Native Plasmid Research Articles

Methylome and Complete Genome Sequence of Parageobacillus toebii DSM 14590T, a Thermophilic Bacterium.

Here, we present the first complete genome assembly of the thermophilic bacterium Parageobacillus toebii DSM 14590T The P. toebii DSM 14590T genome consists of a 3,270,071-bp circular chromosome and a 52,989-bp native plasmid.

Influence of zinc on CTX-M-1 β-lactamase expression in Escherichia coli

ObjectiveZinc oxide is used to prevent post-weaning diarrhoea in pigs as an alternative to antimicrobial growth promoters. This study aims to determine if the use of zinc oxide selects for extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and affects the expression of blaCTX-M-1 in E. coli. MethodsUsing an in vitro faecal micro-cosmos model, the selective properties of zinc were investigated using an E. coli strain with blaCTX-M-1 encoded by a natural IncI1 resistance plasmid (MG1655/pTF2) and another strain where the same gene was located on the chromosome (MG1655::blaCTX-M-1). The micro-cosmos was seeded with faecal material containing an increasing concentration of zinc (0–8 mM). Outcome measurements consisted of colony-forming units (CFU) of the inoculated ESBL E. coli and naturally occurring coliforms as determined by plate counting on MacConkey with and without 5 mg/L cefotaxime as well as total viable bacteria determined on Luria agar without cefotaxime. Expression of blaCTX-M-1 under the experimental zinc concentrations was determined by quantitative polymerase chain reaction. ResultsThe proportion of MG1655/pTF2 of the total viable bacteria was significantly higher at high zinc concentrations (6 and 8 mM) compared with low concentrations (0–4 mM). The messenger RNA (mRNA) levels of blaCTX-M-1 in the two ESBL strains increased at increasing zinc concentrations and varied with the growth phase. ConclusionThe growth of the inoculated CTX-M-1-encoding E. coli MG1655 strains and naturally occurring coliforms was impacted differently when exposed to zinc oxide. The blaCTX-M-1 mRNA expression levels seemed to increase with increasing zinc concentrations, but varied with growth phase, but not gene location.

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum.

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 103 CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.

Reversal of mcr-1-Mediated Colistin Resistance in Escherichia coli by CRISPR-Cas9 System.

PurposeThe plasmid-borne mobilized colistin resistance gene (mcr-1) was discovered in 2015. Subsequently, the rapid horizontal transfer of mcr-1 gene to diverse bacterial species poses a serious threat to public health, which urgently needs the introduction of novel antimicrobial strategies. Therefore, the purpose of this study is to sensitize bacteria to colistin and reduce the propagation of mcr-1 gene by curing mcr-1-harboring plasmid in Escherichia coli (E. coli) using the CRISPR-Cas9 system.MethodsTwo sgRNAs specific to mcr-1 gene were designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-mcr was transformed into E. coli carrying pUC19-mcr-1 or pHNSHP45, separately. The elimination efficiency in strains was evaluated by PCR and quantitative real-time PCR (qPCR). The antimicrobial susceptibility test was performed using the broth microdilution method.ResultsIn this study, we constructed the high copy number plasmid pUC19-mcr-1 and recombinant plasmid pCas9-m1 or pCas9-m2, which contain 20 nt or 30 nt sgRNA sequences targeted to mcr-1, respectively. PCR and qPCR results showed that mcr-1-harboring plasmids could be efficiently eliminated, and there was no significant correlation between sgRNA lengths and curing efficiency. However, when comparing restructured high copy number plasmid (pUC19-mcr-1) to natural resistance plasmid (pHNSHP45) in eliminating efficiency, we found that the content of plasmid backbone had an influence on efficiency. Furthermore, the conjugation assays verified that the engineered CRISPR-Cas9 system in bacteria or in bacteria genome can protect the recipient from plasmid-borne mcr-1 transfer via conjugation. Additionally, sequence analysis showed that three different types of defects in CRISPR-Cas9 system lead to escape mutants.ConclusionWe presented a method that only one plasmid-mediated CRISPR-Cas9 system can be used to efficiently resensitize E. coli to colistin. Moreover, this system provided a great potentiality to counteract the propagation of mcr-1 among bacterial pathogens.

Blind killing of both male and female Drosophila embryos by a natural variant of the endosymbiotic bacterium Spiroplasma poulsonii.

Spiroplasma poulsonii is a vertically transmitted endosymbiont of Drosophila melanogaster that causes male‐killing, that is the death of infected male embryos during embryogenesis. Here, we report a natural variant of S. poulsonii that is efficiently vertically transmitted yet does not selectively kill males, but kills rather a subset of all embryos regardless of their sex, a phenotype we call ‘blind‐killing’. We show that the natural plasmid of S. poulsonii has an altered structure: Spaid, the gene coding for the male‐killing toxin, is deleted in the blind‐killing strain, confirming its function as a male‐killing factor. Then we further investigate several hypotheses that could explain the sex‐independent toxicity of this new strain on host embryos. As the second non‐male‐killing variant isolated from a male‐killing original population, this new strain raises questions on how male‐killing is maintained or lost in fly populations. As a natural knock‐out of Spaid, which is unachievable yet by genetic engineering approaches, this variant also represents a valuable tool for further investigations on the male‐killing mechanism.

The application of the CRISPR-Cas9 system in Pseudomonas syringae pv. actinidiae.

Introduction. Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world.Aim. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species.Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa.Results. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of S. pyogenes, which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site.Conclusion. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.

Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.

Comparative Analysis of Whole-Genome and Methylome Profiles of a Smooth and a Rough Mycobacterium abscessus Clinical Strain.

Mycobacterium abscessus is a fast growing Mycobacterium species mainly causing skin and respiratory infections in human. M. abscessus is resistant to numerous drugs, which is a major challenge for the treatment. In this study, we have sequenced the genomes of two clinical M. abscessus strains having rough and smooth morphology, using the single molecule real-time and Illumina HiSeq sequencing technology. In addition, we reported the first comparative methylome profiles of a rough and a smooth M. abscessus clinical strains. The number of N4-methylcytosine (4mC) and N6-methyladenine (6mA) modified bases obtained from smooth phenotype were two-fold and 1.6 fold respectively higher than that of rough phenotype. We have also identified 4 distinct novel motifs in two clinical strains and genes encoding antibiotic-modifying/targeting enzymes and genes associated with intracellular survivability having different methylation patterns. To our knowledge, this is the first report about genome-wide methylation profiles of M. abscessus strains and identification of a natural linear plasmid (15 kb) in this critical pathogen harboring methylated bases. The pan-genome analysis of 25 M. abscessus strains including two clinical strains revealed an open pan genome comprises of 7596 gene clusters. Likewise, structural variation analysis revealed that the genome of rough phenotype strain contains more insertions and deletions than the smooth phenotype and that of the reference strain. A total of 391 single nucleotide variations responsible for the non-synonymous mutations were detected in clinical strains compared to the reference genome. The comparative genomic analysis elucidates the genome plasticity in this emerging pathogen. Furthermore, the detection of genome-wide methylation profiles of M. abscessus clinical strains may provide insight into the significant role of DNA methylation in pathogenicity and drug resistance in this opportunistic pathogen.

Natural Plasmids in a Swiss Fermented Milk and Colostrum Product assessed by Microbiome Array

Natural Plasmids in a Swiss Fermented Milk and Colostrum Product assessed by Microbiome Array

WASPS: web-assisted symbolic plasmid synteny server.

MotivationComparative plasmid genome analyses require complex tools, the manipulation of large numbers of sequences and constitute a daunting task for the wet bench experimentalist. Dedicated plasmid databases are sparse, only comprise bacterial plasmids and provide exclusively access to sequence similarity searches.ResultsWe have developed Web-Assisted Symbolic Plasmid Synteny (WASPS), a web service granting protein and DNA sequence similarity searches against a database comprising all completely sequenced natural plasmids from bacterial, archaeal and eukaryal origin. This database pre-calculates orthologous protein clustering and enables WASPS to generate fully resolved plasmid synteny maps in real time using internal and user-provided DNA sequences.Availability and implementationWASPS queries befit all current browsers such as Firefox, Edge or Safari while the best functionality is achieved with Chrome. Internet Explorer is not supported. WASPS is freely accessible at https://archaea.i2bc.paris-saclay.fr/wasps/.Supplementary information Supplementary data are available at Bioinformatics online.

Establishment and application of a CRISPR\u2013Cas12a assisted genome-editing system in Zymomonas mobilis

BackgroundEfficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking.ResultsUsing Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed.ConclusionsThis study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

Identification of Genes Essential for Antibiotic-Induced Up-Regulation of Plasmid-Transfer-Genes in Cephalosporin Resistant Escherichia coli.

Bacterial conjugation is one of the most important mechanisms for spread of antibiotic resistance among bacteria. We have previously demonstrated that cefotaxime (CTX) exposure up-regulates expression of Type-IV conjugation transfer genes, and that this leads to increased transfer of a blaCTX–M–1 encoding IncI1 resistance plasmid pTF2 in Escherichia coli. To elucidate the underlying mechanisms, a search for genes that are essential for the up-regulated expression of the transfer (tra) genes in the presence of CTX was undertaken. We constructed a reporter gene-fusion strain MG1655/pTF2 ΔtraF:lacZ where the promoter region of the traF-gene of the plasmid pTF2 was fused with a lacZ on the native plasmid. Random mutagenesis mediated by Tn5 transposon was carried out in the strain, and seven genes (rfaH, yhiN, waaP, waaQ, gnd, pgl, and ISEcp1) were identified where insertion prevented CTX-induced up regulation of traF. Site-specific mutagenesis was carried out, and for all seven mutants, gene deletions abolished the CTX induced up-regulation of traF, and the increased conjugation transfer of the plasmid in the presence of CTX was no longer observed. In addition, the deletion of the genes also abolished CTX induced expression of the blaCTX–M–1 gene. Our results suggested that through CTX induced induction of the identified genes, blaCTX–M–1 expression increased, which led to up-regulation of traF and plasmid transfer. These data reveal that a number of chromosomally encoded genes contribute to the antibiotic induced up-regulation of the conjugation machinery of plasmids, and such genes may be future targets to prevent antibiotic induced spread of resistance plasmids.

Evidence for miR-548c-5p regulation of FOXC2 transcription through a distal genomic target site in human podocytes.

Podocytes are highly differentiated epithelial cells outlining the glomerular vessels. FOXC2 is a transcription factor essential for inducing podocyte differentiation, development and maturation, and is considered to be the earliest podocyte marker. miRNA prediction analysis revealed a full-length target site for the primate-specific miR-548c-5p at a genomic region > 8kb upstream of FOXC2. We hypothesised that the transcription rates of FOXC2 during podocyte differentiation might be tuned by miR-548c-5p through this target site. Experiments were performed with cultured human podocytes, transfected with luciferase reporter constructs bearing this target site region within an enhancer element of the native plasmid. The results confirmed a seed region-driven targeting potential by the miRNA, with mimics downregulating and inhibitors enhancing luciferase activity. Introducing mutations into the miRNA target seed region abolished the expected response. In cultured podocytes, FOXC2 mRNA and protein levels responded to miR-548c-5p abundance in a coordinated manner before and after induction of differentiation, with high statistical significance. Ago-ChIP experiments revealed occupancy of the miRNA target site by miRNA/RISC in undifferentiated cells and its release when differentiation is initiated, allowing its interaction with the gene's promoter region to amplify FOXC2 expression, as shown by chromosome conformation capture and qRT-PCR. Moreover, the expression pattern of FOXC2 during podocyte differentiation seems to be affected by miR-548c-5p, as removal of either endogenous or mimic miR-548c-5p results in increased FOXC2 protein levels and cells resembling those undergoing differentiation. Collectively, results indicate a well-orchestrated regulatory model of FOXC2 expression by a remote upstream target site for miR-548c-5p.

A Closed Chemobrionic System as a Biochemical Delivery Platform.

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.

Selection and propagation of IncP conjugative plasmids following long-term anthropogenic metal pollution in river sediments

For a century, the MetalEurop foundry released metals into the river “La Deûle”. Previous work revealed higher microbial diversity in metal impacted sediments, and horizontal gene transfer mediated by conjugative plasmids was suggested to drive the community adaptation to metals. We used an integrative state-of-the-art molecular approach coupling quantitative PCR, conjugation assays, flow cytometry, fluorescence activated cell sorting and 16S rRNA gene amplicon sequencing to investigate the presence of conjugative plasmids and their propagation patterns in sediment microbiomes. We highlighted the existence of a native broad-host range IncP conjugative plasmid population in polluted sediments, confirming their ecological importance for microbial adaptation. However, despite incompatibilities and decreased transfer frequencies with our own alien IncP plasmid, we evidenced that a wide diversity of bacterial members was still prone to uptake the plasmid, indicating that sediment microbial communities are still inclined to receive conjugative plasmids from the same group. We observed that metal pollution favoured exogenous plasmid transfer to specific metal-selected bacteria, which are likely coming from upstream sources (e.g. wastewater treatment plant, farms…). Altogether, our results suggest that MetalEurop sediments are hotspots for gene transfer via plasmids, acting as an “environmental reservoir” for microbes and mobile elements released by human activities.

Sequence Determinants Spanning -10 Motif and Spacer Region Implicated in Unique Ehrlichia chaffeensis Sigma 32-Dependent Promoter Activity of dnaK Gene.

Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium that causes human monocytic ehrlichiosis. Studying Ehrlichia gene regulation is challenge, as this and related rickettsiales lack natural plasmids and mutagenesis experiments are of a limited scope. E. chaffeensis contains only two sigma factors, σ32 and σ70. We previously developed Escherichia coli surrogate system to study transcriptional regulation from RNA polymerase (RNAP) containing Ehrlichia σ32 or σ70. We reported that RNAP binding motifs of E. chaffeensis genes recognized by σ32 or σ70 share extensive homology and that transcription may be initiated by either one of the sigma factors, although transcriptional efficiencies differ. In the current study, we investigated mapping the E. chaffeensis dnaK gene promoter using the pathogen σ32 expressed in E. coli lacking its native σ32. The E. coli surrogate system and our previously described in vitro transcription system aided in defining the unique −10 motif and spacer sequence of the dnaK promoter. We also mapped σ32 amino acids/domains engaged in its promoter regulation in E. chaffeensis. The data reported in this study demonstrate that the −10 and −35 motifs and spacer sequence located between the two motifs of dnaK promoter are critical for the RNAP function. Further, we mapped the importance of all six nucleotide positions of the −10 motif and identified critical determinants within it. In addition, we reported that the lack of C-rich sequence upstream to the −10 motif is unique in driving the pathogen-specific transcription by its σ32 from dnaK gene promoter. This is the first study in defining an E. chaffeensis σ32-dependent promoter and it offers insights about how this and other related rickettsial pathogens regulate stress response genes.

Bacteria from natural populations transfer plasmids mostly towards their kin.

Plasmids play a key role in microbial ecology and evolution, yet the determinants of plasmid transfer rates are poorly understood. Particularly, interactions between donor hosts and potential recipients are understudied. Here, we investigate the importance of genetic similarity between naturally co-occurring Escherichia coli isolates in plasmid transfer. We uncover extensive variability, spanning over five orders of magnitude, in the ability of isolates to donate and receive two different plasmids, R1 and RP4. Overall, transfer is strongly biased towards clone-mates, but not correlated to genetic distance when donors and recipients are not clone-mates. Transfer is limited by the presence of a functional restriction-modification system in recipients, suggesting sharing of strain-specific defence systems contributes to bias towards kin. Such restriction of transfer to kin sets the stage for longer-term coevolutionary interactions leading to mutualism between plasmids and bacterial hosts in natural communities.

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system.

To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

Emergence of plasmid stability under non-selective conditions maintains antibiotic resistance

Plasmid acquisition is an important mechanism of rapid adaptation and niche expansion in prokaryotes. Positive selection for plasmid-coded functions is a major driver of plasmid evolution, while plasmids that do not confer a selective advantage are considered costly and expected to go extinct. Yet, plasmids are ubiquitous in nature, and their persistence remains an evolutionary paradox. Here, we demonstrate that non-mobile plasmids persist over evolutionary timescales without selection for the plasmid function. Evolving a minimal plasmid encoding for antibiotics resistance in Escherichia coli, we discover that plasmid stability emerges in the absence of antibiotics and that plasmid loss is determined by transcription-replication conflicts. We further find that environmental conditions modulate these conflicts and plasmid persistence. Silencing the transcription of the resistance gene results in stable plasmids that become fixed in the population. Evolution of plasmid stability under non-selective conditions provides an evolutionary explanation for the ubiquity of plasmids in nature.

Non-intertwined strands of plasmid DNA contradicts the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although the W/C model is accepted widely, a number of researchers have raised questions against it and proposed alternative structures for DNA. In the present study, the W/C model was examined using plasmid DNA. It was hypothesized that two strands of plasmid DNA will remain intertwined and not separate from each other under denaturing conditions, if it follows the W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by gel electrophoresis. It was observed that addition of NaOH to pUC19 and pBR322 plasmids resulted in new form of DNA with higher electrophoretic mobility in agarose gel. DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was single-stranded and circular, indicating the separation of two strands of pUC19 plasmid. Next, we examined whether hmP19 DNA can re-anneal to form native pUC19 plasmid. It was observed that neutralization of NaOH resulted in the appearance of native pUC19 plasmid in denatured DNA. Native pUC19 was also formed by hmP19 DNA extracted from agarose gel and was found to be digestible with Hind III. Ability to confer ampicillin resistance in transformed Escherichia coli demonstrated the functionality of pUC19 plasmid formed by extracted hmP19 DNA. Reversible separation of two strands of plasmid into single-stranded circular DNA shows that DNA strands are not intertwined with each other and contradicts the W/C model of DNA structure.