Bacterial Cytoskeleton: Not Your Run-of-the-Mill Tubulin

Large plasmids of some Bacillus species encode a distinct tubulin homolog, TubZ, implicated in maintenance of the host plasmid. A recent study has shown that TubZ polymers exhibit treadmilling behavior in vivo, suggesting that they are involved in mitotic activity

Bacterial Shape: Concave Coiled Coils Curve Caulobacter

AbstractBacterial cells exhibit a wide variety of shapes. Recent results indicate that the characteristic crescent shape of Caulobacter crescentus depends upon an inter-mediate filament-like protein that localizes to the concave side of the cell

Bacteriophage Tubulins: Carrying Their Own Cytoskeleton Key

SummaryCytoskeletal elements are well known to be widespread in eukaryotes and prokaryotes, providing important, diverse functions for cells large and small. Two new studies report that some bacteriophages encode their own tubulin homologs to facilitate phage reproduction within the host cell

Adenine nucleotide-dependent regulation of assembly of bacterial tubulin-like FtsZ by a hypermorph of bacterial actin-like FtsA.

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

AbstractCytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division

ZipA Uses a Two-Pronged FtsZ-Binding Mechanism Necessary for Cell Division

In most bacteria, cell division is centrally organized by the FtsZ protein, which assembles into dynamic filaments at the division site along the cell membrane that interact with other key cell division proteins. In gammaproteobacteria such as Escherichia coli, FtsZ filaments are anchored to the cell membrane by two essential proteins, FtsA and ZipA. Canonically, this interaction was believed to be mediated solely by the FtsZ C-terminal peptide (CTP) domain that interacts with these and several other regulatory proteins. However, we now provide evidence of a second interaction between FtsZ and ZipA. Using site-specific photoactivated cross-linking, we identified a noncanonical FtsZ-binding site on ZipA on the opposite side from the FtsZ CTP-binding pocket. Cross-linking at this site was unaffected by the truncation of the FtsZ linker and CTP domains, indicating that this noncanonical site must interact directly with the globular core domain of FtsZ. Mutations introduced into either the canonical or noncanonical binding sites on ZipA disrupted photo-cross-linking with FtsZ and normal ZipA function in cell division, suggesting that both binding modes are important for normal cell growth and division. One mutation at the noncanonical face was also found to suppress defects of several other canonical and noncanonical site mutations in ZipA, suggesting there is some interdependence between the two sites. Taken together, these results suggest that ZipA employs a two-pronged FtsZ-binding mechanism

Visualization of the type III secretion sorting platform of Shigella flexneri

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress

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