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.     

Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells with a single plasmid focus, the focus located preferentially at mid-cell. In cells with two foci, these located at quarter-cell positions. In the absence of ParB and parC1/parC2, ParA-GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid and subsequently separate them into daughter cells.     

Altered ParA partition proteins of plasmid P1 act via the partition site to block plasmid propagation

The partition system of the P1 plasmid, P1 par consists of the ParA and ParB proteins and a cis -acting site, parS . It is responsible for the orderly segregation of plasmid copies to daughter cells. Plasmids with null mutations in parA or parB replicate normally, but missegregate. ParB binds specifically to the parS site, but the role of ParA and its ATPase activity in partition is unclear. We describe a novel class of parA mutants that cannot be established or maintained as plasmids unless complemented by the wild-type gene. One, parAM314I , is conditional: it can be maintained in cells in minimal medium but cannot be established in cells growing in L broth. The lack of plasmid propagation in L broth-grown cells was shown to be caused by a ParB-dependent activity of the mutant ParA protein that blocks plasmid propagation by an interaction at the parS site. Thus, ParA acts to modify the ParB– parS complex, probably by binding to it. Partition is thought to involve selection of pairs of plasmids before segregation, either by physical pairing of copies or by binding of copies to paired host sites. We suggest that ParA is involved in this reaction and that the mutant ParA protein forms paired complexes that cannot unpair.     

Partitioning of P1 plasmids by gradual distribution of the ATPase ParA

Recently, it has been reported that prokaryotes also have a mitotic-like apparatus in which polymerized fibres govern the bipolar movement of chromosomes and plasmids. Here, we show evidence that a non-mitotic-like apparatus that does not form polymerized filaments carries out plasmid partitioning. P1 ParA, which is a DNA-binding ATPase protein, was found to be distributed through the whole nucleoid and formed a dense spot at the centre of the nucleoid. The fluorescent intensity of the ParA spot blinked, and then the spot gradually migrated from the midcell to a cell quarter position. Such distribution was not observed in anucleate cells, suggesting that the nucleoid could be a matrix for gradual distribution of ParA. Plasmid DNA constantly colocalized at the spot of ParA and migrated according to spot migration and separation. Thus, the gradient distribution of ParA determines the destination of partitioning plasmids and may direct plasmids to the cell quarters.     

Structural biology of plasmid segregation proteins

DNA segregation, or partition, ensures stable genome transmission during cell division. In prokaryotes, partition is best understood for plasmids, which serve as tractable model systems to decipher the molecular underpinnings of this process. Plasmid partition is mediated by par systems, composed of three essential elements: a centromere-like site and the proteins ParA and ParB. In the first step, ParB binds the centromere to form a large segrosome. Subsequently, ParA, an ATPase, binds the segrosome and mediates plasmid separation. Recently determined ParB-centromere structures have revealed key insights into segrosome assembly, whereas ParA structures have shed light on the mechanism of plasmid separation. These structures represent important steps in elucidating the molecular details of plasmid segregation.     

What is the mechanism of ParA‐mediated DNA movement?

The stable maintenance of low‐copy‐number plasmids requires active partitioning, with the most common mechanism in prokaryotes involving the ATPase ParA. ParA proteins undergo intricate spatiotemporal relocations across the nucleoid, dynamics that function to position plasmids at equally spaced intervals. This spacing naturally guarantees equal partitioning of plasmids to each daughter cell. However, the fundamental mechanism linking ParA dynamics with regular plasmid positioning has proved difficult to dissect. In this issue of Molecular Microbiology, Vecchiarelli et al. report on a time‐delay mechanism that allows a slow cycling between the nucleoid‐bound and unbound forms of ParA. The authors also propose a mechanism for plasmid movement that does not rely on ParA polymerization.     

Competing ParA Structures Space Bacterial Plasmids Equally over the Nucleoid

Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These minimal components space plasmids equally over the nucleoid, yet the underlying mechanism is not understood. Here we investigate a model where ParA-ATP can dynamically associate to the nucleoid and is hydrolyzed by plasmid-associated ParB, thereby creating nucleoid-bound, self-organizing ParA concentration gradients. We show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. Such positioning can be achieved regardless of the exact mechanism of plasmid movement, including plasmid diffusion with ParA-mediated immobilization or directed plasmid motion induced by ParB/parC-stimulated ParA structure disassembly. However, we find experimentally that parABC from Escherichia coli plasmid pB171 increases plasmid mobility, inconsistent with diffusion/immobilization. Instead our observations favor directed plasmid motion. Our model predicts less oscillatory ParA dynamics than previously believed, a prediction we verify experimentally. We also show that ParA localization and plasmid positioning depend on the underlying nucleoid morphology, indicating that the chromosomal architecture constrains ParA structure formation. Our directed motion model unifies previously contradictory models for plasmid segregation and provides a robust mechanistic basis for self-organized plasmid spacing that may be widely applicable.     

ParA‐mediated plasmid partition driven by protein pattern self‐organization

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low‐copy plasmids, such as the plasmids P1 and F, employ a three‐component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker‐type ATPase, typically called ParA, which also binds non‐specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP‐driven patterning is involved in partition is unknown. We reconstituted and visualized ParA‐mediated plasmid partition inside a DNA‐carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB‐stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion‐ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.     

Prokaryotic DNA segregation by an actin-like filament

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with properties expected for a force-generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyperfilamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.     

Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.     

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.     

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.     

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.     

Plasmid and chromosome partitioning: surprises from phylogeny

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par-encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter-cell positions). Two types of partitioning ATPases are known: the Walker-type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin-like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid-encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome-encoded ATPases, including members from both Gram-negative and Gram-positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.     

A mechanism for ParB-dependent waves of ParA,a protein related to DNA segregation during cell division in prokaryotes

Prokaryotic plasmids encode partitioning (par) loci involved in segregation of DNA to daughter cells at cell division. A functional fusion protein consisting of Walker-type ParA ATPase and green fluorescent protein (Gfp) oscillates back and forth within nucleoid regions with a wave period of about 20 minutes. A model is discussed which is based on cooperative non-specific binding of ParA to the nucleoid, and local ParB initiated generation of ParA oligomer degradation products, which act autocatalytically on the degradation reaction. The model yields self-initiated spontaneous pattern formation, based on Turing's mechanism, and these patterns are destroyed by the degradation products, only to initiate a new pattern at the opposite nucleoid region. A recurrent wave thus emerges. This may be a particular example of a more general class of pattern forming mechanisms, based on protein oligomerization upon a template (membranes, DNA a.o.) with resulting enhanced NTPase function in the oligomer state, which may bring the oligomer into an unstable internal state. An effector initializes destabilization of the oligomer to yield degradation products, which act as seeds for further degradation in an autocatalytic process. We discuss this mechanism in relation to recent models for MinDE oscillations in E.coli and to microtubule degradation in mitosis. The study points to an ancestral role for the presented pattern types in generating bipolarity in prokaryotes and eukaryotes.     

Structural analysis of the ParR/parC plasmid partition complex

Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 A crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon-helix-helix (RHH)2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition.     

Effects of the P1 plasmid centromere on expression of P1 partition genes

The partition operon of P1 plasmid encodes two proteins, ParA and ParB, required for the faithful segregation of plasmid copies to daughter cells. The operon is followed by a centromere analog, parS, at which ParB binds. ParA, a weak ATPase, represses the par promoter most effectively in its ADP-bound form. ParB can recruit ParA to parS, stimulate its ATPase, and significantly stimulate the repression. We report here that parS also participates in the regulation of expression of the par genes. A single chromosomal parS was shown to augment repression of several copies of the par promoter by severalfold. The repression increase was sensitive to the levels of ParA and ParB and to their ratio. The increase may be attributable to a conformational change in ParA mediated by the parS-ParB complex, possibly acting catalytically. We also observed an in cis effect of parS which enhanced expression of parB, presumably due to a selective modulation of the mRNA level. Although ParB had been earlier found to spread into and silence genes flanking parS, silencing of the par operon by ParB spreading was not significant. Based upon analogies between partitioning and septum placement, we speculate that the regulatory switch controlled by the parS-ParB complex might be essential for partitioning itself.     

ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli

Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the ‘Min’ proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.     

Partition-associated incompatibility caused by random assortment of pure plasmid clusters

Summary Bacterial plasmids and chromosomes encode centromere-like partition loci that actively segregate DNA before cell division. The molecular mechanism behind DNA segregation in bacteria is largely unknown. Here we analyse the mechanism of partition-associated incompatibility for plasmid pB171, a phenotype associated with all known plasmid-encoded centromere loci. An R1 plasmid carrying par2 from plasmid pB171 was destabilized by the presence of an F plasmid carrying parC1, parC2 or the entire par2 locus of pB171. Strikingly, cytological double-labelling experiments revealed no evidence of long-lived pairing of plasmids. Instead, pure R1 and F foci were positioned along the length of the cell, and in a random order. Thus, our results raise the possibility that partition-mediated plasmid incompatibility is not caused by pairing of heterologous plasmids but instead by random positioning of pure plasmid clusters along the long axis of the cell. The strength of the incompatibility was correlated with the capability of the plasmids to compete for the mid-cell position.