virions at successive stages of bacterial infection. Adsorption and genome ejection are fundamental to the bacteriophage life cycle, yet their molecular mechanisms are not well understood. We used cryo–electron tomography to capture T7 virions at successive stages of infection of Escherichia coli minicells at ~4-nm resolution. The six phage tail fibers were folded against the capsid, extending and orienting symmetrically only after productive adsorption to the host cell surface. Receptor binding by the tail triggered conformational changes resulting in the insertion of an extended tail, which functions as the DNA ejection conduit into the cell cytoplasm. After ejection, the extended phage tail collapsed or disassembled, which allowed resealing of the infected cell membrane. These structural studies provide a detailed series of intermediates during phage infection.

Cell cycle arrest in Era GTPase mutants: a potential growth rate‐regulated checkpoint in Escherichia coli

Era is a low‐molecular‐weight GTPase essential for Escherichia coli viability. The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild‐type Era become arrested in the cell cycle at the predivisional two‐cell stage. The partially defective Era GTPase mutation (era1) suppresses several temperature‐sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division. Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis. Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells.

The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z‐ring to drive septation. Spatial and temporal control of Z‐ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well‐studied Min system, less is known about the anti‐DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)‐mediated NO. SlmA contains a TetR‐like DNA‐binding fold, and chromatin immunoprecipitation analyses show that SlmA‐binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA–FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti‐parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA–DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z‐ring formation at the correct place and time to effect NO.