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BioEssays : News and Reviews in... Sep 2021Plasmids are a major type of mobile genetic elements (MGEs) that mediate horizontal gene transfer. The stable maintenance of plasmids plays a critical role in the...
Plasmids are a major type of mobile genetic elements (MGEs) that mediate horizontal gene transfer. The stable maintenance of plasmids plays a critical role in the functions and survival for microbial populations. However, predicting and controlling plasmid persistence and abundance in complex microbial communities remain challenging. Computationally, this challenge arises from the combinatorial explosion associated with the conventional modeling framework. Recently, a plasmid-centric framework (PCF) has been developed to overcome this computational bottleneck. This framework enables the derivation of a simple metric, the persistence potential, to predict plasmid persistence and abundance. Here, we discuss how PCF can be extended to account for plasmid interactions. We also discuss how such model-guided predictions of plasmid fates can benefit from the development of new experimental tools and data-driven computational methods.
Topics: Gene Transfer, Horizontal; Microbiota; Plasmids
PubMed: 34278591
DOI: 10.1002/bies.202100084 -
Plasmid Jan 2021Conjugative, broad host-range plasmids of the L/M complex have been associated with antibiotic resistance since the 1970s. They are found in Gram-negative bacterial...
Conjugative, broad host-range plasmids of the L/M complex have been associated with antibiotic resistance since the 1970s. They are found in Gram-negative bacterial genera that cause human infections and persist in hospital environments. It is crucial that these plasmids are typed accurately so that their clinical and global dissemination can be traced in epidemiological studies. The L/M complex has previously been divided into L, M1 and M2 subtypes. However, those types do not encompass all diversity seen in the group. Here, we have examined 148 complete L/M plasmid sequences in order to understand the diversity of the complex and trace the evolution of distinct lineages. The backbone sequence of each plasmid was determined by removing translocatable genetic elements and reversing their effects in silico. The sequence identities of replication regions and complete backbones were then considered for typing. This supported the distinction of L and M plasmids and revealed that there are five L and eight M types, where each type is comprised of further sub-lineages that are distinguished by variation in their backbone and translocatable element content. Regions containing antibiotic resistance genes in L and M sub-lineages have often formed by initial rare insertion events, followed by insertion of other translocatable elements within the inceptive element. As such, islands evolve in situ to contain genes conferring resistance to multiple antibiotics. In some cases, different plasmid sub-lineages have acquired the same or related resistance genes independently. This highlights the importance of these plasmids in acting as vehicles for the dissemination of emerging resistance genes. Materials are provided here for typing plasmids of the L/M complex from complete sequences or draft genomes. This should enable rapid identification of novel types and facilitate tracking the evolution of existing lineages.
Topics: Anti-Bacterial Agents; Drug Resistance, Microbial; Gram-Negative Bacteria; Humans; Plasmids
PubMed: 32781088
DOI: 10.1016/j.plasmid.2020.102528 -
Methods in Molecular Biology (Clifton,... 2020Plasmid conjugation is intimately linked to the transmission of antibiotic resistances, and many naturally occurring plasmids carry antibiotic resistance genes. Here we...
Plasmid conjugation is intimately linked to the transmission of antibiotic resistances, and many naturally occurring plasmids carry antibiotic resistance genes. Here we describe classical methods based on the transmission of antibiotic resistance determinants routinely used to quantify plasmid conjugation under laboratory conditions. Methods described here are suitable for most plasmid incompatibility groups from Proteobacteria and can be readily adapted to other bacterial species.
Topics: Anti-Bacterial Agents; Bacteria; Conjugation, Genetic; Drug Resistance, Microbial; Plasmids
PubMed: 31584156
DOI: 10.1007/978-1-4939-9877-7_6 -
Plasmid May 2017Plasmids play essential roles in bacterial metabolism, evolution, and pathogenesis. The maintenance of plasmids is of great importance both scientifically and... (Review)
Review
Plasmids play essential roles in bacterial metabolism, evolution, and pathogenesis. The maintenance of plasmids is of great importance both scientifically and practically. In this mini-review, I look at the problem from a slightly different point of view and focus on the spatial distribution of high copy number plasmids, for which no active segregation mechanism has been identified. I review several distribution models and summarize the direct and indirect evidence in the literature, including the most recent progress on measuring the spatial distribution of high copy number plasmids using emerging super-resolution fluorescence microscopy. It is concluded that many open questions remain in the field and that in-depth studies on the spatial distribution of plasmids could shed light on the understanding of the maintenance of plasmids in bacteria.
Topics: Bacteria; DNA Copy Number Variations; DNA Replication; DNA, Bacterial; Microscopy, Fluorescence; Multigene Family; Plasmids
PubMed: 28263761
DOI: 10.1016/j.plasmid.2017.02.005 -
Methods in Molecular Biology (Clifton,... 2022Investigations on gene essentiality have important implications in several fields of basic and applied research. A variety of strategies have been developed over the...
Investigations on gene essentiality have important implications in several fields of basic and applied research. A variety of strategies have been developed over the years to identify essential genes. Here, we describe an implemented plasmid shuffling method useful to assess the essentiality of overlapped genes under very stringent conditions. A host strain harboring the chromosomal deletion of the genes of interest is complemented by a thermosensitive plasmid carrying the copy of gene 1, gene 2, and rpsL allele, conferring streptomycin sensitivity to an otherwise resistant strain. A compatible plasmid harboring a different selectable marker and the copy of gene 2 only is transformed into the host strain, resulting in the coexistence of two plasmids. These cells are grown at high temperatures in a medium containing streptomycin. Under such conditions, viable cells are expected to contain only the incoming plasmid and to carry suppressor mutation(s) that bypass the loss of the essential gene 1. The system may thus represent a valuable tool to identify interactions between essential proteins and cell pathways.
Topics: Alleles; Genes, Essential; Mutation; Plasmids; Streptomycin; Suppression, Genetic
PubMed: 36151490
DOI: 10.1007/978-1-0716-2581-1_3 -
BMC Biotechnology Mar 2021The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning...
BACKGROUND
The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes.
RESULTS
We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated.
CONCLUSIONS
Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.
Topics: Cloning, Molecular; DNA Primers; Escherichia coli; Genetic Vectors; Plasmids; Polymerase Chain Reaction
PubMed: 33722223
DOI: 10.1186/s12896-021-00679-6 -
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 -
Nature Reviews. Microbiology Aug 2023
Topics: Archaea; Plasmids
PubMed: 37322123
DOI: 10.1038/s41579-023-00927-w -
Plasmid Mar 2019The four plasmids in E. coli 39R861 have been used as size standards for closed-circular plasmid DNA since the 1980s. However, their sizes had only been estimated. Here,...
The four plasmids in E. coli 39R861 have been used as size standards for closed-circular plasmid DNA since the 1980s. However, their sizes had only been estimated. Here, the complete sequence of the 61.3 kb cryptic FII plasmid p39R861-3 is reported, and the sizes of p39R861-4, pSa and NTP168 were determined to be 155.8, 40.1 and 6.8 kb, respectively. An improved size standard, 39R861+, was created by adding two sequenced, compatible plasmids to 39R861 via conjugation. The 94.8 kb conjugative B/O plasmid p838B-R mobilised the cryptic, 2.0 kb pBuzz. The six plasmids in 39R861+ are all stably maintained.
Topics: Base Sequence; DNA, Bacterial; Plasmids
PubMed: 30738800
DOI: 10.1016/j.plasmid.2019.01.002 -
Current Opinion in Microbiology Apr 2024Horizontal transfer of plasmids by conjugation is a fundamental mechanism driving the widespread dissemination of drug resistance among bacterial populations. The... (Review)
Review
Horizontal transfer of plasmids by conjugation is a fundamental mechanism driving the widespread dissemination of drug resistance among bacterial populations. The successful colonization of a new host cell necessitates the plasmid to navigate through a series of sequential steps, each dependent on specific plasmid or host factors. This review explores recent advancements in comprehending the cellular and molecular mechanisms that govern plasmid transmission, establishment, and long-term maintenance. Adopting a plasmid-centric perspective, we describe the critical steps and bottlenecks in the plasmid's journey toward a new host cell, encompassing exploration and contact initiation, invasion, establishment and control, and assimilation.
Topics: Conjugation, Genetic; Plasmids; Bacteria
PubMed: 38432159
DOI: 10.1016/j.mib.2024.102449