Species and incompatibility determination within the P1par family of plasmid partition elements

The P1par family of active plasmid partition systems consists of at least six members, broadly distributed in a variety of plasmid types and bacterial genera. Each encodes two Par proteins and contains a cis-acting parS site. Individual par systems can show distinct species specificities; the proteins from one type cannot function with the parS site of another. P1par-versus-P7par specificity resides within two hexamer BoxB repeats encoded by parS that contact the ParB protein near the carboxy terminus. Here, we examine the species specificity differences between Yersinia pestis pMT1parS and Escherichia coli P1 and P7parS. pMT1parS site specificity could be altered to that of either P1 or P7 by point mutation changes in the BoxB repeats. Just one base change in a single BoxB repeat sometimes sufficed. The BoxB sequence appears to be able to adopt a number of forms that define exclusive interactions with different ParB species. The looped parS structure may facilitate this repertoire of interaction specificities. Different P1par family members have different partition-mediated incompatibility specificities. This property defines whether two related plasmids can coexist in the same cell and is important in promoting the evolution of new plasmid species. BoxB sequence changes that switch species specificity between P1, P7, and pMT1 species switched partition-mediated plasmid incompatibility in concert. Thus, there is a direct mechanistic link between species specificity and partition-mediated incompatibility, and the BoxB-ParB interaction can be regarded as a special mechanism for facilitating plasmid evolution.     

Distribution of centromere-like parS sites in bacteria: insights from comparative genomics

Partitioning of low-copy-number plasmids to daughter cells often depends on ParA and ParB proteins acting on centromere-like parS sites. Similar chromosome-encoded par loci likely also contribute to chromosome segregation. Here, we used bioinformatic approaches to search for chromosomal parS sites in 400 prokaryotic genomes. Although the consensus sequence matrix used to search for parS sites was derived from two gram-positive species, putative parS sites were identified on the chromosomes of 69% of strains from all branches of bacteria. Strains that were not found to contain parS sites clustered among relatively few branches of the prokaryotic evolutionary tree. In the vast majority of cases, parS sites were identified in origin-proximal regions of chromosomes. The widespread conservation of parS sites across diverse bacteria suggests that par loci evolved very early in the evolution of bacterial chromosomes and that the absence of parS, parA, and/or parB in certain strains likely reflects the loss of one of more of these loci much later in evolution. Moreover, the highly conserved origin-proximal position of parS suggests par loci are primarily devoted to regulating processes that involve the origin region of bacterial chromosomes. In species containing multiple chromosomes, the parS sites found on secondary chromosomes diverge significantly from those found on their primary chromosomes, suggesting that chromosome segregation of multipartite genomes requires distinct replicon-specific par loci. Furthermore, parS sites on secondary chromosomes are not well conserved among different species, suggesting that the evolutionary histories of secondary chromosomes are more diverse than those of primary chromosomes.     

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.     

Insight into centromere-binding properties of ParB proteins: a secondary binding motif is essential for bacterial genome maintenance

ParB proteins are one of the three essential components of partition systems that actively segregate bacterial chromosomes and plasmids. In binding to centromere sequences, ParB assembles as nucleoprotein structures called partition complexes. These assemblies are the substrates for the partitioning process that ensures DNA molecules are segregated to both sides of the cell. We recently identified the sopC centromere nucleotides required for binding to the ParB homologue of plasmid F, SopB. This analysis also suggested a role in sopC binding for an arginine residue, R219, located outside the helix-turn-helix (HTH) DNA-binding motif previously shown to be the only determinant for sopC-specific binding. Here, we demonstrated that the R219 residue is critical for SopB binding to sopC during partition. Mutating R219 to alanine or lysine abolished partition by preventing partition complex assembly. Thus, specificity of SopB binding relies on two distinct motifs, an HTH and an arginine residue, which define a split DNA-binding domain larger than previously thought. Bioinformatic analysis over a broad range of chromosomal ParBs generalized our findings with the identification of a non-HTH positively charged residue essential for partition and centromere binding, present in a newly identified highly conserved motif. We propose that ParB proteins possess two DNA-binding motifs that form an extended centromere-binding domain, providing high specificity.     

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.     

Prokaryotic ParA-ParB-parS system links bacterial chromosome segregation with the cell cycle

While the essential role of episomal par loci in plasmid DNA partitioning has long been appreciated, the function of chromosomally encoded par loci is less clear. The chromosomal parA-parB genes are conserved throughout the bacterial kingdom and encode proteins homologous to those of the plasmidic Type I active partitioning systems. The third conserved element, the centromere-like sequence called parS, occurs in several copies in the chromosome. Recent studies show that the ParA-ParB-parS system is a key player of a mitosis-like process ensuring proper intracellular localization of certain chromosomal regions such as oriC domain and their active and directed segregation. Moreover, the chromosomal par systems link chromosome segregation with initiation of DNA replication and the cell cycle.     

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.     

The Internal Organization of Mycobacterial Partition Assembly: Does the DNA Wrap a Protein Core?

Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A “DNA outside, protein inside” mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA.     

Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins

What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back‐and‐forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole‐organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self‐organizing oscillator to create motion suitable for chromosome segregation.     

Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional “leaky” boundaries.     

CTP-controlled liquid–liquid phase separation of ParB

The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance by dividing the genetic material equally between daughter cells during cell division. However, the molecular mechanisms underlying the assembly of the partition complex, representing the core of the ParABS system, are still far from being understood. Here we demonstrate that the partition complex is formed via liquid–liquid phase separation. Assembly of the partition complex is initiated by the formation of oligomeric ParB species, which in turn are regulated by CTP-binding. Phase diagrams and in vivo analysis show how the partition complex can further be spatially regulated by parS. By investigating the phylogenetic variation in phase separation and its regulation by CTP, we find a high degree of evolutionary conservation among distantly related prokaryotes. These results advance the understanding of partition complex formation and regulation in general, by confirming and extending recently proposed models.     

The evidence of large-scale DNA-induced compaction in the mycobacterial chromosomal ParB

The bacterial chromosome trafficking apparatus or the segrosome participates in the mitotic-like segregation of the chromosomes prior to cell division in several bacteria. ParB, which is the parS DNA-binding component of the segrosome, polymerizes on the parS-adjacent chromosome to form a nucleoprotein filament of unknown nature for the segregation function. We combined static light scattering, circular dichroism and small-angle X-ray scattering to present evidence that the apo form of the mycobacterial ParB forms an elongated dimer with intrinsically disordered regions as well as folded domains in solution. A comparison of the solution scattering of the apo and the parS-bound ParBs indicates a rather drastic compaction of the protein upon DNA binding. We propose that this binding-induced conformational transition is priming the ParB for polymerization on the DNA template.     

Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27

Background

In low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism.

Results

We generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization.

Conclusions

We show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1523-3) contains supplementary material, which is available to authorized users.     

A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa

Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parS _null) is viable however it demonstrates the phenotype characteristic for parA _null or parB _null mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parS _null phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parS _null mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation.     

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.     

Specificity switching of the P1 plasmid centromere-like site.

The P1 plasmid partition site acts like a centromere, promoting accurate segregation of copies to daughter cells. A 34 bp segment is essential for partition and binds the plasmid ParB protein. Additional sequences act as specificity elements that direct the choice of copies for partition. They include a second ParB binding site and a site for the host integration host factor protein. Sites lacking one or more of these additional elements are switched to a different specificity. Defined mutants were scored for partition specificity and protein binding. The results suggest that the wild-type site is folded in a specific DNA-protein complex. Disruption of the complex leads to an open configuration which, while still active in partition, has altered recognition specificity.     

Regional Control of Chromosome Segregation in Pseudomonas aeruginosa

Chromosome segregation in bacteria occurs concomitantly with DNA replication, and the duplicated regions containing the replication origin oriC are generally the first to separate and migrate to their final specific location inside the cell. In numerous bacterial species, a three-component partition machinery called the ParABS system is crucial for chromosome segregation. This is the case in the gammaproteobacterium Pseudomonas aeruginosa, where impairing the ParABS system is very detrimental for growth, as it increases the generation time and leads to the formation of anucleate cells and to oriC mispositioning inside the cell. In this study, we investigate in vivo the ParABS system in P. aeruginosa. Using chromatin immuno-precipitation coupled with high throughput sequencing, we show that ParB binds to four parS site located within 15 kb of oriC in vivo, and that this binding promotes the formation of a high order nucleoprotein complex. We show that one parS site is enough to prevent anucleate cell formation, therefore for correct chromosome segregation. By displacing the parS site from its native position on the chromosome, we demonstrate that parS is the first chromosomal locus to be separated upon DNA replication, which indicates that it is the site of force exertion of the segregation process. We identify a region of approximatively 650 kb surrounding oriC in which the parS site must be positioned for chromosome segregation to proceed correctly, and we called it “competence zone” of the parS site. Mutant strains that have undergone specific genetic rearrangements allow us to propose that the distance between oriC and parS defines this “competence zone”. Implications for the control of chromosome segregation in P. aeruginosa are discussed.