Plasmid Multimers Research Articles

Analytical and Functional Characterization of Plasmid DNA Topological Forms and Multimers.

Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (∼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.

Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column.

Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column's physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.

Regional dissemination of carbapenem-resistant Enterobacteriaceae accompanying with enhanced resistance in Northern Osaka, Japan

With the rapid spread of multidrug-resistant bacteria, carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide as a major concern because of limited treatment options. Carbapenem resistance is mainly due to carbapenem-ase, a carbapenem-degrading enzyme, which is mainly encoded on a plasmid to spread across bacterial species. However, there have been only small-scale attempts to determine the similarities or accommodations of the plasmids disseminating regionwide. We analysed the 230 CRE isolates carrying blaIMP from 43 medical facilities in the northern Osaka area focusing on the plasmids, the main carriers of the drug resistance genes. Combination of whole genome sequencing and Southern blotting revealed the predominant dissemination of blaIMP-6 by the pKPI-6 plasmid among genetically distinct isolates, as well as the emergences of derivatives that acquired various advantages. We iden-tified heteroresistance likely causing stealth transmissions, which was generated by the transcriptional regu-lation of blaIMP-6, stabilization of blaIMP-6 through chromosomal integration, enhanced carbapenem resistance through plasmid multimerization, or broadened antimicrobial resistance due to a single point mutation in blaIMP-6. In this article, I dis-cussed the mechanisms of regional spread of CRE and enhancement of carbapenem resistance providing the insights to prevent their disseminations.

Enhanced Carbapenem Resistance through Multimerization of Plasmids Carrying Carbapenemase Genes.

ABSTRACTThe worldwide dissemination of carbapenem-resistant Enterobacteriaceae (CRE) poses a critical human health issue by limiting the range of antibiotics that are usable in the treatment of common bacterial infections. Along with CRE, carbapenem heteroresistance has disseminated worldwide, which is described as different levels of carbapenem resistance within a seemingly isogenic bacterial population. Unstable carbapenem resistance will likely lead to unexpected treatment failure due to the enhanced resistance after initiation of treatment, contradicting antimicrobial susceptibility test results. Porin mutation and tandem amplification of the carbapenemase gene have been reported as mechanisms underlying enhanced carbapenem resistance. In this study, we identified multimerization of plasmids carrying carbapenemase genes, by using Southern blotting, whole-genome sequencing, and quantitative PCR (qPCR) analysis for the CRE isolates obtained in our previous surveillance in Osaka, Japan. Plasmids harboring a carbapenemase gene were multimerized by recA, likely through recombination at two consecutive sets of transposase genes of the IS91 family, thereby producing various plasmids of discrete sizes in a single bacterial cell of an Escherichia coli isolate. This multimerization resulted in increased copy numbers of carbapenemase genes, leading to enhanced gene transcription as well as carbapenem resistance. Prior exposure to meropenem further increased the copy number of carbapenemase genes, readily resulting in enhancement of carbapenem resistance. This mechanism may lead to clinical treatment failure by sifting antimicrobial resistance after the treatment initiation.

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase.

To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35h. The specific activity oftyrosine phenol lyase reached 1493U/g dcw, while the volumetric activity increased from 2943 to 14,408U/L for L-DOPA biosynthesis. The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for L-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System, Is Mobilizable among Enterobacteria.

Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl2) nor temperature shift which could induce plasmid DNA uptake.

PKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus.

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6.

This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer.

Genetic analysis of maintenance of pEC156, a naturally occurring Escherichia coli plasmid that carries genes of the EcoVIII restriction–modification system

In the present study the role of the mechanisms responsible for maintenance of a natural plasmid pEC156, that carries genes of the EcoVIII restriction–modification system was investigated. Analysis of this plasmid's genetic content revealed the presence of genetic determinants suggesting two such mechanisms. The first of them relies on site specific recombination utilizing the Xer/cer molecular machinery, while the second involves a restriction–modification system as an addiction module. Our analysis indicated that three factors affect the maintenance of pEC156: (i) a cis-acting cer site involved in resolution of plasmid multimers, (ii) a gene coding for EcoVIII endonuclease, and (iii) plasmid copy number control. The lowest stability was observed with pEC156 derivatives deprived of the cer site. Decreased stability of pEC156 derivatives was also observed in E. coli strains deficient in genes coding for proteins involved in plasmid multimer resolution (XerC, XerD, ArgR and PepA). A similar effect, but to a much lesser extent was observed for the pEC156 derivative without a functional gene coding for EcoVIII endonuclease. Our results indicate that the presence of the cer site is more important for pEC156 stable maintenance than the presence of a functional gene coding for EcoVIII endonuclease. In our work we also tested maintenance of pEC156 possessing a ColE1-type replicon in bacteria belonging to Enterobacteriaceae family. We have found that pEC156 was most stably maintained in Enterobacter cloacae and Klebsiella oxytoca representing coli-type enterobacteria. We have found that in all enterobacteria tested pEC156 derivatives deficient in the cer site were significantly less stably maintained than cer+ variants.

Autonomous and non-autonomous Tn3-family transposons and their role in the evolution of mobile genetic elements.

The Tn3 family of transposons includes diverse elements that encode homologous transposases and contain conserved terminal inverted repeat sequences (IRs). The recent identification of non-autonomous elements, named TIMEs (Tn3-derived Inverted-repeat Miniature Elements), has shed new light on the diversity and evolution of this transposon family. A common feature of TIMEs and other members of this family is their ability to mobilize genomic DNA for transposition as part of composite transposons. These elements significantly influence the structure and properties of plasmids and other mobile genetic elements (MGEs). They may contain and move by transposition (i) plasmid replication systems, (ii) toxin-antitoxin systems and (iii) site-specific recombination modules that can resolve plasmid multimers. Some Tn3 family elements may also transfer large segments of chromosomal DNA into plasmids, which increases the pool of mobile DNA that can take part in horizontal gene transfer.

Transformation of Bacillus subtilis.

Bacillus subtilis has tremendous applications in both academic research and industrial production. However, molecular cloning and transformation of B. subtilis are not as easy as those of Escherichia coli. Here we developed a simple protocol based on super-competent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids generated by prolonged overlap extension-PCR. Super-competent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. This new protocol is simple (e.g., restriction enzyme, phosphatase, and ligase free), fast, and highly efficient (i.e., ~10(7) or ~10(4) transformants per μg of multimeric plasmid or ligated plasmid DNA, respectively). Shuttle vectors for E. coli-B. subtilis are not required.

The topology of plasmid-monomerizing Xer site-specific recombination

Xer site-specific recombination at cer and psi converts bacterial plasmid multimers into monomers so that they can be efficiently segregated to both daughter cells at cell division. Recombination is catalysed by the XerC and XerD recombinases acting at ~30bp core sites, and is regulated by the action of accessory proteins bound to accessory DNA sequences adjacent to the core sites. Recombination normally occurs only between sites in direct repeat in a negatively supercoiled circular DNA molecule, and yields two circular products linked together in a right-handed four-node catenane with antiparallel sites. These and other topological results are explained by a model in which the accessory DNA sequences are interwrapped around the accessory proteins, trapping three negative supercoils so that strand exchange by the XerC and XerD yields the observed four-node catenane.

Cas3 stimulates runaway replication of a ColE1 plasmid in Escherichia coli and antagonises RNaseHI

Cas3 nuclease-helicase is part of CRISPR immunity systems in many bacteria and archaea. In type I CRISPR, Cas3 nuclease degrades invader DNA that has been base-paired to crRNA as an R-loop within a “Cascade” complex. An R-loop is a DNA-RNA hybrid that includes a displaced single-strand DNA loop. Purified Cas3 from E. coli and the archaeon M. thermautrophicus can process R-loops without DNA/RNA sequence specificity and without Cascade. This has potential to affect other aspects of microbial biology that involve R-loops. Regulatory RNAs and host cell proteins modulate replication of ColE1 plasmids (e.g., pUC) from R-loop primers. We observed that Cas3 could override endogenous control of a ColE1 replicon, stimulating uncontrolled (“runaway”) replication and resulting in much higher plasmid yields. This effect was absent when using helicase-defective Cas3 (Cas3K320L) or a non-ColE1 plasmid, and was dependent on RNaseHI. Cas3 also promoted formation of plasmid multimers or concatemers, a phenotype consistent with deregulated ColE1 replication and typical of cells lacking RNaseHI. These effects of Cas3 on ColE1 plasmids are inconsistent with it unwinding R-loops in vivo, at least in this assay. We discuss a model of how Cas3 might be able to regulate RNA molecules in vivo, unless it is targeted to CRISPR defense by Cascade, or kept in check by RecG and RNaseHI.

Characterization of the Streptococcus suis XerS recombinase and its unconventional cleavage of the difSL site

XerC and XerD are members of the tyrosine recombinase family and mediate site-specific recombination that contributes to the stability of circular chromosomes in bacteria by resolving plasmid multimers and chromosome dimers to monomers prior to cell division. Homologues of xerC/xerD genes have been found in many bacteria, and in the lactococci and streptococci, a single recombinase called XerS can perform the functions of XerC and XerD. The xerS gene of Streptococcus suis was cloned, overexpressed and purified as a maltose-binding protein (MBP) fusion. The purified MBP-XerS fusion showed specific DNA-binding activity to both halves of the dif site of S.suis, and covalent protein-DNA complexes were also detected with dif site suicide substrates. These substrates were also cleaved in a specific fashion by MBP-XerS, generating cleavage products separated by an 11-bp spacer region, unlike the traditional 6-8-bp spacer observed in most tyrosine recombinases. Furthermore, xerS mutants of S.suis showed significant growth and morphological changes.

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.

Purification of transcriptionally active multimeric plasmid DNA using zwitterionic detergent and carbonate apatite nano-particles

Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a ‘protein aggregate’ using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the ‘protein aggregate’ for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit.

Complete sequence and analysis of the stability functions of pPSX, a vector that allows stable cloning and expression of Streptomycete genes in Escherichia coli K12

The broad host range, cloning and expression vector pPSX has been completely sequenced and analysed. pPSX is 14.7 kb in length and contains the fusion of two continuous segments of the parental 34 kb, IncW plasmid pR388. pPSX appears to have retained at least three sets of gene/s which contribute in different ways to plasmid stability. The first of these parB, is a known participant in the partitioning of low-copy number plasmids. While the adjoining gene, orf35, has high homology with kfrA, a putative plasmid nucleoid organiser that is often associated with the ParAB family of proteins. The second set of genes; orfs 18, 19, 20, whose exact functions are not clear, have homology to the stability operons of both IncW and IncN plasmids. The third is the resolvase, resP, which may resolve plasmid multimers that can lead to plasmid instability. pPSX is a small, stable cloning vector good for cloning and expression of a wide range of genes, including those from streptomycetes.

Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA·carAB and PepA·cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by ∼235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the –35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers.