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Journal of Global Antimicrobial... Mar 2024This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella...
OBJECTIVES
This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism.
METHODS
We employed whole-genome sequencing to decipher the genetic architecture of pS130-4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests.
RESULTS
pS130-4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both bla and hypervirulence genes. Our conjugation experiments established that pS130-4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10 and 10. Remarkably, pS130-4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130-4's virulence remained intact postconjugation.
CONCLUSION
A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid.
Topics: Animals; Genes, MDR; Klebsiella pneumoniae; Larva; Plasmids
PubMed: 38307249
DOI: 10.1016/j.jgar.2024.01.010 -
Nucleic Acids Research Jul 2023PlasMapper 3.0 is a web server that allows users to generate, edit, annotate and interactively visualize publication quality plasmid maps. Plasmid maps are used to plan,...
PlasMapper 3.0 is a web server that allows users to generate, edit, annotate and interactively visualize publication quality plasmid maps. Plasmid maps are used to plan, design, share and publish critical information about gene cloning experiments. PlasMapper 3.0 is the successor to PlasMapper 2.0 and offers many features found only in commercial plasmid mapping/editing packages. PlasMapper 3.0 allows users to paste or upload plasmid sequences as input or to upload existing plasmid maps from its large database of >2000 pre-annotated plasmids (PlasMapDB). This database can be searched by plasmid names, sequence features, restriction sites, preferred host organisms, and sequence length. PlasMapper 3.0 also supports the annotation of new or never-before-seen plasmids using its own feature database that contains common promoters, terminators, regulatory sequences, replication origins, selectable markers and other features found in most cloning plasmids. PlasMapper 3.0 has several interactive sequence editors/viewers that allow users to select and view plasmid regions, insert genes, modify restriction sites or perform codon optimization. The graphics for PlasMapper 3.0 have also been substantially upgraded. It now offers an interactive, full-color plasmid viewer/editor that allows users to zoom, rotate, re-color, linearize, circularize, edit annotated features and modify plasmid images or labels to improve the esthetic qualities of their plasmid map and textual displays. All the plasmid images and textual displays are downloadable in multiple formats. PlasMapper 3.0 is available online at https://plasmapper.ca.
Topics: Software; User-Computer Interface; Plasmids; Computers; Base Sequence; Internet
PubMed: 37099365
DOI: 10.1093/nar/gkad276 -
Trends in Biochemical Sciences Nov 1998The rolling-circle mechanism of DNA replication is used by small prokaryotic genomes, such as single-stranded phages and plasmids. However, phages and plasmids have... (Review)
Review
The rolling-circle mechanism of DNA replication is used by small prokaryotic genomes, such as single-stranded phages and plasmids. However, phages and plasmids have adapted the rolling-circle mechanism differently to suit their contrasting biological needs. The phi X174 phage uses a monomeric initiator protein catalytically, displays incomplete termination and recycles the initiator protein, in order to mass-produce phage progeny. By contrast, to control replication precisely, the pT181 plasmid uses a dimeric initiator protein stochiometrically, completes termination and inactivates the initiator after each replication cycle. The phi X174 phage and the pT181 plasmid represent paradigmatic adaptations of the rolling-circle mechanism and could provide models for other replicons.
Topics: Bacteriophage phi X 174; Bacteriophages; Catalysis; DNA Replication; Dimerization; Models, Genetic; Plasmids
PubMed: 9852762
DOI: 10.1016/s0968-0004(98)01302-4 -
MBio May 2021Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial...
Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plasmid coexistence was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids. Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful function, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during periods of strong selection for the useful plasmid-encoded function.
Topics: Adaptation, Physiological; Bacteria; Genetic Fitness; Genome, Bacterial; Phenotype; Plasmids; Selection, Genetic
PubMed: 33975933
DOI: 10.1128/mBio.00558-21 -
Thoracic Cancer Nov 2020Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular...
BACKGROUND
Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one-step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10-15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo.
METHODS
The pEGFP-N1-HA plasmid was constructed by one-step PCR and transformation. Cells were transfected with pEGFP-N1-HA and pEGFP-N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA-GFP fusion protein was detected by confocal microscopy.
RESULTS
The pEGFP-N1-HA plasmid was successfully constructed and HA expression in cells.
CONCLUSIONS
Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction.
KEY POINTS
Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids.
Topics: Cloning, Molecular; DNA; Humans; Plasmids; Polymerase Chain Reaction
PubMed: 33015950
DOI: 10.1111/1759-7714.13660 -
Microbial Genomics Jan 2023For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this...
Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical and isolates in Belgium.
For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated. The combination with long-read sequencing offers a potential solution, as it allows hybrid assemblies. Nevertheless, its use in surveillance has so far been limited. This study aimed to demonstrate its added value for AMR surveillance based on a case study of extended-spectrum beta-lactamases (ESBLs). ESBL genes have been reported to occur also on plasmids. To gain insight into the diversity and genetic context of ESBL genes detected in clinical isolates received by the Belgian National Reference Center between 2013 and 2018, 100 ESBL-producing and 31 ESBL-producing were sequenced with MiSeq and a representative selection of 20 and six isolates additionally with MinION technology, allowing hybrid assembly. The gene was found to be responsible for a rapid rise in the ESBL phenotype from 2017. This gene was mostly detected on multi-resistance-carrying IncFII plasmids. Based on clustering, these plasmids were determined to be distinct from the circulating plasmids before 2017. They were spread to different species and within between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing for which only clonal expansion occurred. Matches of up to 99 % to plasmids of various bacterial hosts from all over the world were found, but global alignments indicated that direct or recent ESBL-plasmid transfers did not occur. It is most likely that travellers introduced these in Belgium and subsequently spread them domestically. However, a clear link to a specific country could not be made. Moreover, integration of in the chromosome of two isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in , ESBL genes were only found on plasmids, of which and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical and isolates. The hybrid assembly data allowed us to check the accuracy of plasmid prediction tools. MOB-suite showed the highest accuracy. However, these tools cannot replace the accuracy of long-read and hybrid assemblies. This study illustrates the added value of hybrid assemblies for AMR surveillance and shows that a strategy where even just representative isolates of a collection used for hybrid assemblies could improve international AMR surveillance as it allows plasmid tracking.
Topics: Belgium; beta-Lactamases; Microbial Sensitivity Tests; Plasmids; Shigella; Salmonella
PubMed: 36748573
DOI: 10.1099/mgen.0.000925 -
Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies' MinION device.BMC Bioinformatics Mar 2023Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through...
BACKGROUND
Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.
RESULTS
Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies' MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.
CONCLUSIONS
Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.
Topics: Sequence Analysis, DNA; Nanopores; Plasmids; DNA; High-Throughput Nucleotide Sequencing
PubMed: 36964503
DOI: 10.1186/s12859-023-05226-y -
Nucleic Acids Research Jul 2021Controlled gene expression is fundamental for the study of gene function and our ability to engineer bacteria. However, there is currently no easy-to-use genetics...
Controlled gene expression is fundamental for the study of gene function and our ability to engineer bacteria. However, there is currently no easy-to-use genetics toolbox that enables controlled gene expression in a wide range of diverse species. To facilitate the development of genetics systems in a fast, easy, and standardized manner, we constructed and tested a plasmid assembly toolbox that will enable the identification of well-regulated promoters in many Proteobacteria and potentially beyond. Each plasmid is composed of four categories of genetic parts (i) the origin of replication, (ii) resistance marker, (iii) promoter-regulator and (iv) reporter. The plasmids can be efficiently assembled using ligation-independent cloning, and any gene of interest can be easily inserted in place of the reporter. We tested this toolbox in nine different Proteobacteria and identified regulated promoters with over fifty-fold induction range in eight of these bacteria. We also constructed variant libraries that enabled the identification of promoter-regulators with varied expression levels and increased inducible fold change relative to the original promoter. A selection of over 50 plasmids, which contain all of the toolbox's genetic parts, are available for community use and will enable easy construction and testing of genetics systems in both model and non-model bacteria.
Topics: Bioengineering; Gene Expression Regulation; Plasmids; Promoter Regions, Genetic; Proteobacteria
PubMed: 34125913
DOI: 10.1093/nar/gkab496 -
Microbiology Spectrum Aug 2023Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments....
Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.
Topics: Indian Ocean; Plasmids; Anti-Bacterial Agents; Metagenomics; Metagenome
PubMed: 37395658
DOI: 10.1128/spectrum.00400-23 -
Plasmid May 2017Conjugative plasmids are widespread and play an important role in bacterial evolution by accelerating adaptation through horizontal gene transfer. However, explaining...
Conjugative plasmids are widespread and play an important role in bacterial evolution by accelerating adaptation through horizontal gene transfer. However, explaining the long-term stability of plasmids remains challenging because segregational loss and the costs of plasmid carriage should drive the loss of plasmids though purifying selection. Theoretical and experimental studies suggest two key evolutionary routes to plasmid stability: First, the evolution of high conjugation rates would allow plasmids to survive through horizontal transmission as infectious agents, and second, compensatory evolution to ameliorate the cost of plasmid carriage can weaken purifying selection against plasmids. How these two evolutionary strategies for plasmid stability interact is unclear. Here, we summarise the literature on the evolution of plasmid stability and then use individual based modelling to investigate the evolutionary interplay between the evolution of plasmid conjugation rate and cost amelioration. We find that, individually, both strategies promote plasmid stability, and that they act together to increase the likelihood of plasmid survival. However, due to the inherent costs of increasing conjugation rate, particularly where conjugation is unlikely to be successful, our model predicts that amelioration is the more likely long-term solution to evolving stable bacteria-plasmid associations. Our model therefore suggests that bacteria-plasmid relationships should evolve towards lower plasmid costs that may forestall the evolution of highly conjugative, 'infectious' plasmids.
Topics: Bacteria; Biological Evolution; Chromosomes, Bacterial; Conjugation, Genetic; Gene Expression Regulation, Bacterial; Gene Transfer, Horizontal; Genetic Fitness; Genetic Loci; Models, Statistical; Mutagenesis, Insertional; Plasmids; Selection, Genetic
PubMed: 28461121
DOI: 10.1016/j.plasmid.2017.04.003