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.     

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.     

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.     

Promiscuous stimulation of ParF protein polymerization by heterogeneous centromere binding factors

The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.     

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.     

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.     

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.     

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.     

P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.     

Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor.

The P1 plasmid partition system is responsible for segregation of daughter plasmids during division of the Escherichia coli host cell. The P1-encoded elements consist of two essential proteins, ParA and ParB, and the cis-acting incB region. The incB region determines partition-mediated incompatibility and contains the centromere-like site parS. We have isolated and purified the two proteins. ParB binds specifically to the incB region in vitro. DNase I footprinting assays place a strong binding site over the 35-bp parS sequence previously shown to be sufficient for partition when the Par proteins are supplied in trans. A weaker site lies within the incB region in sequences that are important for specifying incompatibility, but are not essential for partition. Gel band retardation assays show that a host factor binds specifically to the incB sequence. The factor strongly stimulates binding of ParB. Cutting the region at a site between the two ParB binding sites yields two fragments that can bind ParB but not host factor. Thus, information for host-factor binding lies in the region determining the specificity of plasmid incompatibility. The roles of parB and the host factor in partition and the specificity of plasmid incompatibility are discussed.     

ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB-parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein-DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein-DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.     

The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells

The P1 ParA protein is an ATPase that recognizes the parA promoter region where it acts to autoregulate the P1 parA–parB operon. The ParB protein is essential for plasmid partition and recognizes the cis -acting partition site parS . The regulatory role of ParA is also essential because a controlled level of ParB protein is critical for partition. However, we show that this regulatory activity is not the only role for ParA in partition. Efficient partition can be achieved without autoregulation as long as Par protein levels are kept within a range of low values. The properties of ParA mutants in these conditions showed that ParA is essential for some critical step in the partition process that is independent of par operon regulation. The putative nucleotide-binding site for the ParA ATPase was identified and disrupted by mutation. The resulting mutant was substantially defective for autoregulation and completely inactive for partition in a system in which the need for autoregulation is abolished. Thus, the ParA nucleotide-binding site appears to be necessary both for the repressor activity of ParA and for some essential step in the partition process itself. We propose that the nucleotide-bound form of the enzyme adopts a configuration that favours binding to the operator, but that the ATPase activity of ParA is required for some energetic step in partition of the plasmid copies to daughter cells.     

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.     

Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1 par Family of Plasmid Partition Elements

The P1, P7, and pMT1 par systems are members of the P1 par family of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes the parS partition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts two parS motifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.