Plasmid Partitioning Research Articles

Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation. The N-terminal domain of a cytoplasmic chemoreceptor encoded next to ppfA is also required for segregation, probably functioning as a ParB analog to control PpfA ATPase activity. An orphan ParA involved in segregating protein clusters therefore uses a similar mechanism to plasmid-segregating ParA/B systems and requires a partner protein for function. Given the large number of genomes that encode orphan ParAs, this may be a common mechanism regulating segregation of proteins and protein complexes.

Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coli can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coli homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60–70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH.

Requirements for Borrelia burgdorferi plasmid maintenance

Borrelia burgdorferi has multiple linear and circular plasmids that are faithfully replicated and partitioned as the bacterium grows and divides. The low copy number of these replicons implies that active partitioning contributes to plasmid stability. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated by the complexity of the genome and the possibility that products may act in trans. Consequently, we have studied the replication–partition region (bbb10–13) of the B. burgdorferi 26kb circular plasmid (cp26) in Escherichia coli, by fusion with a partition-defective miniF plasmid. Our analysis demonstrated that bbb10, bbb11, and bbb13 are required for stable miniF maintenance, whereas bbb12 is dispensable. To validate these results, we attempted to inactivate two of these genes in B. burgdorferi. bbb12 mutants were obtained at a typical frequency, suggesting that the bbb12 product is dispensable for cp26 maintenance as well. We could not directly measure cp26 stability in the bbb12 mutant, because cp26 carries essential genes, and bacteria that have lost cp26 are inviable. Conversely, we were unable to inactivate bbb10 on cp26 of B. burgdorferi. Our results suggest that bbb12 is dispensable for cp26 maintenance, whereas bbb10, bbb11, and bbb13 play crucial roles in that process.

Measuring the Plasmid Copy Number of Single E. Coli by Digital PCR: Are Plasmids Partitioned in Clusters during Cell Division?

The presence of plasmids is important for the behavior of bacteria, as they often carry antibiotic resistance genes and genes involved in virulence of pathogenic strains. Furthermore plasmids are often responsible for horizontal gene transfer. In order to remain in the bacteria the plasmids have to provide complex regulation mechanisms and often inhere active partitioning during cell division. Fingerprints of the resulting plasmid stability can be found in the plasmid copy number distribution. However, these distributions have been rarely reported, since they have been experimentally difficult to obtain.We performed single cell measurements that allow to piece together the plasmid copy number distribution in a bacterial population. We use fluorescent activated cell sorting (FACS) to isolate single bacteria and subsequently utilize the single molecule resolution of digital PCR to determine the plasmid copy number of single cells.Interestingly, the measurement of the plasmid pZE which has a ColE1 like origin of replication showed a rather broad distribution. An unexpectedly high percentage of the cells had very low plasmid copy numbers, despite the high mean copy number of 120. This characteristic however disappeared for a mutant which additionally contains the active partitioning system parABC. The instability of the pZE copy number is unlikely to be the result of random plasmid partitioning; we explain the findings by clustered partitioning, i.e. clusters of plasmids are partitioned to the daughter cells rather than the individual plasmids. Results are high statistical fluctuations of the plasmid copy number. Other groups showed evidence for clustering of plasmids in E. Coli and also showed, that these clusters get dissolved if the plasmids contain parABC genes. Furthermore, theoretical models which employ clustered partitioning fits our data quantitatively.

Single-Molecule Studies of the Bacterial Segregosome Component Spo0J

The ParAB system is a broadly conserved module involved in bacterial plasmid partitioning and chromosome segregation. The core DNA binding component of the system, ParB, binds to consensus parS sites in plasmid or chromosomal DNA but also interacts with adjacent DNA non-sequence-specifically. We have used single molecule techniques, including a new “DNA motion capture” assay, to characterize the interactions of the Bacillus subtilis ParB homolog Spo0J with double-stranded DNA. In contrast to previous models of ParB polymerization on DNA to form a filament, our results suggest that Spo0J forms higher-order complexes by trapping DNA loops.

Dynamic Self-Organization of Bacterial DNA Segregation Machinery in a Cell-Free Reaction

Bacterial plasmids have evolved segregation machineries to partition replicated DNA to the daughter cells at cell division. P1 phage lysogenizes as a low-copy-number plasmid in Escherichia coli. Its partition system consists of three components, a centromere-like region, parS, an adaptor protein that binds to the centromere, ParB and a partition ATPase, ParA. In general, it is known that a ParB/parS partition complex is formed when ParB oligomerize onto the centromere. This large nucleoprotein complex interacts with ParA and is thought to couple ATP hydrolysis to drive the movement and segregation of plasmids to opposite cell-halves. To understand ATP-driven DNA segregation, we reconstituted the P1 plasmid partition system in a cell-free reaction and visualized the spatiotemporal dynamics using TIRF microscopy. We coated a flow cell surface with non-specific DNA to mimic the bacterial nucleoid surface and flowed in the three-component reaction system. We found that ParA coats the artificial nucleoid creating a reference scaffold for plasmid movement. ParA assembles onto the ParB/parS complexes and anchors them onto the ParA-coated nucleoid surface. ParB stimulates ParA disassembly leading to vigorous Brownian motion of the plasmid as the complex loses bridging interactions with the nucleoid. The plasmid detaches from the nucleoid surface leaving a hole devoid of ParA, which is refilled rapidly with ParA rebinding onto the nucleoid. FRAP experiments demonstrate the dynamic exchange of proteins on the nucleoid surface and the partition complex. We present a Par partition model of ParB-stimulated ParA assembly/disassembly triggering dynamic instability leading to plasmid segregation and movement.

Bacterial plasmid partition machinery: a minimalist approach to survival

The accurate segregation or partition of replicated DNA is essential for ensuring stable genome transmission. Partition of bacterial plasmids requires only three elements: a centromere-like DNA site and two proteins, a partition NTPase, and a centromere-binding protein (CBP). Because of this simplicity, partition systems have served as tractable model systems to study the fundamental molecular mechanisms required for DNA segregation at an atomic level. In the last few years, great progress has been made in this endeavor. Surprisingly, these studies have revealed that although the basic partition components are functionally conserved between three types of plasmid partition systems, these systems employ distinct mechanisms of DNA segregation. This review summarizes the molecular insights into plasmid segregation that have been achieved through these recent structural studies.

Analysis of two marine metagenomes reveals the diversity of plasmids in oceanic environments

Plasmid diversity is still poorly understood in pelagic marine environments. Metagenomic approaches have the potential to reveal the genetic diversity of microbes actually present in an environment and the contribution of mobile genetic elements such as plasmids. By searching metagenomic datasets from flow cytometry-sorted coastal California seawater samples dominated by cyanobacteria (SynMeta) and from the Global Ocean Survey (GOS) putative marine plasmid sequences were identified as well as their possible hosts in the same samples. Based on conserved plasmid replication protein sequences predicted from the SynMeta metagenomes, PCR primers were designed for amplification of one plasmid family and used to confirm that metagenomic contigs of this family were derived from plasmids. These results suggest that the majority of plasmids in SynMeta metagenomes were small and cryptic, encoding mostly their own replication proteins. In contrast, probable plasmid sequences identified in the GOS dataset showed more complexity, consistent with a much more diverse microbial population, and included genes involved in plasmid transfer, mobilization, stability and partitioning. Phylogenetic trees were constructed based on common replication protein functional domains and, even within one replication domain family, substantial diversity was found within and between different samples. However, some replication protein domain families appear to be rare in the marine environment.

ParAB-mediated intermolecular association of plasmid P1 parS Sites

The P1 plasmid partition system depends on ParA–ParB proteins acting on centromere-like parS sites for a faithful plasmid segregation during the Escherichia coli cell cycle. In vivo we placed parS into host E. coli chromosome and on a Sop + F plasmid and found that the stability of a P1 plasmid deleted for parA–parB could be partially restored when parB was expressed in trans. In vitro, parS, conjugated to magnetic beads could capture free parS DNA fragment in presence of ParB. In vitro, ParA stimulated ParB-mediated association of intermolecular parS sites in an ATP-dependent manner. However, in the presence of ADP, ParA reduced ParB-mediated pairing to levels below that seen by ParB alone. ParB of P1 pairs the parS sites of plasmids in vivo and fragments in vitro. Our findings support a model whereby ParB complexes P1 plasmids, ParA–ATP stimulates this interaction and ParA–ADP inhibits ParB pairing activity in a parS-independent manner.

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.

Association of a centromere specific nucleosome with the yeast plasmid partitioning locus

The genetically defined point centromeres of budding yeasts and the epigenetically specified regional centromeres of all other eukaryotes harbor a common epigenetic mark in the form of a non-standard nucleosome. Although, the composition of the protein core of the centromere specific nucleosome and the nature of the DNA wrap around it are at present controversial, there is no doubt that this specialized nucleosome harbors a variant of the standard histone H3 (cenH3). The association of cenH3, called Cse4 in Saccharomyces cerevisiae, with the partitioning locus (STB) of the high copy selfish plasmid 2 micron circle that resides in the yeast nucleus and propagates itself stably is intriguing. Recent observations are consistent with Cse4 being a nucleosome component at STB. A common nucleosome identity for the partitioning loci of the chromosomes and the plasmid of yeast support arguments based on evolutionary considerations that the origin of the unusual point centromere of budding yeasts may be traced to the STB locus of an ancestral plasmid.

RepC protein of the octopine‐type Ti plasmid binds to the probable origin of replication within repC and functions only in cis

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere

The Saccharomyces cerevisiae 2-μm plasmid is a multicopy selfish genome that resides in the nucleus. The genetic organization of the plasmid is optimized for stable, high-copy propagation in host-cell populations. The plasmid's partitioning system poaches host factors, including the centromere-specific histone H3-variant Cse4 and the cohesin complex, enabling replicated plasmid copies to segregate equally in a chromosome-coupled fashion. We have characterized the in vivo chromatin topology of the plasmid partitioning locus STB in its Cse4-associated and Cse4-nonassociated states. We find that the occupancy of Cse4 at STB induces positive DNA supercoiling, with a linking difference (ΔLk) contribution estimated between +1 and +2 units. One plausible explanation for this contrary topology is the presence of a specialized Cse4-containing nucleosome with a right-handed DNA writhe at a functional STB, contrasted by a standard histone H3-containing nucleosome with a left-handed DNA writhe at a nonfunctional STB. The similarities between STB and centromere in their nucleosome signature and DNA topology would be consistent with the potential origin of the unusual point centromere of budding yeast chromosomes from the partitioning locus of an ancestral plasmid.

The replication origin of a repABC plasmid

BackgroundrepABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression.ResultsTo identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d.ConclusionsRepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides within the repC gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in promoting this phenotype.

Alterations of RNA maps of IncP-7 plasmid pCAR1 in various Pseudomonas bacteria

RNA transcripts from 199-kb incompatibility P-7 plasmid pCAR1 were analyzed using microarrays with evenly tiled probes with a nine-nucleotide offset in six different Pseudomonas host strains. We re-annotated 12 ORFs based on their RNA maps and on the comparisons with other sequenced IncP-7 plasmids. Ninety six of two hundred ORFs were identified on the IncP-7 backbone related to basic functions of the plasmid (replication, partition and conjugative transfer). More than 90% ORFs on the backbone were transcribed in each host strain, suggesting their importance for the plasmid survival in the host strains. Genes related to partition and conjugative transfer were differentially transcribed host by host, whereas the repA gene encoding replication initiation protein was transcribed at comparable level in each host. As for other plasmid ‘accessory genes’ of pCAR1 encoding carbazole degradation, putative transporter, or transposase were also differentially transcribed among different host strains. These differences may have resulted in distinct behaviors of the plasmid or of its host strain, and RNA maps of pCAR1 give us important information to understand the plasmid behaviors in different environments.

Centromere binding specificity in assembly of the F plasmid partition complex

The segregation of plasmid F of Escherichia coli is highly reliable. The Sop partition locus, responsible for this stable maintenance, is composed of two genes, sopA and sopB and a centromere, sopC, consisting of 12 direct repeats of 43 bp. Each repeat carries a 16-bp inverted repeat motif to which SopB binds to form a nucleoprotein assembly called the partition complex. A database search for sequences closely related to sopC revealed unexpected features that appeared highly conserved. We have investigated the requirements for specific SopB–sopC interactions using a surface plasmon resonance imaging technique. We show that (i) only 10 repeats interact specifically with SopB, (ii) no base outside the 16-bp sopC sites is involved in binding specificity, whereas five bases present in each arm are required for interactions, and (iii) the A-C central bases contribute to binding efficiency by conforming to a need for a purine–pyrimidine dinucleotide. We have refined the SopB–sopC binding pattern by electro-mobility shift assay and found that all 16 bp are necessary for optimal SopB binding. These data and the model we propose, define the basis of the high binding specificity of F partition complex assembly, without which, dispersal of SopB over DNA would result in defective segregation.

Prevalence and Significance of Plasmid Maintenance Functions in the Virulence Plasmids of Pathogenic Bacteria

Virulence functions of pathogenic bacteria are often encoded on large extrachromosomal plasmids. These plasmids are maintained at low copy number to reduce the metabolic burden on their host. Low-copy-number plasmids risk loss during cell division. This is countered by plasmid-encoded systems that ensure that each cell receives at least one plasmid copy. Plasmid replication and recombination can produce plasmid multimers that hinder plasmid segregation. These are removed by multimer resolution systems. Equitable distribution of the resulting monomers to daughter cells is ensured by plasmid partition systems that actively segregate plasmid copies to daughter cells in a process akin to mitosis in higher organisms. Any plasmid-free cells that still arise due to occasional failures of replication, multimer resolution, or partition are eliminated by plasmid-encoded postsegregational killing systems. Here we argue that all of these three systems are essential for the stable maintenance of large low-copy-number plasmids. Thus, they should be found on all large virulence plasmids. Where available, well-annotated sequences of virulence plasmids confirm this. Indeed, virulence plasmids often appear to contain more than one example conforming to each of the three system classes. Since these systems are essential for virulence, they can be regarded as ubiquitous virulence factors. As such, they should be informative in the search for new antibacterial agents and drug targets.

Cse4 (CenH3) Association with the Saccharomyces cerevisiae Plasmid Partitioning Locus in Its Native and Chromosomally Integrated States: Implications in Centromere Evolution

The histone H3 variant Cse4 specifies centromere identity in Saccharomyces cerevisiae by its incorporation into a special nucleosome positioned at CEN DNA and promotes the assembly of the kinetochore complex, which is required for faithful chromosome segregation. Our previous work showed that Cse4 is also associated with the partitioning locus STB of the 2μm circle--a multicopy plasmid that resides in the yeast nucleus and propagates itself stably. Cse4 is essential for the functional assembly of the plasmid partitioning complex, including the recruitment of the yeast cohesin complex at STB. We have located Cse4 association strictly at the origin-proximal subregion of STB. Three of the five directly repeated tandem copies of a 62-bp consensus sequence element constituting this region are necessary and sufficient for the recruitment of Cse4. The association of Cse4 with STB is dependent on Scm3, the loading factor responsible for the incorporation of Cse4 into the CEN nucleosome. A chromosomally integrated copy of STB confers on the integration site the capacity for Cse4 association as well as cohesin assembly. The localization of Cse4 in chromatin digested by micrococcal nuclease is consistent with the potential assembly of one Cse4-containing nucleosome, but not more than two, at STB. The remarkable ability of STB to acquire a very specialized, and strictly regulated, chromosome segregation factor suggests its plausible evolutionary kinship with CEN.

Reconstitution of Bacterial Plasmid Segregation and its Dynamic Pattern Formation

The segregation of genetic material prior to cell division is an essential process for all organisms. In order to maintain faithful inheritance of genetic information, chromosome copies must be partitioned to the daughter cells before cell division. The bacterial P1 plasmid in Escherichia coli is a simple yet useful model system to study the mechanism of chromosome segregation in prokaryotes as they encode only three components that are necessary for plasmid partitioning: a parS DNA sequence on the plasmid acting as the partition site and two partition proteins, ParA and ParB. In vivo studies have shown that the dynamics of ParA, an ATPase, is involved in the positioning of the plasmid copies with ParB bound at parS sites prior to cell division, but how these three components work together to drive plasmid segregation remains unanswered [1]. To better understand the mechanism of P1 plasmid partitioning, we have previously characterized ATP-dependent DNA binding process of ParA [2]. In this work, we reconstituted the three-component partition system in a cell-free reaction. We present the spatio-temporal dynamics of the ParA/B/S partition system observed real-time using TIRF microscopy.[1] Gerdes K, Howard M, Szardenings F, Cell. 2010; 141(6): 927-42.[2] Vecchiarelli AG, Han YW, Tan X, Mizuuchi M, Ghirlando R, Biertumpfel C, Funnell BE, Mizuuchi K., Mol. Microbiol. 2010; 78(1): 78-91.