Plasmid Partitioning Research Articles

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

Two birds with one stone: SGI1 can stabilize itself and expel the IncC helper by hijacking the plasmid parABS system.

The SGI1 family integrative mobilizable elements, which are efficient agents in distribution of multidrug resistance in Gammaproteobacteria, have a complex, parasitic relationship with their IncC conjugative helper plasmids. Besides exploiting the transfer apparatus, SGI1 also hijacks IncC plasmid control mechanisms to time its own excision, replication and expression of self-encoded T4SS components, which provides advantages for SGI1 over its helpers in conjugal transfer and stable maintenance. Furthermore, SGI1 destabilizes its helpers in an unknown, replication-dependent way when they are concomitantly present in the same host. Here we report how SGI1 exploits the helper plasmid partitioning system to displace the plasmid and simultaneously increase its own stability. We show that SGI1 carries two copies of sequences mimicking the parS sites of IncC plasmids. These parS-like elements bind the ParB protein encoded by the plasmid and increase SGI1 stability by utilizing the parABS system of the plasmid for its own partitioning, through which SGI1 also destabilizes the helper plasmid. Furthermore, SGI1 expresses a small protein, Sci, which significantly strengthens this plasmid-destabilizing effect, as well as SGI1 maintenance. The plasmid-induced replication of SGI1 results in an increased copy-number of parS-like sequences and Sci expression leading to strong incompatibility with the helper plasmid.

VirB, a transcriptional activator of virulence in Shigella flexneri, uses CTP as a cofactor

VirB is a transcriptional activator of virulence in the gram-negative bacterium Shigella flexneri encoded by the large invasion plasmid, pINV. It counteracts the transcriptional silencing by the nucleoid structuring protein, H-NS. Mutations in virB lead to loss of virulence. Studies suggested that VirB binds to specific DNA sequences, remodels the H-NS nucleoprotein complexes, and changes DNA supercoiling. VirB belongs to the superfamily of ParB proteins which are involved in plasmid and chromosome partitioning often as part of a ParABS system. Like ParB, VirB forms discrete foci in Shigella flexneri cells harbouring pINV. Our results reveal that purified preparations of VirB specifically bind the ribonucleotide CTP and slowly but detectably hydrolyse it with mild stimulation by the virS targeting sequences found on pINV. We show that formation of VirB foci in cells requires a virS site and CTP binding residues in VirB. Curiously, DNA stimulation of clamp closure appears efficient even without a virS sequence in vitro. Specificity for entrapment of virS DNA is however evident at elevated salt concentrations. These findings suggest that VirB acts as a CTP-dependent DNA clamp and indicate that the cellular microenvironment contributes to the accumulation of VirB specifically at virS sites.

The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation.

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

Characterisation and comparative genomics of three new Varanus-associated Borrelia spp. from Indonesia and Australia

BackgroundBorrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia, once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards (Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia.MethodsWe used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus-associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date.ResultsThe genomic architecture of Varanus-associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage.ConclusionsIn this work we present four new genomes from three novel Borrelia, and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii, ‘Candidatus Borrelia undatumii’, and ‘Candidatus Borrelia rubricentralis’ for them.Graphical

Partitioning and migration of antibiotic resistance genes at soil-water-air interface mediated by plasmids

The partitioning and migration of antibiotic resistance genes (ARGs) at the interfaces of soil, water, and air play a critical role in the environmental transmission of antibiotic resistance. This study investigated the partitioning and migration of resistant plasmids as representatives of extracellular-ARGs (eARGs) in artificially constructed soil-water-air systems. Additionally, it quantitatively studied the influence of soil pH, clay mineral content, organic matter content, and simulated rainfall on the migration of eARGs via orthogonal experiments. The findings revealed that the sorption equilibrium between eARGs and soil can be attained within 3 h, following the two-compartment first-order kinetic model. The average partition ratio of eARGs in soil, water, and air is 7:2:1, and soil pH and clay mineral content are identified as the main influencing factors. The proportion of eARGs migrating from soil to water and air is 8.05% and 0.52%, respectively. Correlation and significance analyses showed that soil pH has a significant impact on the soil-water and soil-air mobility of eARGs, while clay content affects the percentage of peaks during migration. Moreover, rainfall exerts a noticeable impact on the timing of peaks during migration. This study provided quantitative insights into the proportion of eARGs in soil, water, and air and elucidated the key factors influencing the partitioning and migration of eARGs from the perspectives of the sorption mechanism.

The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function.

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.

Distinct architectural requirements for the parS centromeric sequence of the pSM19035 plasmid partition machinery.

Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS-DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParBpSM of the ribbon-helix-helix (RHH) family. We show that parSpSM with four or more contiguous ParBpSM-binding sequence repeats is required to assemble a stable ParApSM-ParBpSM complex and efficiently activate the ParApSM-ATPase, stimulating complex disassembly. Disruption of the contiguity of the parSpSM sequence array destabilizes the ParApSM-ParBpSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParApSM-ATP.

Preparing Plasmid DNA from Bacteria.

The most common method for isolating plasmid DNA is derived from an alkaline lysis procedure. The procedure exploits the differential partitioning of plasmid and chromosomal DNA when denatured by alkali and subsequently renatured by neutralization of the medium. The circular covalently closed nature of plasmid DNA allows the denatured DNA strands to quickly find each other and reanneal during the renaturation step. This is not the case for chromosomal DNA, which, upon neutralization, aggregates with denatured proteins through hydrophobic interactions. As a result, plasmid DNA remains in solution and can be easily separated from most of the other macromolecules that coprecipitate. For the subsequent purification step, one can use the silica membrane technology integrated in many commercial kits. This technology exploits the ability of DNA to bind to silica in the presence of chaotropic salts. DNA is retained by a silica-based column, whereas most of the polysaccharides and proteins flow through. After wash steps to eliminate residual contaminants and salts, DNA is selectively eluted under low-salt conditions. A kit-free but relatively more cumbersome alternative to this procedure is the traditional phenol-chloroform extraction method followed by ethanol precipitation. Both methods are detailed here.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

Deletion of a Genetic Region of lp17 Affects Plasmid Copy Number in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease pathogen, is maintained in its enzootic life cycle through complex gene regulatory pathways encoded on its uniquely fragmented genome. This genome consists of over 20 plasmids, and the regulatory mechanisms of plasmid maintenance and replication are largely unknown. The bbd21 gene, encoded on lp17 and a member of the paralogous family 32 proteins, was originally proposed to be a putative parA orthologue involved with plasmid partitioning; however, this function has not been confirmed to date. To determine the role of bbd21 in B. burgdorferi, we utilized targeted gene deletion and discovered bbd21 and bbd22 are co-transcribed. The effects of bbd21 and bbd22 deletion on plasmid copy number and mammalian infectivity were assessed. By qPCR, lp17 copy number did not differ amongst strains during mid-exponential and stationary growth phases. However, after in vitro passaging, the mutant strain demonstrated an 8-fold increase in lp17 copies, suggesting a cumulative defect in plasmid copy number regulation. Additionally, we compared lp17 copy number between in vitro and mammalian host-adapted conditions. Our findings showed 1) lp17 copy number was significantly different between these growth conditions for both the wild type and bbd21-bbd22 deletion mutant and 2) under mammalian host-adapted cultivation, the absence of bbd21-bbd22 resulted in significantly decreased copies of lp17. Murine infection studies using culture and qPCR demonstrated bbd21-bbd22 deletion resulted in a tissue colonization defect, particularly in the heart. Lastly, we showed bbd21 transcription appears to be independent of direct rpoS regulation based on similar expression levels in wild type and ΔrpoS. Altogether, our findings indicate the bbd21-bbd22 genetic region is involved with regulation of lp17 plasmid copy number. Furthermore, we propose the possibility that lp17 plasmid copy number is important for microbial pathogenesis by the Lyme disease spirochete.

Functional activity changes in ParB protein by a KCK-tag

Faithful chromosome segregation during cell division is fundamental for all living organisms. The bacterial parABS partitioning system is central to chromosome segregation and plasmid partitioning. The Bacillus subtilis ParB (BsParB) bacterial DNA-binding protein specifically recognizes parS DNA sites near the replication origin. BsParB spreads 10-20 kb from parS sites flanking the region, promoting recruitment of additional BsParB molecules to form higher-order nucleoprotein complexes essential for faithful chromosome segregation.

Single-molecule studies of the role of CTP in the action of ParB protein

ParB protein is a component of ParABS system and plays an essential role in plasmid partitioning and bacterial chromosome segregation. It specifically recognizes short parS DNA sequences and spreads to vicinity regions. Recent discoveries indicate that ParB proteins are novel enzymes that bind and hydrolyze cytidine triphosphate (CTP) in their actions, but their working mechanisms are still elusive. We aimed to reveal how the presence of CTP affects the function of Bacillus subtilis ParB (BsParB or Spo0J) protein by utilizing single-molecule flow-stretching assays.

A role for the last C-terminal helix of the F plasmid segregating protein SopA in nucleoid binding and plasmid maintenance

The rapid emergence and spread of antibiotic resistance is a growing global burden. Antibiotic resistance is often associated with large single or low copy number plasmids, which rely upon cytoskeletal proteins for their stable maintenance. While the mechanism of plasmid partitioning has been well established for the R plasmids, the molecular details by which the F plasmid is maintained is only beginning to emerge. The partitioning function of the F plasmid depends upon a ParA/ MinD family of proteins known as SopA. SopA, by virtue of its ATP-dependent non-specific DNA binding activity and association with the bacterial nucleoid, drives the segregation of the F plasmid into the daughter cells. This function further depends upon the stimulation of the ATPase activity of SopA by the SopBC complex. Here, we report that several residues in the last C-terminal helix in SopA play a crucial but distinct role in SopA function and plasmid maintenance. While the deletion of the last five residues in SopA does not affect its ability to bind the nucleoid or SopB, they severely affect the plasmid partitioning function. Further, we show that while mutations in certain polar residues in the C-terminal helix only mildly affect its localisation to the nucleoid, others cause defects in nsDNA binding and disrupt plasmid maintenance functions.

Molecular Analysis of Psk1 Par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids

Molecular Analysis of Psk1 Par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids

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.

Mathematical Models of Plasmid Population Dynamics.

With plasmid-mediated antibiotic resistance thriving and threatening to become a serious public health problem, it is paramount to increase our understanding of the forces that enable the spread and maintenance of drug resistance genes encoded in mobile genetic elements. The relevance of plasmids as vehicles for the dissemination of antibiotic resistance genes, in addition to the extensive use of plasmid-derived vectors for biotechnological and industrial purposes, has promoted the in-depth study of the molecular mechanisms controlling multiple aspects of a plasmids’ life cycle. This body of experimental work has been paralleled by the development of a wealth of mathematical models aimed at understanding the interplay between transmission, replication, and segregation, as well as their consequences in the ecological and evolutionary dynamics of plasmid-bearing bacterial populations. In this review, we discuss theoretical models of plasmid dynamics that span from the molecular mechanisms of plasmid partition and copy-number control occurring at a cellular level, to their consequences in the population dynamics of complex microbial communities. We conclude by discussing future directions for this exciting research topic.

Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems.

Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEG–amine) or conjugation with positively charged amino acids (PEG–lysine, PEG–arginine, and PEG–histidine) were studied to increase the selectivity of PEG–dextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEG–dextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination.

Three ParA Dimers Cooperatively Assemble on Type Ia Partition Promoters.

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by partition systems composed of two proteins, ParA and ParB, and a centromere site. Auto-regulation of Par operon expression is important for efficient partitioning and is primarily mediated by ParA for type Ia plasmid partition systems. For the F-plasmid, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface-plasmon-resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. Molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter. We propose that three ParAF dimers bind cooperatively to overlapping motifs, thus covering the promoter region. A similar organization is found on closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning.

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.