206195134
Robust Min‐system oscillation in the presence of internal photosynthetic membranes in cyanobacteria
The oscillatory Min system of Escherichia coli defines the cell division plane by regulating the site of FtsZ‐ring formation and represents one of the best‐understood examples of emergent protein self‐organization in nature. The oscillatory patterns of the Min‐system proteins MinC, MinD and MinE (MinCDE) are strongly dependent on the geometry of membranes they bind. Complex internal membranes within cyanobacteria could disrupt this self‐organization by sterically occluding or sequestering MinCDE from the plasma membrane. Here, it was shown that the Min system in the cyanobacterium Synechococcus elongatus PCC 7942 oscillates from pole‐to‐pole despite the potential spatial constraints imposed by their extensive thylakoid network. Moreover, reaction‐diffusion simulations predict robust oscillations in modeled cyanobacterial cells provided that thylakoid network permeability is maintained to facilitate diffusion, and suggest that Min proteins require preferential affinity for the plasma membrane over thylakoids to correctly position the FtsZ ring. Interestingly, in addition to oscillating, MinC exhibits a midcell localization dependent on MinD and the DivIVA‐like protein Cdv3, indicating that two distinct pools of MinC are coordinated in S. elongatus. Our results provide the first direct evidence for Min oscillation outside of E. coli and have broader implications for Min‐system function in bacteria and organelles with internal membrane systems.
33431021
Assembly dynamics of the bacterial MinCDE system and spatial regulation of the Z ring.
The positioning of a cytoskeletal element that dictates the division plane is a fundamental problem in biology. The assembly and positioning of this cytoskeletal element has to be coordinated with DNA segregation and cell growth to ensure that equal-sized progeny cells are produced, each with a copy of the chromosome. In most prokaryotes, cytokinesis involves positioning a Z ring assembled from FtsZ, the ancestral homologue of tubulin. The position of the Z ring is determined by a gradient of negative regulators of Z-ring assembly. In Escherichia coli, the Min system consists of three proteins that cooperate to position the Z ring through a fascinating oscillation, which inhibits the formation of the Z ring away from midcell. Additional gradients of negative regulators of FtsZ assembly are used by E. coli and other bacteria to achieve spatial control of Z-ring assembly.
6574911
Bacterial cell division: assembly, maintenance and disassembly of the Z ring
Bacterial cell division is orchestrated by a tubulin homologue, FtsZ, which polymerizes to form a ring-like structure that is both a scaffold for the assembly of the bacterial cytokinetic machinery and, at least in part, a source of the energy for constriction. FtsZ assembly is tightly regulated, and a diverse repertoire of accessory proteins contributes to the formation of a functional division machine that is responsive to cell cycle status and environmental stress. In this Review, we describe the interaction of these proteins with FtsZ and discuss recent advances in our understanding of Z ring assembly.
8079091
Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division.
Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells.
13947768
The bacterial Min system
A mother cell giving rise to offspring usually needs to choose the site of cytokinesis carefully, as this will determine the size and shape of the daughter cells. Rod-shaped bacteria that divide by binary fission, such as Escherichia coli, often mark their cell division sites at their cell midpoint so that daughter cells are roughly equivalent in size and shape. So how does E. coli know where its middle is? Its cell poles are defined by the previous cell division, but, because E. coli grows by incorporating new cell wall and membrane uniformly along its length, the future cell division site at mid-cell is newly made and has no known pre-existing markers. One way to select the new mid-cell site would be to measure the distance from the two opposing cell poles, using a system that could recognize markers at those poles and define the spot furthest from both markers. This would require that both polar markers act negatively on cell division at equivalent intensities. The result would be a concentration gradient, with the lowest concentration of the negative regulator at the cell midpoint, the greatest distance from both cell poles. It turns out that E. coli and some other rod-shaped bacteria select their cell midpoint using such a negatively acting morphogen gradient, set up by the Min system, which is the focus of this Primer. As is true for many fascinating molecular mechanisms, the first inkling came from the behavior of cells in which this system was broken.
40701584
Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts
MinD is a widely conserved ATPase that has been demonstrated to play a pivotal role in selection of the division site in eubacteria and chloroplasts. It is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif. MinD localizes to the cytoplasmic face of the inner membrane in Escherichia coli, and its association with the inner membrane is a prerequisite for membrane recruitment of the septation inhibitor MinC. However, the mechanism by which MinD associates with the membrane has proved enigmatic; it seems to lack a transmembrane domain and the amino acid sequence is devoid of hydrophobic tracts that might predispose the protein to interaction with lipids. In this study, we show that the extreme C-terminal region of MinD contains a highly conserved 8- to 12-residue sequence motif that is essential for membrane localization of the protein. We provide evidence that this motif forms an amphipathic helix that most likely mediates a direct interaction between MinD and membrane phospholipids. A model is proposed whereby the membrane-targeting motif mediates the rapid cycles of membrane attachment–release–reattachment that are presumed to occur during pole-to-pole oscillation of MinD in E. coli.
10857161
Negative membrane curvature as a cue for subcellular localization of a bacterial protein
Bacterial proteins often localize to distinct sites within the cell, but the primary cues that dictate localization are largely unknown. Recent evidence has shown that positive membrane curvature can serve as a cue for localization of a peripheral membrane protein. Here we report that localization of the peripheral membrane protein DivIVA is determined in whole or in part by recognition of negative membrane curvature and that regions of the protein near the N and C terminus are important for localization. DivIVA, which is a cell division protein in Bacillus subtilis, localizes principally as a ring at nascent septa and secondarily to the less negatively curved, inside surface of the hemispherical poles of the cell. When cytokinesis is prevented, DivIVA redistributes itself to, and becomes markedly enriched at, the poles. When the rod-shaped cells are converted into spheres (protoplasts) by treatment with lysozyme, DivIVA adopts a largely uniform distribution around the cell. Recognition of membrane curvature by peripheral membrane proteins could be a general strategy for protein localization in bacteria.
20126604
Features critical for membrane binding revealed by DivIVA crystal structure
DivIVA is a conserved protein in Gram‐positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N‐terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled‐coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low‐resolution crystal structure of the C‐terminal (Ct) domain displays a curved tetramer made from two parallel coiled‐coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.
18974843
Cellular Architecture Mediates DivIVA Ultrastructure and Regulates Min Activity in Bacillus subtilis
ABSTRACT The assembly of the cell division machinery at midcell is a critical step of cytokinesis. Many rod-shaped bacteria position septa using nucleoid occlusion, which prevents division over the chromosome, and the Min system, which prevents division near the poles. Here we examined the in vivo assembly of the Bacillus subtilis MinCD targeting proteins DivIVA, a peripheral membrane protein that preferentially localizes to negatively curved membranes and resembles eukaryotic tropomyosins, and MinJ, which recruits MinCD to DivIVA. We used structured illumination microscopy to demonstrate that both DivIVA and MinJ localize as double rings that flank the septum and first appear early in septal biosynthesis. The subsequent recruitment of MinCD to these double rings would separate the Min proteins from their target, FtsZ, spatially regulating Min activity and allowing continued cell division. Curvature-based localization would also provide temporal regulation, since DivIVA and the Min proteins would localize to midcell after the onset of division. We use time-lapse microscopy and fluorescence recovery after photobleaching to demonstrate that DivIVA rings are highly stable and are constructed from newly synthesized DivIVA molecules. After cell division, DivIVA rings appear to collapse into patches at the rounded cell poles of separated cells, with little or no incorporation of newly synthesized subunits. Thus, changes in cell architecture mediate both the initial recruitment of DivIVA to sites of cell division and the subsequent collapse of these rings into patches (or rings of smaller diameter), while curvature-based localization of DivIVA spatially and temporally regulates Min activity. IMPORTANCE The Min systems of Escherichia coli and Bacillus subtilis both inhibit FtsZ assembly, but one key difference between these two species is that whereas the E. coli Min proteins localize to the poles, the B. subtilis proteins localize to nascent division sites by interaction with DivIVA and MinJ. It is unclear how MinC activity at midcell is regulated to prevent it from interfering with FtsZ engaged in medial cell division. We used superresolution microscopy to demonstrate that DivIVA and MinJ, which localize MinCD, assemble double rings that flank active division sites and septa. This curvature-based localization mechanism holds MinCD away from the FtsZ ring at midcell, and we propose that this spatial organization is the primary mechanism by which MinC activity is regulated to allow division at midcell. Curvature-based localization also conveys temporal regulation, since it ensures that MinC localizes after the onset of division. The Min systems of Escherichia coli and Bacillus subtilis both inhibit FtsZ assembly, but one key difference between these two species is that whereas the E. coli Min proteins localize to the poles, the B. subtilis proteins localize to nascent division sites by interaction with DivIVA and MinJ. It is unclear how MinC activity at midcell is regulated to prevent it from interfering with FtsZ engaged in medial cell division. We used superresolution microscopy to demonstrate that DivIVA and MinJ, which localize MinCD, assemble double rings that flank active division sites and septa. This curvature-based localization mechanism holds MinCD away from the FtsZ ring at midcell, and we propose that this spatial organization is the primary mechanism by which MinC activity is regulated to allow division at midcell. Curvature-based localization also conveys temporal regulation, since it ensures that MinC localizes after the onset of division.
10487926
Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli.
Accurate placement of the division septum at the midpoint of Escherichia coli cells requires the combined action of a general division inhibitor (MinC), a site-specific suppressor of division inhibition (MinE), and an ATPase (MinD) that is required for proper functioning of both MinC and MinE. We previously showed that a functional MinE-Gfp fusion accumulates in a ring structure at/near the middle of cells. Here we show that functional Gfp-MinD accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle imposed by MinE. The results indicate that MinD represents a novel type of dynamic cellular element in bacteria, with multiple roles in directing the division apparatus to the middle of the cell.
7961846
Direct MinE–membrane interaction contributes to the proper localization of MinDE in E. coli
Dynamic oscillation of the Min system in Escherichia coli determines the placement of the division plane at the midcell. In addition to stimulating MinD ATPase activity, we report here that MinE can directly interact with the membrane and this interaction contributes to the proper MinDE localization and dynamics. The N‐terminal domain of MinE is involved in direct contact between MinE and the membranes that may subsequently be stabilized by the C‐terminal domain of MinE. In an in vitro system, MinE caused liposome deformation into membrane tubules, a property similar to that previously reported for MinD. We isolated a mutant MinE containing residue substitutions in R10, K11 and K12 that was fully capable of stimulating MinD ATPase activity, but was deficient in membrane binding. Importantly, this mutant was unable to support normal MinDE localization and oscillation, suggesting that direct MinE interaction with the membrane is critical for the dynamic behavior of the Min system.
30014235
Min protein patterns emerge from rapid rebinding and membrane interaction of MinE
In Escherichia coli, the pole-to-pole oscillation of the Min proteins directs septum formation to midcell, which is required for symmetric cell division. In vitro, protein waves emerge from the self-organization of MinD, a membrane-binding ATPase, and its activator MinE. For wave propagation, the proteins need to cycle through states of collective membrane binding and unbinding. Although MinD presumably undergoes cooperative membrane attachment, it is unclear how synchronous detachment is coordinated. We used confocal and single-molecule microscopy to elucidate the order of events during Min wave propagation. We propose that protein detachment at the rear of the wave, and the formation of the E-ring, are accomplished by two complementary processes: first, local accumulation of MinE due to rapid rebinding, leading to dynamic instability; and second, a structural change induced by membrane-interaction of MinE in an equimolar MinD–MinE (MinDE) complex, which supports the robustness of pattern formation.
11377456
The Min Oscillator Uses MinD-Dependent Conformational Changes in MinE to Spatially Regulate Cytokinesis
In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded β sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded β sheet MinE dimer with the released β strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.
16755355
Dynamic structures in Escherichia coli: Spontaneous formation of MinE rings and MinD polar zones
In Escherichia coli, division site selection is regulated in part by the Min-protein system. Oscillations of the Min proteins from pole to pole every ≈40 sec have been revealed by in vivo studies of GFP fusions. The dynamic oscillatory structures produced by the Min proteins, including a ring of MinE protein, compact polar zones of MinD, and zebra-striped oscillations in filamentous cells, remain unexplained. We show that the Min oscillations, including mutant phenotypes, can be accounted for by in vitro-observed interactions involving MinD and MinE, with a crucial role played by the rate of nucleotide exchange. Recent discoveries suggest that protein oscillations may play a general role in proper chromosome and plasmid partitioning.
30836343
Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE
Selection of the division site in Escherichia coli is regulated by the min system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE. In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles. We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicles into tubes. Stimulation of the MinD ATPase by addition of MinE led to disassembly of the tubes and the release of MinD from the vesicles. It is proposed that this MinE-regulated dynamic assembly of MinD underlies MinD oscillation.
43749461
Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE
Using a bacterial two‐hybrid system and a combination of in vivo and in vitro assays that take advantage of the green fluorescent reporter protein (GFP), we have investigated the localization and the protein–protein interaction of several key components of the cytokinetic machinery of cyanobacteria (i.e. the progenitor of chloroplast). We demonstrate that (i) the ftsZ and zipN genes are essential for the viability of the model cyanobacterium Synechocystis sp. PCC 6803, whereas the minCDE cluster is dispensable for cell growth; (ii) the GTP‐binding domain of FtsZ is crucial to FtsZ assembly into the septal ring at mid‐cell; (iii) the Z‐ring of deeply constricted daughter cells is oriented perpendicularly to the mother Z‐ring, showing that Synechocystis divides in alternating perpendicular planes; (iv) the MinCDE system affects the morphology of the cell, as well as the position and the shape of FtsZ structures; and (v) MinD is targeted to cell membranes in a process involving its C‐terminal amphipathic helix, but not its ATP‐binding region. Finally, we have also characterized a novel Z‐interacting protein, ZipN, the N‐terminal DnaJ domain of which is critical to the decoration of the Z‐ring, and we report that this process is independent of MinCDE.
6109008
Division accuracy in a stochastic model of Min oscillations in Escherichia coli.
Accurate cell division in Escherichia coli requires the Min proteins MinC, MinD, and MinE as well as the presence of nucleoids. MinD and MinE exhibit spatial oscillations, moving from pole to pole of the bacterium, resulting in an average MinD concentration that is low at the center of the cell and high at the poles. This concentration minimum is thought to signal the site of cell division. Deterministic models of the Min oscillations reproduce many observed features of the system, including the concentration minimum of MinD. However, there are only a few thousand Min proteins in a bacterium, so stochastic effects are likely to play an important role. Here, we show that Monte Carlo simulations with a large number of proteins agree well with the results from a deterministic treatment of the equations. The location of minimum local MinD concentration is too variable to account for cell division accuracy in wild-type, but is consistent with the accuracy of cell division in cells without nucleoids. This finding confirms the need to include additional mechanisms, such as reciprocal interactions with the cell division ring or positioning of the nucleoids, to explain wild-type accuracy.
17602857
An experimentalist's guide to computational modelling of the Min system
Spatio‐temporal oscillations of the Min proteins are essential for selecting the cell division site in Escherichia coli. These oscillations are a key example of a biological phenomenon that can only be understood on a systems level rather than on the level of its individual components. Here, we review the key concepts that mathematical modelling has added to our understanding of the Min system. While several different mechanisms have been proposed, in all cases the oscillations emerge from a dynamic instability of a uniform protein distribution. To generate this instability, however, the various mechanisms rely on different features of Min protein interactions and transport. We critically evaluate these mechanisms in light of recent experimental evidence. We also review the effects of fluctuations caused by low cellular concentration of Min proteins, and describe how stochastic effects may potentially influence Min protein dynamics.
1520981
The Min System as a General Cell Geometry Detection Mechanism: Branch Lengths in Y-Shaped Escherichia coli Cells Affect Min Oscillation Patterns and Division Dynamics
ABSTRACT In Escherichia coli, division site placement is regulated by the dynamic behavior of the MinCDE proteins, which oscillate from pole to pole and confine septation to the centers of normal rod-shaped cells. Some current mathematical models explain these oscillations by considering interactions among the Min proteins without recourse to additional localization signals. So far, such models have been applied only to regularly shaped bacteria, but here we test these models further by employing aberrantly shaped E. coli cells as miniature reactors. The locations of MinCDE proteins fused to derivatives of green fluorescent protein were monitored in branched cells with at least three conspicuous poles. MinCDE most often moved from one branch to another in an invariant order, following a nonreversing clockwise or counterclockwise direction over the time periods observed. In cells with two short branches or nubs, the proteins oscillated symmetrically from one end to the other. The locations of FtsZ rings were consistent with a broad MinC-free zone near the branch junctions, and Min rings exhibited the surprising behavior of moving quickly from one possible position to another. Using a reaction-diffusion model that reproduces the observed MinCD oscillations in rod-shaped and round E. coli, we predict that the oscillation patterns in branched cells are a natural response of Min behavior in cellular geometries having different relative branch lengths. The results provide further evidence that Min protein oscillations act as a general cell geometry detection mechanism that can locate poles even in branched cells.
27134918
Spatial Regulators for Bacterial Cell Division Self-Organize into Surface Waves in Vitro
In the bacterium Escherichia coli, the Min proteins oscillate between the cell poles to select the cell center as division site. This dynamic pattern has been proposed to arise by self-organization of these proteins, and several models have suggested a reaction-diffusion type mechanism. Here, we found that the Min proteins spontaneously formed planar surface waves on a flat membrane in vitro. The formation and maintenance of these patterns, which extended for hundreds of micrometers, required adenosine 5′-triphosphate (ATP), and they persisted for hours. We present a reaction-diffusion model of the MinD and MinE dynamics that accounts for our experimental observations and also captures the in vivo oscillations.
6332184
Changes in the Min Oscillation Pattern before and after Cell Birth
ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.
18713536
Reconstitution of self-organizing protein gradients as spatial cues in cell-free systems
Intracellular protein gradients are significant determinants of spatial organization. However, little is known about how protein patterns are established, and how their positional information directs downstream processes. We have accomplished the reconstitution of a protein concentration gradient that directs the assembly of the cell division machinery in E.coli from the bottom-up. Reconstituting self-organized oscillations of MinCDE proteins in membrane-clad soft-polymer compartments, we demonstrate that distinct time-averaged protein concentration gradients are established. Our minimal system allows to study complex organizational principles, such as spatial control of division site placement by intracellular protein gradients, under simplified conditions. In particular, we demonstrate that FtsZ, which marks the cell division site in many bacteria, can be targeted to the middle of a cell-like compartment. Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development. DOI: http://dx.doi.org/10.7554/eLife.03949.001
15940436
Oscillations of Min-proteins in micropatterned environments: a three-dimensional particle-based stochastic simulation approach.
The Min-proteins from E. coli and other bacteria are the best characterized pattern forming system in cells and their spatiotemporal oscillations have been successfully reconstituted in vitro. Different mathematical and computational models have been used to better understand these oscillations. Here we use particle-based stochastic simulations to study Min-oscillations in patterned environments. We simulate a rectangular box of length 10 μm and width 5 μm that is filled with grid or checkerboard patterns of different patch sizes and distances. For this geometry, we find different stable oscillation patterns, typically pole-to-pole oscillations along the minor axis and striped oscillations along the major axis. The Min-oscillations can switch from one pattern to the other, either effected by changes in pattern geometry or stochastically. By automatic analysis of large-scale computer simulations, we show quantitatively how the perturbing effect of increased patch distance can be rescued by increased patch size. We also show that striped oscillations occur robustly in arbitrarily shaped filamentous E. coli cells. Our results highlight the robustness and variability of Min-oscillations, put limits on the effect of putative division sites, and provide a powerful computational framework for future studies of protein self-organization in patterned environments.
16914422
Symmetry and scale orient Min protein patterns in shaped bacterial sculptures
The boundary of a cell defines the shape and scale for its subcellular organisation. However, the effects of the cell’s spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shape Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2x1x1 to 11x6x1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe, and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale in adaptation to the cell size within a characteristic length range of 3–6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling, and an adaptive range, when and only when facilitated by the three-dimensional confinement of cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.
55663758
Cyanobacteria in Coral Reef Ecosystems: A Review
Cyanobacteria have dominated marine environments and have been reef builders on Earth for more than three million years (myr). Cyanobacteria still play an essential role in modern coral reef ecosystems by forming a major component of epiphytic, epilithic, and endolithic communities as well as of microbial mats. Cyanobacteria are grazed by reef organisms and also provide nitrogen to the coral reef ecosystems through nitrogen fixation. Recently, new unicellular cyanobacteria that express nitrogenase were found in the open ocean and in coral reef lagoons. Furthermore, cyanobacteria are important in calcification and decalcification. All limestone surfaces have a layer of boring algae in which cyanobacteria often play a dominant role. Cyanobacterial symbioses are abundant in coral reefs; the most common hosts are sponges and ascidians. Cyanobacteria use tactics beyond space occupation to inhibit coral recruitment. Cyanobacteria can also form pathogenic microbial consortia in association with other microbes on living coral tissues, causing coral tissue lysis and death, and considerable declines in coral reefs. In deep lagoons, coccoid cyanobacteria are abundant and are grazed by ciliates, heteroflagellates, and the benthic coral reef community. Cyanobacteria produce metabolites that act as attractants for some species and deterrents for some grazers of the reef communities.
5361675
Engineering cyanobacteria to generate high-value products.
Although many microorganisms have been used for the bioindustrial generation of valuable metabolites, the productive potential of cyanobacterial species has remained largely unexplored. Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements, tolerance of marginal agricultural environments, rapid genetics, and carbon-neutral applications that could be leveraged to address global climate change concerns. Here, we review recent research involving the engineering of cyanobacterial species for the production of valuable bioindustrial compounds, including natural cyanobacterial products (e.g. sugars and isoprene), biofuels (e.g. alcohols, alkanes and hydrogen), and other commodity chemicals. Biological and economic obstacles to scaled cyanobacterial production are highlighted, and methods for increasing cyanobacterial production efficiencies are discussed.
30458113
Plastid evolution.
The ancestors of modern cyanobacteria invented O(2)-generating photosynthesis some 3.6 billion years ago. The conversion of water and CO(2) into energy-rich sugars and O(2) slowly transformed the planet, eventually creating the biosphere as we know it today. Eukaryotes didn't invent photosynthesis; they co-opted it from prokaryotes by engulfing and stably integrating a photoautotrophic prokaryote in a process known as primary endosymbiosis. After approximately a billion of years of coevolution, the eukaryotic host and its endosymbiont have achieved an extraordinary level of integration and have spawned a bewildering array of primary producers that now underpin life on land and in the water. No partnership has been more important to life on earth. Secondary endosymbioses have created additional autotrophic eukaryotic lineages that include key organisms in the marine environment. Some of these organisms have subsequently reverted to heterotrophic lifestyles, becoming significant pathogens, microscopic predators, and consumers. We review the origins, integration, and functions of the different plastid types with special emphasis on their biochemical abilities, transfer of genes to the host, and the back supply of proteins to the endosymbiont.
30757951
Identification of cyanobacterial cell division genes by comparative and mutational analyses
We performed comparative and mutational analyses to define more comprehensively the repertoire of genes involved in cyanobacterial cell division. Genes ftsE, ftsI, ftsQ, ftsW, and (previously recognized) ftsZ, minC, minD, minE and sulA were identified as homologues of cell division genes of Gram‐negative and Gram‐positive bacteria. Transposon mutagenesis of Synechococcus elongatus PCC 7942 identified five additional genes, cdv1, cdv2, cdv3, ftn6 and cikA, involved in cell division. cdv1 encodes a presumptive periplasmic peptidyl‐prolyl cis‐trans isomerase. cdv2 has similarity to ylmF which, like divIVA, lies within the Gram‐positive bacterial ylm gene cluster whose members have functions associated with division. Conservation of other ylm genes in cyanobacteria suggests that cyanobacteria and Gram‐positive bacteria share specific division proteins. Two ylm homologues are also found in algal and plant genomes. cdv3 has low but significant similarity to divIVA, suggesting that minE and cdv3 both mediate division‐site determination in cyanobacteria. In contrast, Gram‐positive bacteria lack minE, and (Gram‐negative) proteobacteria lack divIVA. ftn6, of unknown function, and the circadian input kinase, cikA, are specific to cyanobacteria. In S. elongatus, unlike in other bacteria, FtsZ rings are formed at sites occupied by nucleoids. Thus, the division machinery of cyanobacteria differs in its composition and regulation from that of Gram‐negative and Gram‐positive bacteria.
8730819
Conservation and differences of the Min system in the chloroplast and bacterial division site placement
Chloroplasts are descended from a cyanobacterial endosymbiont and divide by binary fission. Reminiscent of the process in their bacterial ancestor, chloroplast division involves a part of cyanobacteria-derived division machineries in addition to those acquired during chloroplast evolution.1,2 In both bacterial and chloroplast division, formation of the FtsZ ring at the mid position is required for subsequent constriction and fission at the mid division site.1-4 As in bacteria, positioning of the FtsZ ring at the mid-chloroplast is mediated by the Min system.1,2 Recently, we identified the MCD1 protein, a plant-specific component of the Min system in Arabidopsis thaliana chloroplasts.5 Unlike other division components that have been acquired after endosymbiosis and function outside of the chloroplasts (i.e. in/on the outer envelope membrane),6-9 MCD1 functions inside the chloroplast. Since we already discussed about the function and significance of MCD1 as a division component of plant origin,5 here we focus on and discuss about the diversity and evolution of the Min system.
205367392
ZipN, an FtsA‐like orchestrator of divisome assembly in the model cyanobacterium Synechocystis PCC6803
We pursued the characterization of the divisome of the spherical‐celled cyanobacterium Synechocystis PCC6803, through deletion, site‐directed mutagenesis, GFP tagging, two‐hybrid and co‐immunoprecipitation assays. We presently report that the DivIVA‐like protein Cdv3 is essential to both cell growth and division, whereas the AmiC, AmpH, FtsE, FtsN, SpoIID, YlmD, YlmE and YlmG proteins are dispensable. With the exception of the self‐interacting protein YlmD, none of these dispensable factors appeared to interact with ZipN, the crucial cytokinetic factor we previously characterized. By contrast, we found that ZipN interacts with itself and the self‐interacting protein Cdv3, as well as with all other crucial cytokinetic factors we previously characterized, namely: FtsZ, FtsI, FtsQ, SepF and ZipS. We also identified ZipN amino acids selectively involved in ZipN interaction with one of its following partners, Cdv3, FtsQ or SepF. Finally, we found no direct interaction between Cdv3, SepF and ZipS. Collectively, these results indicate that ZipN is a central player of divisome assembly in cyanobacteria, similarly to the FtsA protein of E. coli that is absent in cyanobacteria and chloroplast.
15971701
Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes
BackgroundIn spite of their abundance and importance, little is known about cyanobacterial cell biology and their cell cycle. During each cell cycle, chromosomes must be separated into future daughter cells, i.e. into both cell halves, which in many bacteria is achieved by an active machinery that operates during DNA replication. Many cyanobacteria contain multiple identical copies of the chromosome, but it is unknown how chromosomes are segregated into future daughter cells, and if an active or passive mechanism is operative. In addition to an outer and an inner cell membrane, cyanobacteria contain internal thylakoid membranes that carry the active photosynthetic machinery. It is unclear whether thylakoid membranes are invaginations of the inner cell membrane, or an independent membrane system.ResultsWe have used different fluorescent dyes to study the organization of chromosomes and of cell and thylakoid membranes in live cyanobacterial cells. FM1-43 stained the outer and inner cytoplasmic membranes but did not enter the interior of the cell. In contrast, thylakoid membranes in unicellular Synechocystis cells became visible through a membrane-permeable stain only. Furthermore, continuous supply of the fluorescent dye FM1-43 resulted in the formation of one to four intracellular fluorescent structures in Synechocystis cells, within occurred within 30 to 60 minutes, and may represent membrane vesicles. Using fluorescent DNA stains, we found that Synechocystis genomic DNA is compacted in the cell centre that is devoid of thylakoid membranes. Nucleoids segregated very late in the cell cycle, just before complete closing of the division septum. In striking contrast to Bacillus subtilis, which possesses an active chromosome segregation machinery, fluorescence intensity of stained nucleoids differed considerably between the two Synechocystis daughter cells soon after cell division.ConclusionOur experiments strongly support the idea that the cytoplasmic and thylakoid membranes are not directly connected, but separate entities, in unicellular cyanobacteria. Our findings suggest that a transport system may exist between the cytoplasmic membrane and thylakoids, which could mediate the extension of thylakoid membranes and possibly also protein transport from the cytoplasmic membrane to thylakoid membranes. The cell cycle studies in Synechocystis sp. PCC 6803 show that the multiple chromosome copies per cell segregate very late in the cell cycle and in a much less stringent manner than in B. subtilis cells, indicating that chromosomes may become segregated randomly and in a passive fashion, possibly through constriction of the division septum.
21316289
Spatial ordering of chromosomes enhances the fidelity of chromosome partitioning in cyanobacteria
Many cyanobacteria have been shown to harbor multiple chromosome copies per cell, yet little is known about the organization, replication, and segregation of these chromosomes. Here, we visualize individual chromosomes in the cyanobacterium Synechococcus elongatus via time-lapse fluorescence microscopy. We find that chromosomes are equally spaced along the long axis of the cell and are interspersed with another regularly spaced subcellular compartment, the carboxysome. This remarkable organization of the cytoplasm along with accurate midcell septum placement allows for near-optimal segregation of chromosomes to daughter cells. Disruption of either chromosome ordering or midcell septum placement significantly increases the chromosome partitioning error. We find that chromosome replication is both asynchronous and independent of the position of the chromosome in the cell and that spatial organization is preserved after replication. Our findings on chromosome organization, replication, and segregation in S. elongatus provide a basis for understanding chromosome dynamics in bacteria with multiple chromosomes.
2175258
Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium*
Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram-negative bacterium with an additional internal membrane system that fulfills the energetic requirements of the cell.
1444330
Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria
Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher‐plant chloroplasts, allowing water‐soluble and lipid‐soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane‐bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.
81776261
Architecture of Thylakoid Membrane Networks
The primary events of oxygenic photosynthesis are carried out within intricate membrane lamellar systems called thylakoid networks. These networks, which are present in cyanobacteria, algae, and higher plants, accommodate all of the molecular complexes necessary for the light-driven reactions of photosynthesis and provide a medium for energy transduction. Here, we describe the ultrastructure of thylakoid membranes and their three-dimensional organization in various organisms along the evolutionary tree. Along the way we discuss issues pertaining to the formation and maintenance of these membranes, the means by which they enable molecular traffic within and across them, and the manner by which they respond to short- and long-term variations in light conditions.
207740915
Division and dynamic morphology of plastids.
Plastid division is fundamental to the biology of plant cells. Division by binary fission entails the coordinated assembly and constriction of four concentric rings, two internal and two external to the organelle. The internal FtsZ ring and external dynamin-like ARC5/DRP5B ring are connected across the two envelopes by the membrane proteins ARC6, PARC6, PDV1, and PDV2. Assembly-stimulated GTPase activity drives constriction of the FtsZ and ARC5/DRP5B rings, which together with the plastid-dividing rings pull and squeeze the envelope membranes until the two daughter plastids are formed, with the final separation requiring additional proteins. The positioning of the division machinery is controlled by the chloroplast Min system, which confines FtsZ-ring formation to the plastid midpoint. The dynamic morphology of plastids, especially nongreen plastids, is also considered here, particularly in relation to the production of stromules and plastid-derived vesicles and their possible roles in cellular communication and plastid functionality.
24646296
Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ
ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.
7650379
A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli
The E. coli minicell locus (minB) was shown to code for three gene products (MinC, MinD, and MinE) whose coordinate action is required for proper placement of the division spetum. Studies of the phenotypic effects of expression of the three genes, alone and in all possible combinations, indicated the following: cell poles contain potential division sites that will support additional septation events unless specifically inactivated; the minC and minD gene products act in concert to form a nonspecific inhibitor of septation that is capable of blocking cell division at all potential division sites; and the minE gene codes for a topological specificity factor that, in wild-type cells, prevents the division inhibitor from acting at internal division sites while permitting it to block septation at polar sites.
17312187
The Phyre2 web portal for protein modeling, prediction and analysis
Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.
21392348
The Switch I and II Regions of MinD Are Required for Binding and Activating MinC
ABSTRACT MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations in minD and carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.
11260994
The N Terminus of MinD Contains Determinants Which Affect Its Dynamic Localization and Enzymatic Activity
ABSTRACT MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including Neisseria gonorrhoeae and Escherichia coli. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in E. coli. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of N. gonorrhoeae MinD (MinDNg) on protein dynamism, localization, and interactions with MinDNg and with MinENg. Deletions or mutations were generated in the first five residues of MinDNg, and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinDNg interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinDNg mutants could still oscillate from pole to pole in E. coli, the GFP-MinDNg oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinDNg proteins lacking the first three residues or with an I5E substitution possessed higher MinENg-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinDNg are implicated in regulating the enzymatic activity and dynamic localization of the protein.
12949569
Analysis of MinD Mutations Reveals Residues Required for MinE Stimulation of the MinD ATPase and Residues Required for MinC Interaction
ABSTRACT The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.
43201775
Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120
ABSTRACT When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N2. Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.
40198864
Specialized techniques for site-directed mutagenesis in cyanobacteria.
Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The "plastic" genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation. Here we describe different approaches to mutagenize the chromosome of S. elongatus, including insertional mutagenesis, hit-and-run allele replacement, rps12-mediated gene replacement, and regulated expression of genes from ectopic sites, the neutral sites of the S. elongatus genome.
45816940
Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in Cyanobacterium Synechococcus elongatus PCC 7942.
The cyanobacterium Synechococcus elongatus PCC 7942 is a major model species for studies of photosynthesis. It is are also a potential cell factory for the production of renewable biofuels and valuable chemicals. We employed engineered riboswitches to control translational initiation of target genes in this cyanobacterium. A firefly luciferase reporter assay revealed that three theophylline riboswitches performed as expected in the cyanobacterium. Riboswitch-E* exhibited very low leaky expression of luciferase and superior and dose-dependent on/off regulation of protein expression by theophylline. The maximum magnitude of the induction vs. basal level was ∼190-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 2 mM, facilitating the fine-tuning of luciferase expression. We adapted this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC gene product is controlled by the theophylline concentration in the culture medium. The results demonstrated that the adequately adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic mutant. This theophylline-dependent riboswitch system has potential for various applications as a useful genetic tool in cyanobacteria.
5018724
A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
793158
A stochastic model of Min oscillations in Escherichia coli and Min protein segregation during cell division.
The Min system in Escherichia coli directs division to the centre of the cell through pole-to-pole oscillations of the MinCDE proteins. We present a one-dimensional stochastic model of these oscillations which incorporates membrane polymerization of MinD into linear chains. This model reproduces much of the observed phenomenology of the Min system, including pole-to-pole oscillations of the Min proteins. We then apply this model to investigate the Min system during cell division. Oscillations continue initially unaffected by the closing septum, before cutting off rapidly. The fractions of Min proteins in the daughter cells vary widely, from 50%-50% up to 85%-15% of the total from the parent cell, suggesting that there may be another mechanism for regulating these levels in vivo.
29650415
Characterization of divIVA and Other Genes Located in the Chromosomal Region Downstream of the dcw Cluster in Streptococcus pneumoniae
ABSTRACT We analyzed the chromosome region of Streptococcus pneumoniae located downstream of the division and cell wall (dcw) cluster that contains the homolog of the Bacillus subtilis cell division gene divIVA and some genes of unknown function. Inactivation of divIVA in S. pneumoniae resulted in severe growth inhibition and defects in cell shape, nucleoid segregation, and cell division. Inactivation of the ylm genes resulted in some morphological and/or division abnormalities, depending on the inactivated gene. Transcriptional analysis revealed a relationship between these genes and the ftsA and ftsZ cell division genes, also indicating that the connection between the dcw cluster and the divIVA region is more extensive than just chromosomal position and gene organization.
86145726
JPred4: A protein secondary structure prediction server
JPred4 (http://www.compbio.dundee.ac.uk/jpred4) is the latest version of the popular JPred protein secondary structure prediction server which provides predictions by the JNet algorithm, one of the most accurate methods for secondary structure prediction. In addition to protein secondary structure, JPred also makes predictions of solvent accessibility and coiled-coil regions. The JPred service runs up to 94 000 jobs per month and has carried out over 1.5 million predictions in total for users in 179 countries. The JPred4 web server has been re-implemented in the Bootstrap framework and JavaScript to improve its design, usability and accessibility from mobile devices. JPred4 features higher accuracy, with a blind three-state ( -helix, -strand and coil) secondary structure prediction accuracy of 82.0% while solvent accessibility prediction accuracy has been raised to 90% for residues <5% accessible. Reporting of results is enhanced both on the website and through the optional email summaries and batch submission results. Predictions are now presented in SVG format with options to view full multiple sequence alignments with and without gaps and insertions. Finally, the help-pages have been updated and tool-tips added as well as step-by-step tutorials.
335815
Effect of minCD on FtsZ Ring Position and Polar Septation in Bacillus subtilis
ABSTRACT We examined the pattern of FtsZ localization in a Bacillus subtilis minCD mutant. When grown in minimal medium, the majority (∼89%) of the minCD mutant cells with an FtsZ ring had a single, medially positioned FtsZ ring. These results indicate that genes in addition to minCD function to restrict the number and position of FtsZ rings. When grown in rich medium, greater than 50% of the minCD mutant cells had multiple FtsZ rings, indicating significant differences in regulation of FtsZ ring formation based on growth medium.
7145026
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.
97677
The Min system is not required for precise placement of the midcell Z ring in Bacillus subtilis
In bacteria, the Min system plays a role in positioning the midcell division site by inhibiting the formation of the earliest precursor of cell division, the Z ring, at the cell poles. However, whether the Min system also contributes to establishing the precise placement of the midcell Z ring is unresolved. We show that the Z ring is positioned at midcell with a high degree of precision in Bacillus subtilis, and this is completely maintained in the absence of the Min system. Min is therefore not required for correct midcell Z ring placement in B. subtilis. Our results strongly support the idea that the primary role of the Min system is to block Z ring formation at the cell poles and that a separate mechanism must exist to ensure cell division occurs precisely at midcell.
33634175
SlmA, a nucleoid-associated, FtsZ binding protein required for blocking septal ring assembly over Chromosomes in E. coli.
Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.
8177148
The Min system and other nucleoid-independent regulators of Z ring positioning
Rod-shaped bacteria such as E. coli have mechanisms to position their cell division plane at the precise center of the cell, to ensure that the daughter cells are equal in size. The two main mechanisms are the Min system and nucleoid occlusion (NO), both of which work by inhibiting assembly of FtsZ, the tubulin-like scaffold that forms the cytokinetic Z ring. Whereas NO prevents Z rings from constricting over unsegregated nucleoids, the Min system is nucleoid-independent and even functions in cells lacking nucleoids and thus NO. The Min proteins of E. coli and B. subtilis form bipolar gradients that inhibit Z ring formation most at the cell poles and least at the nascent division plane. This article will outline the molecular mechanisms behind Min function in E. coli and B. subtilis, and discuss distinct Z ring positioning systems in other bacterial species.
25569726
The divIVA minicell locus of Bacillus subtilis
The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB- mutant. The DivIVA-mutants, but not the DivIVB- mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.
3448555
Protein-Protein Interaction Domains of Bacillus subtilis DivIVA
ABSTRACT DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent protein-tagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.
45022694
A Novel Gene That Bears a DnaJ Motif Influences Cyanobacterial Cell Division
ABSTRACT Transposon Tn5-692 mutagenizes Synechococcus sp. strain PCC 7942 efficiently. The predicted product of the gene mutated in the Tn5-692-derived cell division mutant FTN2 has an N-terminal DnaJ domain, as have its cyanobacterial and plant orthologs. Anabaena sp. strain PCC 7120, when mutated in genes orthologous to ftn2 and ftn6, forms akinete-like cells.
14878163
ARC6 Is a J-Domain Plastid Division Protein and an Evolutionary Descendant of the Cyanobacterial Cell Division Protein Ftn2 Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.013292.
Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6–green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site–determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.
206327221
Functional Analysis of the Chloroplast Division Complex Using Schizosaccharomyces pombe as a Heterologous Expression System
Abstract Chloroplast division is driven by a macromolecular complex that assembles at the midplastid. The FtsZ ring (Z ring) is the central structure in this complex, and is composed of the functionally distinct cytoskeletal proteins FtsZ1 and FtsZ2. Recent studies in the heterologous Schizosaccharomyces pombe system showed that Arabidopsis FtsZ1 and FtsZ2 filaments have distinct assembly and turnover characteristics. To further analyze these FtsZs, we employed this system to compare the assembly and dynamic properties of FtsZ1 and FtsZ2 lacking their N- and/or C-termini with those of their full-length counterparts. Our data provide evidence that the N-terminus of FtsZ2 is critical for its structural dominance over FtsZ1, and that the N- and C-termini promote polymer bundling and turnover of both FtsZs and contribute to their distinct behaviors. We also assessed how ARC6 affects FtsZ2 filament dynamics, and found that it interacts with and stabilizes FtsZ2 filaments in S. pombe independent of its presumed Z-ring tethering function in planta. Finally, we generated FtsZ1-FtsZ2 coexpression constructs to facilitate reconstitution of more complex interaction networks. Our experiments yield new insight into factors influencing FtsZ behavior and highlight the utility of S. pombe for analyzing chloroplast FtsZs and their assembly regulators.
35974981
FtsZ1/FtsZ2 Turnover in Chloroplasts and the Role of ARC3
Abstract Chloroplast division requires filamentation temperature-sensitive Z (FtsZ), a tubulin-like GTPase of cyanobacterial endosymbiotic origin. Plants and algae possess two distinct FtsZ protein families, FtsZ1 and FtsZ2 that co-assemble into a ring (Z-ring) at the division site. Z-ring assembly and disassembly and division site positioning is controlled by both positive and negative factors via their specific interactions with FtsZ1 and FtsZ2. Here we present the in planta analysis of Arabidopsis FtsZ1 and FtsZ2 turnover in the context of a native chloroplast division machinery. Fluorescence recovery after photobleaching analysis was conducted using fluorescently tagged FtsZ at wild-type (WT)-like levels. Rapid photobleaching, low signal-to-noise ratio, and phototropic movements of chloroplasts were overcome by (i) using progressive intervals in time-lapse imaging, (ii) analyzing epidermal rather than stromal chloroplasts, and (iii) employing image stack alignment during postprocessing. In plants of WT background, fluorescence recovery half-times averaged 117 and 325 s for FtsZ1 and FtsZ2, respectively. In plants lacking ARC3, the key negative regulator of FtsZ assembly, the turnover was threefold slower. The findings are discussed in the context of previous results conducted in a heterologous system.
207629333
Proteomic analysis of plasma membranes of cyanobacterium Synechocystis sp. Strain PCC 6803 in response to high pH stress.
Cyanobacteria are unique prokaryotes possessing plasma-, outer- and thylakoid membranes. The plasma membrane of a cyanobacterial cell serves as a crucial barrier against its environment and is essential for biogenesis of cyanobacterial photosystems. Previously, we have identified 79 different proteins in the plasma membrane of Synechocystis sp. Strain PCC 6803 based on 2D- and 1D- gels and MALDI-TOF MS. In this work, we have performed a proteomic study screening for high-pH-stress proteins in Synechocystis. 2-D gel profiles of plasma membranes isolated from both control and high pH-treated cells were constructed and compared quantitatively based on different protein staining methods including DIGE analysis. A total of 55 differentially expressed protein spots were identified using MALDI-TOF MS and MALDI-TOF/TOF MS, corresponding to 39 gene products. Twenty-five proteins were enhanced/induced and 14 reduced by high pH. One-third of the enhanced/induced proteins were transport and binding proteins of ABC transporters including 3 phosphate transport proteins. Other proteins include MinD involved in cell division, Cya2 in signaling and proteins involved in photosynthesis and respiration. Furthermore, among these proteins regulated by high pH, eight were found to be hypothetical proteins. Functional significance of the high-pH-stress proteins is discussed integrating current knowledge on cyanobacterial cell physiology.
2698405
Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly1[OPEN]
A plant-specific chloroplast division protein has multiple functions in coordinating and influencing the assembly of internal and external contractile rings across the two envelope membranes. Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division.
10983302
PARC6, a novel chloroplast division factor, influences FtsZ assembly and is required for recruitment of PDV1 during chloroplast division in Arabidopsis.
Chloroplast division in plant cells is accomplished through the coordinated action of the tubulin-like FtsZ ring inside the organelle and the dynamin-like ARC5 ring outside the organelle. This coordination is facilitated by ARC6, an inner envelope protein required for both assembly of FtsZ and recruitment of ARC5. Recently, we showed that ARC6 specifies the mid-plastid positioning of the outer envelope proteins PDV1 and PDV2, which have parallel functions in dynamin recruitment. PDV2 positioning involves direct ARC6-PDV2 interaction, but PDV1 and ARC6 do not interact indicating that an additional factor functions downstream of ARC6 to position PDV1. Here, we show that PARC6 (paralog of ARC6), an ARC6-like protein unique to vascular plants, fulfills this role. Like ARC6, PARC6 is an inner envelope protein with its N-terminus exposed to the stroma and Arabidopsis parc6 mutants exhibit defects of chloroplast and FtsZ filament morphology. However, whereas ARC6 promotes FtsZ assembly, PARC6 appears to inhibit FtsZ assembly, suggesting that ARC6 and PARC6 function as antagonistic regulators of FtsZ dynamics. The FtsZ inhibitory activity of PARC6 may involve its interaction with the FtsZ-positioning factor ARC3. A PARC6-GFP fusion protein localizes both to the mid-plastid and to a single spot at one pole, reminiscent of the localization of ARC3, PDV1 and ARC5. Although PARC6 localizes PDV1, it is not required for PDV2 localization or ARC5 recruitment. Our findings indicate that PARC6, like ARC6, plays a role in coordinating the internal and external components of the chloroplast division complex, but that PARC6 has evolved distinct functions in the division process.
30154828
Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803
Purified thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803 were used for the first time in proteomic studies. The membranes were prepared by a combination of sucrose density centrifugation and aqueous polymer two‐phase partitioning. In total, 76 different proteins were identified from 2‐ and 1‐D gels by MALDI‐TOF MS analysis. Twelve of the identified proteins have a predicted Sec/Tat signal peptide. Fourteen of the proteins were known, or predicted to be, integral membrane proteins. Among the proteins identified were subunits of the well‐characterized thylakoid membrane constituents Photosystem I and II, ATP synthase, cytochrome b6f‐complex, NADH dehydrogenase, and phycobilisome complex. In addition, novel thylakoid membrane proteins, both integral and peripheral were found, including enzymes involved in protein folding and pigment biosynthesis. The latter were the chlorophyll biosynthesis enzymes, light‐dependent protochlorophyllide reductase and geranylgeranyl reductase as well as phytoene desaturase involved in carotenoid biosynthesis and a water‐soluble carotenoid‐binding protein. Interestingly, in view of the protein sorting mechanism in cyanobacteria, one of the two signal peptidases type I of Synechocystis was found in the thylakoid membrane, whereas the second one has been identified previously in the plasma membrane. Sixteen proteins are hypothetical proteins with unknown function.
19483290
Effects of Phospholipid Composition on MinD-Membrane Interactions in Vitro and in Vivo*
The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic α-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693–15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.
1219735
MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli*
Background: Understanding the bacterial division machinery is essential to decoding cellular physiology. Results: The cell division proteins MinD/MinE bind tightly to anionic lipids, which reduces ATPase activity. Conclusion: MinD and MinE interact preferably with anionic lipids that are positioned at the cell pole. Significance: These results provide insight into a mechanism that regulates bacterial cell division and may present a useful target for antimicrobial development. The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (Kd) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The Kd for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (kon). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.
21253095
Differential affinities of MinD and MinE to anionic phospholipid influence Min patterning dynamics in vitro
The E. coli Min system forms a cell‐pole‐to‐cell‐pole oscillator that positions the divisome at mid‐cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid‐cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no‐flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD‐independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning.
12073103
Bacterial membrane lipids: diversity in structures and pathways.
For many decades, Escherichia coli was the main model organism for the study of bacterial membrane lipids. The results obtained served as a blueprint for membrane lipid biochemistry, but it is clear now that there is no such thing as a typical bacterial membrane lipid composition. Different bacterial species display different membrane compositions and even the membrane composition of cells belonging to a single species is not constant, but depends on the environmental conditions to which the cells are exposed. Bacterial membranes present a large diversity of amphiphilic lipids, including the common phospholipids phosphatidylglycerol, phosphatidylethanolamine and cardiolipin, the less frequent phospholipids phosphatidylcholine, and phosphatidylinositol and a variety of other membrane lipids, such as for example ornithine lipids, glycolipids, sphingolipids or hopanoids among others. In this review, we give an overview about the membrane lipid structures known in bacteria, the different metabolic pathways involved in their formation, and the distribution of membrane lipids and metabolic pathways across taxonomical groups.
40312655
Characterization of the Fad12 mutant of Synechocystis that is defective in delta 12 acyl-lipid desaturase activity.
The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for delta 12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the delta 12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the delta 12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.
19113695
Membrane potential is important for bacterial cell division
Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, we report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. We show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects observed with certain antimicrobial compounds.
9620507
Evolution of multicellularity coincided with increased diversification of cyanobacteria and the Great Oxidation Event
Cyanobacteria are among the most diverse prokaryotic phyla, with morphotypes ranging from unicellular to multicellular filamentous forms, including those able to terminally (i.e., irreversibly) differentiate in form and function. It has been suggested that cyanobacteria raised oxygen levels in the atmosphere around 2.45–2.32 billion y ago during the Great Oxidation Event (GOE), hence dramatically changing life on the planet. However, little is known about the temporal evolution of cyanobacterial lineages, and possible interplay between the origin of multicellularity, diversification of cyanobacteria, and the rise of atmospheric oxygen. We estimated divergence times of extant cyanobacterial lineages under Bayesian relaxed clocks for a dataset of 16S rRNA sequences representing the entire known diversity of this phylum. We tested whether the evolution of multicellularity overlaps with the GOE, and whether multicellularity is associated with significant shifts in diversification rates in cyanobacteria. Our results indicate an origin of cyanobacteria before the rise of atmospheric oxygen. The evolution of multicellular forms coincides with the onset of the GOE and an increase in diversification rates. These results suggest that multicellularity could have played a key role in triggering cyanobacterial evolution around the GOE.
15200638
Circadian Rhythms in Cyanobacteria
SUMMARY Life on earth is subject to daily and predictable fluctuations in light intensity, temperature, and humidity created by rotation of the earth. Circadian rhythms, generated by a circadian clock, control temporal programs of cellular physiology to facilitate adaptation to daily environmental changes. Circadian rhythms are nearly ubiquitous and are found in both prokaryotic and eukaryotic organisms. Here we introduce the molecular mechanism of the circadian clock in the model cyanobacterium Synechococcus elongatus PCC 7942. We review the current understanding of the cyanobacterial clock, emphasizing recent work that has generated a more comprehensive understanding of how the circadian oscillator becomes synchronized with the external environment and how information from the oscillator is transmitted to generate rhythms of biological activity. These results have changed how we think about the clock, shifting away from a linear model to one in which the clock is viewed as an interactive network of multifunctional components that are integrated into the context of the cell in order to pace and reset the oscillator. We conclude with a discussion of how this basic timekeeping mechanism differs in other cyanobacterial species and how information gleaned from work in cyanobacteria can be translated to understanding rhythmic phenomena in other prokaryotic systems.
24058881
Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours.
To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density. We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity. The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression. Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.
2662536
Elevated ATPase Activity of KaiC Applies a Circadian Checkpoint on Cell Division in Synechococcus elongatus
A circadian clock coordinates physiology and behavior in diverse groups of living organisms. Another major cyclic cellular event, the cell cycle, is regulated by the circadian clock in the few cases where linkage of these cycles has been studied. In the cyanobacterium Synechococcus elongatus, the circadian clock gates cell division by an unknown mechanism. Using timelapse microscopy, we confirm the gating of cell division in the wild-type and demonstrate the regulation of cytokinesis by key clock components. Specifically, a state of the oscillator protein KaiC that is associated with elevated ATPase activity closes the gate by acting through a known clock output pathway to inhibit FtsZ ring formation at the division site. An activity that stimulates KaiC phosphorylation independently of the KaiA protein was also uncovered. We propose a model that separates the functions of KaiC ATPase and phosphorylation in cell division gating and other circadian behaviors.
1351008
Enzymatic assembly of DNA molecules up to several hundred kilobases
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
2945597
Detailed Simulations of Cell Biology with Smoldyn 2.1
Most cellular processes depend on intracellular locations and random collisions of individual protein molecules. To model these processes, we developed algorithms to simulate the diffusion, membrane interactions, and reactions of individual molecules, and implemented these in the Smoldyn program. Compared to the popular MCell and ChemCell simulators, we found that Smoldyn was in many cases more accurate, more computationally efficient, and easier to use. Using Smoldyn, we modeled pheromone response system signaling among yeast cells of opposite mating type. This model showed that secreted Bar1 protease might help a cell identify the fittest mating partner by sharpening the pheromone concentration gradient. This model involved about 200,000 protein molecules, about 7000 cubic microns of volume, and about 75 minutes of simulated time; it took about 10 hours to run. Over the next several years, as faster computers become available, Smoldyn will allow researchers to model and explore systems the size of entire bacterial and smaller eukaryotic cells.