Mechanism of the asymmetric activation of the MinD ATPase by MinE

MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE; however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE; however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE-stimulated ATPase activity and suggest that it is a key residue affected by MinE binding.     

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.     

The partition system of multidrug resistance plasmid TP228 includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily

The segregational stability of bacterial, low-copy-number plasmids is promoted primarily by active partition. The plasmid-specified components of the prototypical P1 plasmid partition system consist of two proteins, ParA (44.3 kDa) and ParB (38.5 kDa), which, in conjunction with integration host factor, form a nucleoprotein complex at the plasmid partition site, parS. This complex is the probable substrate for the directed temporal and spatial intracellular movement of plasmids before cell division. The genetic organization of the partition cassette of the multidrug resistance plasmid TP228 differs markedly from that of the P1 paradigm. The TP228 system includes a novel member (ParF; 22.0 kDa) of the ParA superfamily of ATPases, of which the P1 ParA protein is the archetype. However, the ParF protein and its immediate relatives form a discrete subgroup of the ParA superfamily, which evolutionarily is more related to the MinD subgroup of cell division proteins than to ParA of P1. The TP228 and P1 partition modules differ further in that the former does not include a parB homologue, but does specify a protein (ParG; 8.6 kDa) unrelated to ParB. Homologues of the parF gene are widely disseminated on eubacterial genomes, suggesting that ParF-mediated partition may be a common mechanism by which plasmid segregational stability is achieved.     

MinD and role of the deviant Walker A motif,dimerization and membrane binding in oscillation

The ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli. It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA). All members of the subgroup have a deviant Walker A motif which contains a conserved 'signature' lysine that characterizes this subgroup. In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis. ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer. Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient. In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable. Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane. The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.     

A family of ATPases involved in active partitioning of diverse bacterial plasmids

Low copy-number bacterial plasmids F (the classical Escherichia coli sex factor) and prophage P1 encode partitioning functions which may provide fundamental insights into the active processes which ensure that bacterial genomes are segregated to both daughter cells prior to cell division. These partitioning systems involve two proteins: ParA and ParB. We report that incC from the broad host-range plasmid RK2 is a member of the family of ParA partitioning proteins and that these proteins (as well as related proteins encoded by plasmids from Agrobacterium tumefaciens and Chlamydia trachomatis) contain type I nucleotide-binding motifs. Also, we show that the cell division inhibitor MinD is homologous to members of the ParA family. Sequence comparisons of ParB proteins suggest that they may contain sites for phosphorylation. We propose that ATP hydrolysis by the ParA protein may result in phosphorylation of the ParB protein, thereby causing a conformational shift necessary to separate paired plasmid molecules at the cell division plane.     

Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171

Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions without the involvement of partition-specific host cell receptors and is thus consistent with the striking observation that partition loci can function in heterologous host organisms.     

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created 'super-repressors', in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA's repressor activity and suggest that ATP hydrolysis is essential for plasmid localization.     

The N terminus of MinD contains determinants which affect its dynamic localization and enzymatic activity

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 (MinD(Ng)) on protein dynamism, localization, and interactions with MinD(Ng) and with MinE(Ng). Deletions or mutations were generated in the first five residues of MinD(Ng), and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinD(Ng) interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinD(Ng) mutants could still oscillate from pole to pole in E. coli, the GFP-MinD(Ng) oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinD(Ng) proteins lacking the first three residues or with an I5E substitution possessed higher MinE(Ng)-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinD(Ng) are implicated in regulating the enzymatic activity and dynamic localization of the protein.     

ParA ATPases can move and position DNA and subcellular structures

Prokaryotic chromosomes and plasmids can be actively segregated by partitioning (par) loci. The common ParA-encoding par loci segregate plasmids by arranging them in regular arrays over the nucleoid by an unknown mechanism. Recent observations indicate that ParA moves plasmids and chromosomes by a pulling mechanism. Even though ParAs form filaments in vitro it is not known whether similar structures are present in vivo. ParA of P1 forms filaments in vitro at very high concentrations only and filament-like structures have not been observed in vivo. Consequently, a 'diffusion-ratchet' mechanism was suggested to explain plasmid movement by ParA of P1. We compare this mechanism with our previously proposed filament model for plasmid movement by ParA. Remarkably, ParA homologues have been discovered to arrange subcellular structures such as carboxysomes and chemotaxis sensory receptors in a regular manner very similar to those of the plasmid arrays.     

Towards understanding the molecular basis of bacterial DNA segregation

Bacteria ensure the fidelity of genetic inheritance by the coordinated control of chromosome segregation and cell division. Here, we review the molecules and mechanisms that govern the correct subcellular positioning and rapid separation of newly replicated chromosomes and plasmids towards the cell poles and, significantly, the emergence of mitotic-like machineries capable of segregating plasmid DNA. We further describe surprising similarities between proteins involved in DNA partitioning (ParA/ParB) and control of cell division (MinD/MinE), suggesting a mechanism for intracellular positioning common to the two processes. Finally, we discuss the role that the bacterial cytoskeleton plays in DNA partitioning and the missing link between prokaryotes and eukaryotes that is bacterial mechano-chemical motor proteins.     

The bacterial ParA-ParB partitioning proteins

A pair of genes designated parA and parB are encoded by many low copy number plasmids and bacterial chromosomes. They work with one or more cis-acting sites termed centromere-like sequences to ensure better than random predivisional partitioning of the DNA molecule that encodes them. The centromere-like sequences nucleate binding of ParB and titrate sufficient protein to create foci, which are easily visible by immuno-fluorescence microscopy. These foci normally follow the plasmid or the chromosomal replication oriC complexes. ParA is a membrane-associated ATPase that is essential for this symmetric movement of the ParB foci. In Bacillus subtilis ParA oscillates from end to end of the cell as does MinD of E. coli, a relative of the ParA family. ParA may facilitate ParB movement along the inner surface of the cytoplasmic membrane to encounter and become tethered to the next replication zone. The ATP-bound form of ParA appears to adopt the conformation needed to drive partition. Hydrolysis to create ParA-ADP or free ParA appears to favour a form that is not located at the pole and binds to DNA rather than the partition complex. Definition of the protein domains needed for interaction with membranes and the conformational changes that occur on interaction with ATP/ADP will provide insights into the partitioning mechanism and possible targets for inhibitors of partitioning.     

Operational Principles for the Dynamics of the In Vitro ParA-ParB System

In many bacteria the ParA-ParB protein system is responsible for actively segregating DNA during replication. ParB proteins move by interacting with DNA bound ParA-ATP, stimulating their unbinding by catalyzing hydrolysis, that leads to rectified motion due to the creation of a wake of depleted ParA. Recent in vitro experiments have shown that a ParB covered magnetic bead can move with constant speed over a DNA covered substrate that is bound by ParA. It has been suggested that the formation of a gradient in ParA leads to diffusion-ratchet like motion of the ParB bead but how it forms and generates a force is still a matter of exploration. Here we develop a deterministic model for the in vitro ParA-ParB system and show that a ParA gradient can spontaneously form due to any amount of initial spatial noise in bound ParA. The speed of the bead is independent of this noise but depends on the ratio of the range of ParA-ParB force on the bead to that of removal of surface bound ParA by ParB. We find that at a particular ratio the speed attains a maximal value. We also consider ParA rebinding (including cooperativity) and ParA surface diffusion independently as mechanisms for ParA recovery on the surface. Depending on whether the DNA covered surface is undersaturated or saturated with ParA, we find that the bead can accelerate persistently or potentially stall. Our model highlights key requirements of the ParA-ParB driving force that are necessary for directed motion in the in vitro system that may provide insight into the in vivo dynamics of the ParA-ParB system.     

Gonococcal MinD affects cell division in Neisseria gonorrhoeae and Escherichia coli and exhibits a novel self-interaction

The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae (MinD(Ng)) in this mutant. Hence, MinD(Ng) is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD(Ng) is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC(Ng) and MinD(Ng) from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC(Ng) or MinD(Ng) alone did not display noticeable morphological changes. These studies suggest that MinD(Ng) is involved in inhibiting gonococcal cell division, likely in conjunction with MinC(Ng). The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD(Ng) in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD(Ng) revealed a novel MinD(Ng) self-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinD(Ng). These results indicate that MinD(Ng) is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.     

MinC/MinD copolymers are not required for Min function

In Escherichia coli, precise placement of the cytokinetic Z ring at midcell requires the concerted action of the three Min proteins. MinD activates MinC, an inhibitor of FtsZ, at least in part, by recruiting it to the membrane and targeting it to the Z ring, while MinE stimulates the MinD ATPase inducing an oscillation that directs MinC/MinD activity away from midcell. Recently, MinC and MinD were shown to form copolymers of alternating dimers of MinC and MinD, and it was suggested that these copolymers are the active form of MinC/MinD. Here, we use MinD mutants defective in binding MinC to generate heterodimers with wild‐type MinD that are unable to form MinC/MinD copolymers. Similarly, MinC mutants defective in binding to MinD were used to generate heterodimers with wild‐type MinC that are unable to form copolymers. Such heterodimers are active and in the case of MinC were shown to mediate spatial regulation of the Z ring demonstrating that MinC/MinD copolymer formation is not required. Our results are consistent with a model in which a membrane anchored MinC/MinD complex is targeted to the Z ring through the conserved carboxy tail of FtsZ leading to breakage of FtsZ filaments.     

MinJ (YvjD) is a topological determinant of cell division in Bacillus subtilis

In Bacillus subtilis, FtsZ ring formation and cell division is favoured at the midcell because the inhibitor proteins MinC and MinD are indirectly restricted to the cell poles by the protein DivIVA. Here we identify MinJ, a topological determinant of medial FtsZ positioning that acts as an intermediary between DivIVA and MinD. Due to unrestricted MinD activity, cells mutated for minJ exhibited pleiotropic defects in homologous recombination, swarming motility and cell division. MinJ restricted MinD activity by localizing MinD to the cell poles through direct protein-protein interaction. MinJ itself localized to cell poles in a manner that was dependent on DivIVA. MinJ is conserved in other low G+C Gram-positive bacteria and may be an important component of cell division site selection in these organisms.     

Selection of the midcell division site in Bacillus subtilis through MinD-dependent polar localization and activation of MinC

Bacterial cell division commences with the assembly of the tubulin-like protein, FtsZ, at midcell to form a ring. Division site selection in rod-shaped bacteria is mediated by MinC and MinD, which form a division inhibitor. Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at midcell. We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles. This localization is reminiscent of the pattern previously described for MinD. Localization of MinC requires both early (FtsZ) and late (PbpB) division proteins, and it is completely dependent on MinD. The effects of a divIVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after division, rather than recruitment to nascent division sites. By overexpressing minC or minD, we show that both proteins are required to block division, but that only MinD needs to be in excess of wild-type levels. The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor.     

Architecture of the ParF*ParG protein complex involved in prokaryotic DNA segregation

The mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATP-binding activity of ParF. ParG interacts in a sequence-specific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.     

Mutual effects of MinD-membrane interaction: II. Domain structure of the membrane enhances MinD binding

MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on pre-existing anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.     

叶绿体分裂相关蛋白CrMinD的保守功能

细胞或质体中部正确分裂位点的选择是MinD蛋白与其他Min蛋白（MinC／E）相互作用的结果,MinD蛋白在原核细胞以及植物叶绿体的分裂过程中发挥着重要的作用。细胞中MinD蛋白浓度的明显升高可影响正常细胞的分裂过程而产生丝状体细胞。为了研究叶绿体分裂蛋白CrMinD的保守功能,构建了衣藻CrMinD-gfp的原核表达重组质粒进行了原核功能验证。试验结果表明,衣藻CrMinD蛋白的过量表达严重影响了大肠杆菌的分裂,其在原核细胞中运动和定位与用GFP标记的原核细胞MinD蛋白具有相似性。更进一步证明了叶绿体分裂同源物CrMinD蛋白与原核细胞MinD蛋白有着相似的功能,是一个进化上功能保守的蛋白。同时,这一结果也为研究植物细胞中质体的分裂机制奠定了一定的基础。     

Uncoupling of Nucleotide Hydrolysis and Polymerization in the ParA Protein Superfamily Disrupts DNA Segregation Dynamics

DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog ParF display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere-binding factor augments ParF ATPase activity but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket not only is crucial for optimal ATPase kinetics but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes.