High Copy Number Plasmid Research Articles

Quantitative Localization Microscopy Reveals a Novel Organization of a High-Copy Number Plasmid

The maintenance of high-copy number plasmids within bacteria had been commonly thought to result from free diffusion and random segregation. Recent microscopy experiments, however, observed high-copy number plasmids clustering into discrete foci, which seemed to contradict this model, and hinted at an undiscovered active mechanism, as often found in low-copy number plasmids. We recently investigated the cellular organization of a ColE1-derivative plasmid in Escherichia coli bacteria using quantitative superresolved microscopy based on single-molecule localization in combination with single-molecule fluorescence in situ hybridization (smFISH). We observed that many of the plasmids aggregated into large clusters, although most of the plasmids were randomly distributed throughout the bacteria, minus an excluded volume about the chromosomal DNA. Our results indicate that neither of the previous models completely encompasses the behavior of high-copy number plasmids. We also found many plasmids within the chromosomal volume, providing further evidence that the nucleoid does not fully exclude DNA and RNA.

A roadmap for gene system development in Clostridium

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius.

Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening.

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%-92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (Mw) of 2.7-6.8 × 106 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.

Quantitative Localization Microscopy Combined with DNA smFISH Reveals New Features of the Organization of High-Copy Number Plasmids in Bacteria

The maintenance of high-copy number (hcn) plasmids within a colony of bacteria has been commonly thought to result from simple free diffusion and random segregation. Recent microscopy experiments, however, observed hcn plasmids clustering into discrete foci, which seemed to contradict this model, and hinted at an undiscovered active mechanism, as often found in low-copy number plasmids. To address this issue, we investigated the cellular organization of hcn plasmids (ColE1-like) in bacteria using quantitative localization microscopy in combination with DNA single-molecule fluorescence in situ hybridization (smFISH). We observed that, although many hcn plasmids aggregated into large clusters, a majority of the plasmids were randomly distributed throughout the bacteria, minus an excluded volume about the chromosomal DNA. Our results indicate that neither of the previous models are complete, and suggest a “mixed distribution model” of randomly diffusing plasmids and large clustered aggregates. We also found plasmids within the chromosomal volume of the cell, suggesting that the nucleoid may not fully exclude plasmids, and that the nucleoid may be more amorphous than previously thought.

Investigation of Partition Mechanism of High-Copy Number Bacterial Plasmids via Replication Incompatibility

Bacterial plasmids play important role in gene transport between cells both vertically and horizontally, and are also versatile tools for gene engineering. Their maintenance of inherence in the host cells is believed to depend on the delicate quantity control through the replication origin and the effective management of partition. Without active partition system, high-copy number plasmids are suggested to be distributed as multiple foci but randomly partitioned during cell division. Lack of the direct evidence, the detail mechanism and dynamics of plasmid location and partition in cell is unknown. Taking the advantage of temporary tolerance on the incompatibility of replication origin, two ColE1 derivative plasmids with different antibiotic resists and repressible replication origin are co-transformed and their distributions and motions are observed at single cell level simultaneously. Based on the nature of competition between plasmids, it also provides the opportunity to actually reveal a single plasmid translocation between multiple foci, which is affected by the nucleoids of the dividing cells. Also, the variance of the inhered number of copies for both of two plasmids from parent to daughter cells provides the insight of partition mechanism.

Employing Peptide Nucleic Acids for Enhanced Super-Resolved Imaging of Bacterial DNA

DNA single-molecule fluorescence in situ hybridization (smFISH) is a powerful technique used to visualize intracellular DNA. However, the technique suffers from a fundamental limitation in the annealing (or hybridization) efficiency of the labeled probes. This is largely due to competition from the complementary DNA strand, which can similarly anneal to the target. With the invention of artificial DNA mimics such as peptide nucleic acids (PNAs), which contain a neutral backbone, the hybridization efficiency and specificity of smFISH can be significantly increased due to the reduced electrostatic repulsion between the PNA probes and DNA target. Using super-resolved localization microscopy with PNA smFISH, we are investigating the spatial organization and maintenance of high-copy number plasmids in bacteria. In the future, this technique may also be fruitful in imaging genetic loci within the chromosome and, because PNA can strand invade dsDNA, live-cell imaging.

Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.

BackgroundLeuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc.ResultsUsing an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α–amylase.ConclusionsThe high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824.

BackgroundClostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum.ResultsBoth systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1).ConclusionsThe codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Our study also revealed a surprising number of errors in the ATCC 824 genome sequence, while at the same time emphasising the need to re-sequence commonly used laboratory strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0410-0) contains supplementary material, which is available to authorized users.

Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD’s copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid’s ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 102–105 cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at −4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed that both primer pairs were acceptable and were predicted to have good performance. Those predictions were in agreement with the in vitro test that gave a single band in the PCR product’s electropherogram and a single peak in DNA amplicon’s melting curve with a Tm value of 79.01 ± 0.11°C for the tdk primer and 81.53 ± 0.29°C for the ori primer. The efficiency of each primer was 1.95 and 1.97, respectively. The calculation result of pCAD’s copy number was 13.1 ± 0.3 copies/cell, showing that pCAD’s low copy number has been determined and confirmed. Meanwhile, it was 576.3 ± 91.9 copies/cell for pJExpress414-sod, in accordance with the hypothesis that pUC ori regulates the high copy number plasmid. In conclusion, the designed primers and qPCR conditions used in this study can be used to determine plasmid copy number for plasmids with pBR322 and pUC ori. The method should be tested further on plasmids harboring other type of ori.

Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

Interactions of Escherichia coli molecular chaperone HtpG with DnaA replication initiator DNA.

The bacterial chaperone high-temperature protein G (HtpG), a member of the Hsp90 protein family, is involved in the protection of cells against a variety of environmental stresses. The ability of HtpG to form complexes with other bacterial proteins, especially those involved in fundamental functions, is indicative of its cellular role. An interaction between HtpG and DnaA, the main initiator of DNA replication, was studied both in vivo, using a bacterial two-hybrid system, and in vitro with a modified pull-down assay and by chemical cross-linking. In vivo, this interaction was demonstrated only when htpG was expressed from a high copy number plasmid. Both in vitro assays confirmed HtpG–DnaA interactions.

Solvent resistance pumps of Pseudomonas putida S12: Applications in 1-naphthol production and biocatalyst engineering

The solvent resistance capacity of Pseudomonas putida S12 was applied by using the organism as a host for biocatalysis and through cloning and expressing solvent resistant pump genes into Escherichia coli. P. putida S12 expressing toluene ortho mononooxygenase (TOM-Green) was used for 1-naphthol production in a water-organic solvent biphasic system. Application of P. putida S12 improved 1-naphthol production per gram cell dry weight by approximately 42% compared to E. coli. Moreover, P. putida S12 enabled the use of a less expensive solvent, decanol, for 1-naphthol production. The solvent resistant pump (srpABC) genes of P. putida S12 were cloned into a solvent sensitive E. coli strain to transfer solvent tolerance. Recombinant strains bearing srpABC genes in either a low-copy number or a high-copy number plasmid grew in the presence of saturated concentration of toluene. Both of the recombinant strains were more tolerant to 1% v/v of toxic solvents, decanol and hexane, reaching similar cell density as the no-solvent control. Reverse-transcriptase analysis revealed that the srpABC genes were transcribed in engineered strains. The results demonstrate successful transfer of the proton-dependent solvent resistance mechanism and suggest that the engineered strain could serve as more robust biocatalysts in media with organic solvents.

Production of β-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae.

BackgroundApocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants—like raspberries and roses—by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid β-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP.ResultsIntegration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of β-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased β-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of β-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase.ConclusionsAn efficient β-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene—the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0273-x) contains supplementary material, which is available to authorized users.

A series of medium and high copy number arabinose-inducible Escherichia coli expression vectors compatible with pBR322 and pACYC184

The original pBAD24 plasmid and the derived lower copy number (the pBAD322 series) expression vectors have been widely used in Escherichia coli, Salmonella enterica, and related bacteria. However, a flexible pBAD expression system has been available only in pMB1 (ColE1) vectors. We report a series of pBAD vectors that replicate using the origin of plasmid RSF1030 that are compatible with pMB1 (ColE1) and p15A (pACYC) vectors. Both high (≥pBAD24) and medium (~pBAD322) copy number plasmids encoding resistance to ampicillin, chloramphenicol, kanamycin, tetracycline, spectinomycin/streptomycin, gentamycin, or trimethoprim are available.

Imaging centromere-based incompatibilities: Insights into the mechanism of incompatibility mediated by low-copy number plasmids

In bacteria, low-copy number plasmids are faithfully segregated at cell division by active partition systems that rely on plasmid-specific centromere sequences. When an identical centromere is present on a second plasmid, faithful partition is impaired causing plasmid loss. Depending on the copy number of the co-resident replicon, several mechanisms have been proposed to account for this centromere-based plasmid incompatibility. To gain further insights into these mechanisms, we analyzed the positioning of the F plasmid in the presence of incompatible low- and high-copy number plasmids carrying the F centromere. Our data are fully compatible with the titration hypothesis when extra-centromeres are present on high-copy number plasmids. Interestingly, our plasmids' localization data revealed that the strong incompatibility phenotype, observed when extra centromeres are present on a partition defective low-copy number plasmid, does not directly result from a partition deficiency as previously proposed. We provide a new and simple hypothesis for explaining the strong incompatibility phenotype based on the timing of replication of low-copy number plasmids.

Promoters inducible by aromatic amino acids and γ-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae.

A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.

Genotoxicity of chemical compounds identification and assessment by yeast cells transformed with GFP reporter constructs regulated by the PLM2 or DIN7 promoter.

Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline-N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.

Tuning constitutive recombinant gene expression in Lactobacillus plantarum

BackgroundLactobacillus plantarum constitutes a well-recognized food-grade system for the expression of recombinant proteins in the field of industrial and medical biotechnology. For applications in vivo or in biotechnological processes, the level of expression of e.g. antigens or enzymes is often critical, as expression levels should be of a certain effectiveness, yet, without putting too much strain to the overall system. The key factors that control gene expression are promoter strength, gene copy number and translation efficiency. In order to estimate the impact of these adjusting screws in L. plantarum CD033, we have tested several constitutive promoters in combination with high and low copy number plasmid backbones and varying space between the Shine-Dalgarno sequence and the start-codon.ResultsBy combining strong promoters, such as transcription elongation factor promoters, isolated from L. plantarum CD033 and L. buchneri CD034, a synthetic promoter, originally derived from L. plantarum WCSF1 and a heterologous promoter derived from L. buchneri CD034 with a high and a low copy number origin of replication we demonstrated various expression levels of the model protein mCherry. All promoters were feasible for protein expression and in all cases, the high copy number origin of replication increased expression twofold. We found that the optimal spacer between the Shine-Dalgarno sequence and the start codon in L. plantarum consists of 8 nucleotides and elongation as well as shortening this sequence gradually down-regulates gene expression.ConclusionsWe have evaluated the effects of a set of gene regulatory tools to fine tune recombinant gene expression in L. plantarum CD033. We have thus, provided potential expression vectors useful for constitutive protein expression in lactic acid bacteria ranging from moderate to strong production levels.

Mechanisms of plasmid segregation: Have multicopy plasmids been overlooked?

Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

Plasmid segregation by a moving ATPase gradient

Unlike the mitotic segregation of eukaryotic sister chromatids, DNA partitioning in bacteria is still not well understood. Bacterial high–copy-number plasmids can be stably maintained by random distribution of their copies during cell division. In contrast, the faithful transmission of low–copy-number plasmids and many chromosomes depends on an active process mediated by conserved, tripartite segregation systems (1). A central component of these machineries is a nucleoside triphosphatase driving the partitioning reaction, which can be classified as an actin-like ATPase (ParM), a tubulin-like GTPase (TubZ), or a Walker-type ATPase (ParA). Systems using an actin or tubulin homolog function by means of a filament-based pushing or pulling mechanism (2, 3). Most low–copy-number plasmids and chromosomes are, however, segregated by Walker-type ATPases. Despite extensive research, it is not yet unambiguously established how this third group of proteins harnesses the energy released during ATP hydrolysis for plasmid movement. Based on previous analyses, two competing models have been put forward. In the filament-pulling model, ParA is assumed to form polymers that move DNA by repeated polymerization/depolymerization cycles. In contrast, the diffusion-ratchet model proposes a concentration gradient of ParA dimers on the nucleoid as the driving force for DNA segregation. In PNAS, Vecchiarelli et al. (4) now provide direct evidence in support of the latter model by fully reconstituting in vitro the segregation system of the Escherichia coli F plasmid.