Bacterial Tubulin Research Articles

Lateral interactions between protofilaments of the bacterial tubulin homolog FtsZ are essential for cell division.

The prokaryotic tubulin homolog FtsZ polymerizes into protofilaments, which further assemble into higher-order structures at future division sites to form the Z-ring, a dynamic structure essential for bacterial cell division. The precise nature of interactions between FtsZ protofilaments that organize the Z-ring and their physiological significance remain enigmatic. In this study, we solved two crystallographic structures of a pair of FtsZ protofilaments, and demonstrated that they assemble in an antiparallel manner through the formation of two different inter-protofilament lateral interfaces. Our in vivo photocrosslinking studies confirmed that such lateral interactions occur in living cells, and disruption of the lateral interactions rendered cells unable to divide. The inherently weak lateral interactions enable FtsZ protofilaments to self-organize into a dynamic Z-ring. These results have fundamental implications for our understanding of bacterial cell division and for developing antibiotics that target this key process.

The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro

The bacterial tubulin FtsZ polymerizes to form a discontinuous ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL), and a C-terminal conserved peptide (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The assembly of FtsZ protofilaments into dynamic clusters is critical for cell division, but the interactions between protofilaments and regulatory mechanisms that mediate cluster assembly and dynamics are unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro FtsZ lacking its CTL (ΔCTL) shows a dramatically increased propensity to form long multifilament bundles compared with wild type (WT). ΔCTL also displays a reduced GTP hydrolysis rate compared with WT, but this altered activity does not account for bundle formation, as reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts ΔCTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of the FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between FtsZ-ring structure and function in bacterial cytokinesis.

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis.

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and β-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

A novel gene, cilia flagella associated protein 44, encoding an enzyme cleaving FtsZ and tubulin contributes to the regulation of secretory pathway

We identified a novel gene, encoding the central region of the cilia and flagella associated protein 44 (Cfap44), that regulates trafficking of cellular components and morphology via the cleavage of cellular proteins (particularly β-tubulin). Although Cfap44 is registered in GenBank, the functions of both the central part and full-length protein are unknown except for a polymorphism associated with proprotein convertase 9 activity, the third gene of familiar hyper-cholesterolemia. In mice and humans, both unspliced and spliced RNAs were transcribed, and the spliced form was predominantly transcribed in the brain and embryonic tissues. In transfectants carrying this gene, various cellular processes such as cell division, transport of cellular components, and proteolytic processing of several proteins were found to be affected. The cleavage of β-tubulin was observed. A bacterial tubulin homolog, cell division protein FtsZ, was also cleaved in vivo and in vitro by the spliced form of Cfap44 product. Furthermore, the unspliced form showed proteolytic activity with low substrate specificity. Various biological activities of Cfap44 may be due to a direct effect of cleavage of β-tubulin inhibiting microtubule formation, or an indirect effect with the cross-recognition of the cleavage site between β-tubulin and other molecules.

Bacterial Tubulins: A Eukaryotic-Like Microtubule Cytoskeleton

Ever since their discovery, bacterial tubulins, found in several Prosthecobacter species, have raised curiosity as they are closely related to eukaryotic tubulin. Deng and colleagues now present new evidence for the functional homology of the two cytoskeletal systems where in vitro reconstituted Btub-microtubules display eukaryote-like biochemical and dynamic properties.

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes

Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.

GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis.

The bacterial tubulin FtsZ is the central component of the cell division machinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity. The treadmilling dynamics direct the processive movement of the septal cell wall synthesis machinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall were substantially altered. Thus, FtsZ treadmilling provides a mechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology.

Intermediate Filaments Supporting Cell Shape and Growth in Bacteria.

For years intermediate filaments (IF), belonging to the third class of filamentous cytoskeletal proteins alongside microtubules and actin filaments, were thought to be exclusive to metazoan cells. Structurally these eukaryote IFs are very well defined, consisting of globular head and tail domains, which flank the central rod-domain. This central domain is dominated by an α-helical secondary structure predisposed to form the characteristic coiled-coil, parallel homo-dimer. These elementary dimers can further associate, both laterally and longitudinally, generating a variety of filament-networks built from filaments in the range of 10 nm in diameter. The general role of these filaments with their characteristic mechano-elastic properties both in the cytoplasm and in the nucleus of eukaryote cells is to provide mechanical strength and a scaffold supporting diverse shapes and cellular functions.Since 2003, after the first bacterial IF-like protein, crescentin was identified, it has been evident that bacteria also employ filamentous networks, other than those built from bacterial tubulin or actin homologues, in order to support their cell shape, growth and, in some cases, division. Intriguingly, compared to their eukaryote counterparts, the group of bacterial IF-like proteins shows much wider structural diversity. The sizes of both the head and tail domains are markedly reduced and there is great variation in the length of the central rod-domain. Furthermore, bacterial rod-domains often lack the sub-domain organisation of eukaryote IFs that is the defining feature of the IF-family. However, the fascinating display of filamentous assemblies, including rope, striated cables and hexagonal laces together with the conditions required for their formation both in vitro and in vivo strongly resemble that of eukaryote IFs suggesting that these bacterial proteins are deservedly classified as part of the IF-family and that the current definition should be relaxed slightly to allow their inclusion. The lack of extensive head and tail domains may well make the bacterial proteins more amenable for structural characterisation, which will be essential for establishing the mechanism for their association into filaments. What is more, the well-developed tools for bacterial manipulations provide an excellent opportunity of studying the bacterial systems with the prospect of making significant progress in our understanding of the general underlying principles of intermediate filament assemblies.

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.

One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

FtsZ-Dependent Elongation of a Coccoid Bacterium.

ABSTRACTA mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZG193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZG193D filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci.

A Prophage-Encoded Small RNA Controls Metabolism and Cell Division in Escherichia coli.

Hundreds of small RNAs (sRNAs) have been identified in diverse bacterial species, and while the functions of most remain unknown, some regulate key processes, particularly stress responses. The sRNA DicF was identified over 25years ago as an inhibitor of cell division but since then has remained uncharacterized. DicF consists of 53 nucleotides and is encoded by a gene carried on a prophage (Qin) in the genomes of many Escherichia coli strains. We demonstrated that DicF inhibits cell division via direct base pairing with ftsZ mRNA to repress translation and prevent new synthesis of the bacterial tubulin homolog FtsZ. Systems analysis using computational and experimental methods identified additional mRNA targets of DicF: xylR and pykA mRNAs, encoding the xylose uptake and catabolism regulator and pyruvate kinase, respectively. Genetic analyses showed that DicF directly base pairs with and represses translation of these targets. Phenotypes of cells expressing DicF variants demonstrated that DicF-associated growth inhibition is not solely due to repression of ftsZ, indicating that the physiological consequences of DicF-mediated regulation extend beyond effects on cell division caused by reduced FtsZ synthesis. IMPORTANCE sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E.coli are highly conserved and present in the E.coli core genome. In contrast, the sRNA DicF (identified over 20years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E.coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E.coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages.

New data on structures formed by the FtsZ protein during the cell division process in Escherichia coli obtained using the localization microscopy method

The FtsZ protein, a bacterial tubulin homolog, is one of the key proteins in bacterial cell division, forming a contractile Z-ring in the middle of the dividing cell. In the present study, immunofluorescence staining, in combination with the localization microscopy method, was used for visualization of the structures formed by unlabelled FtsZ in Escherrichia coli cells. The techniques employed allowed reconstruction of the multistep mechanism of formation of FtsZ structures during the cytokinesis process. New data were obtained confirming the hypothesis that FtsZ is a helixlike structure that constricts during the division, producing constriction between the daughter cells.

The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction.

The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery, and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here, we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges, and lyse, resembling treatment with β-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL, however cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wildtype. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment.

Microbiology: A beacon for bacterial tubulin.

The protein FtsZ forms a ring structure that constricts to allow bacterial cells to divide. A second protein, MapZ, has now been found to guide FtsZ to the correct mid-cell position in the bacterium Streptococcus pneumoniae. See Letter p.259 In many bacteria, the correct mid-cell placement of the cell division machinery —including the ring formed by the conserved tubulin homologue FtsZ — is controlled by the Min system, which prevents FtsZ polymerization at the cell poles. This system is absent in the pathogenic bacterium Streptococcus pneumoniae, however. Here Christophe Grangeasse and colleagues identify a placement mechanism that does operate in S. pneumonia. It depends on mid-cell anchored protein Z (MapZ) to form ring structures at the cell equator that separate as the cell elongates, acting as a permanent beacon for the division sites. MapZ interacts with FtsZ to position the Z-ring for division.

Antisense growth inhibition of methicillin-resistant Staphylococcus aureus by locked nucleic acid conjugated with cell-penetrating peptide as a novel FtsZ inhibitor.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are becoming increasingly difficult to treat, owing to acquired antibiotic resistance. The emergence and spread of MRSA limit therapeutic options and require new therapeutic strategies, including novel MRSA-active antibiotics. Filamentous temperature-sensitive protein Z (FtsZ) is a highly conserved bacterial tubulin homologue that is essential for controlling the bacterial cell division process in different species of S. aureus. We conjugated a locked nucleic acid (LNA) that targeted ftsZ mRNA with the peptide (KFF)3K, to generate peptide-LNA (PLNA). The present study aimed to investigate whether PLNA could be used as a novel antibacterial agent. PLNA787, the most active agent synthesized, exhibited promising inhibitory effects on four pathogenic S. aureus strains in vitro. PLNA787 inhibited bacterial growth and resolved lethal Mu50 infections in epithelial cell cultures. PLNA787 also improved the survival rates of Mu50-infected mice and was associated with reductions of bacterial titers in several tissue types. The inhibitory effects on ftsZ mRNA and FtsZ protein expression and inhibition of the bacterial cell division process are considered to be the major mechanisms of PLNA. PLNA787 demonstrated activity against MRSA infections in vitro and in vivo. Our findings suggest that ftsZ mRNA is a promising new target for developing novel antisense antibiotics.

The role of cytoskeletal elements in shaping bacterial cells.

Beginning from the recognition of FtsZ as a bacterial tubulin homolog in the early 1990s, many bacterial cytoskeletal elements have been identified, including homologs to the major eukaryotic cytoskeletal elements (tubulin, actin, and intermediate filament) and the elements unique in prokaryotes (ParA/MinD family and bactofilins). The discovery and functional characterization of the bacterial cytoskeleton have revolutionized our understanding of bacterial cells, revealing their elaborate and dynamic subcellular organization. As in eukaryotic systems, the bacterial cytoskeleton participates in cell division, cell morphogenesis, DNA segregation, and other important cellular processes. However, in accordance with the vast difference between bacterial and eukaryotic cells, many bacterial cytoskeletal proteins play distinct roles from their eukaryotic counterparts; for example, control of cell wall synthesis for cell division and morphogenesis. This review is aimed at providing an overview of the bacterial cytoskeleton, and discussing the roles and assembly dynamics of bacterial cytoskeletal proteins in more detail in relation to their most widely conserved functions, DNA segregation and coordination of cell wall synthesis.

Bacterial tubulin TubZ-Bt transitions between a two-stranded intermediate and a four-stranded filament upon GTP hydrolysis

Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning systems, facilitating faithful segregation of low copy-number plasmids during bacterial cell division. One such protein, TubZ-Bt, is found on the large pBtoxis plasmid in Bacillus thuringiensis, and interacts via its extended C terminus with a DNA adaptor protein TubR. Here, we use cryo-electron microscopy to determine the structure of TubZ-Bt filaments and light scattering to explore their mechanism of polymerization. Surprisingly, we find that the helical filament architecture is remarkably sensitive to nucleotide state, changing from two-stranded to four-stranded depending on the ability of TubZ-Bt to hydrolyze GTP. We present pseudoatomic models of both the two- and four-protofilament forms based on cryo-electron microscopy reconstructions (10.8 Å and 6.9 Å, respectively) of filaments formed under different nucleotide states. These data lead to a model in which the two-stranded filament is a necessary intermediate along the pathway to formation of the four-stranded filament. Such nucleotide-directed structural polymorphism is to our knowledge an unprecedented mechanism for the formation of polar filaments.

Localization of P42 and F 1 -ATPase α-Subunit Homolog of the Gliding Machinery in Mycoplasma mobile Revealed by Newly Developed Gene Manipulation and Fluorescent Protein Tagging

Mycoplasma mobile has a unique mechanism that enables it to glide on solid surfaces faster than any other gliding mycoplasma. To elucidate the gliding mechanism, we developed a transformation system for M. mobile based on a transposon derived from Tn4001. Modification of the electroporation conditions, outgrowth time, and colony formation from the standard method for Mycoplasma species enabled successful transformation. A fluorescent-protein tagging technique was developed using the enhanced yellow fluorescent protein (EYFP) and applied to two proteins that have been suggested to be involved in the gliding mechanism: P42 (MMOB1050), which is transcribed as continuous mRNA with other proteins essential for gliding, and a homolog of the F1-ATPase α-subunit (MMOB1660). Analysis of the amino acid sequence of P42 by PSI-BLAST suggested that P42 evolved from a common ancestor with FtsZ, the bacterial tubulin homologue. The roles of P42 and the F(1)-ATPase subunit homolog are discussed as part of our proposed gliding mechanism.

Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5'-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.