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FEMS Microbiology Ecology Mar 2021Plasmids are common in natural bacterial communities, facilitating bacterial evolution via horizontal gene transfer. Bacterial species vary in their proficiency to host...
Plasmids are common in natural bacterial communities, facilitating bacterial evolution via horizontal gene transfer. Bacterial species vary in their proficiency to host plasmids: whereas plasmids are stably maintained in some species regardless of selection for plasmid-encoded genes, in other species, even beneficial plasmids are rapidly lost. It is, however, unclear how this variation in host proficiency affects plasmid persistence in communities. Here, we test this using multispecies bacterial soil communities comprising species varying in their proficiency to host a large conjugative mercury resistance plasmid, pQBR103. The plasmid reached higher community-level abundance where beneficial and when introduced to the community in a more proficient host species. Proficient plasmid host species were also better able to disseminate the plasmid to a wider diversity of host species. These findings suggest that the dynamics of plasmids in natural bacterial communities depend not only upon the plasmid's attributes and the selective environment but also upon the proficiency of their host species.
Topics: Bacteria; Conjugation, Genetic; Gene Transfer, Horizontal; Host Specificity; Plasmids
PubMed: 33580956
DOI: 10.1093/femsec/fiab026 -
Organic & Biomolecular Chemistry Mar 2005It is now common for bacterial infections to resist the preferred antibiotic treatment. In particular, hospital-acquired infections that are refractory to multiple... (Review)
Review
It is now common for bacterial infections to resist the preferred antibiotic treatment. In particular, hospital-acquired infections that are refractory to multiple antibiotics and ultimately result in death of the patient are prevalent. Many of the bacteria causing these infections have become resistant to antibiotics through the process of lateral gene transfer, with the newly acquired genes encoding a variety of resistance-mediating proteins. These foreign genes often enter the bacteria on plasmids, which are small, circular, extrachromosomal pieces of DNA. This plasmid-encoded resistance has been observed for virtually all classes of antibiotics and in a wide variety of Gram-positive and Gram-negative organisms; many antibiotics are no longer effective due to such plasmid-encoded resistance. The systematic removal of these resistance-mediating plasmids from the bacteria would re-sensitize bacteria to standard antibiotics. As such, plasmids offer novel targets that have heretofore been unexploited clinically. This Perspective details the role of plasmids in multi-drug resistant bacteria, the mechanisms used by plasmids to control their replication, and the potential for small molecules to disrupt plasmid replication and re-sensitize bacteria to antibiotics. An emphasis is placed on plasmid replication that is mediated by small counter-transcript RNAs, and the "plasmid addiction" systems that employ toxins and antitoxins.
Topics: Bacteria; Base Sequence; Cell Death; DNA Replication; Drug Resistance, Bacterial; Molecular Sequence Data; Plasmids
PubMed: 15750634
DOI: 10.1039/b500182j -
STAR Protocols Jun 2021Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a...
Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in for mass sequencing. The concept of this protocol could be applied into any species. For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).
Topics: DNA Damage; DNA Mutational Analysis; DNA, Bacterial; Escherichia coli; Mutagenesis; Mutation; Plasmids
PubMed: 33786464
DOI: 10.1016/j.xpro.2021.100399 -
Journal of Pharmaceutical Sciences Nov 2013This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification...
This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.
Topics: Animals; CHO Cells; Cricetinae; Cricetulus; DNA; Genes, Reporter; Green Fluorescent Proteins; Lipids; Plasmids; Transfection
PubMed: 23996350
DOI: 10.1002/jps.23709 -
Endeavour Mar 1993The mechanisms of nonconjugative plasmid replication (in the single cell) and maintenance (at the population level) are of concern to the microbiologist and to the... (Review)
Review
The mechanisms of nonconjugative plasmid replication (in the single cell) and maintenance (at the population level) are of concern to the microbiologist and to the genetic engineer who wishes to exploit their ability to express cloned genes. This article concentrates mainly on Escherichia coli as the host organism and examines the mechanisms by which both naturally occurring and genetically engineered plasmids persist in populations during periods of growth. Additional strategies to ensure high yields of recombinant product are briefly considered.
Topics: Cell Division; DNA Replication; Microscopy, Electron; Phenotype; Plasmids; Replicon
PubMed: 7686097
DOI: 10.1016/0160-9327(93)90007-p -
Biotechnology Letters Nov 2012Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host...
Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host strains during plasmid preparation, we employed E. coli JM109 and TOP10 cells to prepare four plasmids ranging from 4.7 to 16.8 kb in size. Each plasmid was extracted from JM109 and TOP10 cells using an alkaline lysis mini-preparation method. However, when commercial kits were used to extract the same plasmids from JM109 cells, the large molecular weight plasmids substantially degraded, compared with their smaller counterparts. No degradation was observed when the four plasmids were extracted from E. coli TOP10 cells using the same commercial kit. We conclude, therefore, that the performance of E. coli in high quality plasmid preparations can be affected by plasmid size.
Topics: Analysis of Variance; Cell Line; DNA; Electrophoresis, Agar Gel; Escherichia coli; Green Fluorescent Proteins; HEK293 Cells; Humans; Molecular Weight; Plasmids; Recombinant Proteins; Transfection
PubMed: 22782268
DOI: 10.1007/s10529-012-0994-4 -
Advanced Drug Delivery Reviews Jul 2006The encapsulation of DNA into polymeric depot systems can be used to spatially and temporally control DNA release, leading to a sustained, local delivery of therapeutic... (Review)
Review
The encapsulation of DNA into polymeric depot systems can be used to spatially and temporally control DNA release, leading to a sustained, local delivery of therapeutic factors for tissue regeneration. Prior to encapsulation, DNA may be condensed with cationic polymers to decrease particle size, protect DNA from degradation, promote interaction with cell membranes, and facilitate endosomal release via the proton sponge effect. DNA has been encapsulated with either natural or synthetic polymers to form micro- and nanospheres, porous scaffolds and hydrogels for sustained DNA release and the polymer physical and chemical properties have been shown to influence transfection efficiency. Polymeric depot systems have been applied for bone, skin, and nerve regeneration as well as therapeutic angiogenesis, indicating the broad applicability of these systems for tissue engineering.
Topics: Animals; Delayed-Action Preparations; Drug Delivery Systems; Gene Transfer Techniques; Humans; Plasmids; Polymers; Regenerative Medicine; Tissue Engineering
PubMed: 16759734
DOI: 10.1016/j.addr.2006.03.004 -
Biotechnology Progress Nov 2016Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their...
Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid-bearing and plasmid-free cells. The undesired plasmid-free cells grew 30% faster than the desired plasmid-bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid-bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418-1425, 2016.
Topics: Ampicillin; Bioreactors; Escherichia coli; Escherichia coli Proteins; Plasmids
PubMed: 27593226
DOI: 10.1002/btpr.2341 -
Microbial Genomics Oct 2020Bacterial plasmids play a large role in allowing bacteria to adapt to changing environments and can pose a significant risk to human health if they confer virulence and...
Bacterial plasmids play a large role in allowing bacteria to adapt to changing environments and can pose a significant risk to human health if they confer virulence and antimicrobial resistance (AMR). Plasmids differ significantly in the taxonomic breadth of host bacteria in which they can successfully replicate, this is commonly referred to as 'host range' and is usually described in qualitative terms of 'narrow' or 'broad'. Understanding the host range potential of plasmids is of great interest due to their ability to disseminate traits such as AMR through bacterial populations and into human pathogens. We developed the MOB-suite to facilitate characterization of plasmids and introduced a whole-sequence-based classification system based on clustering complete plasmid sequences using Mash distances (https://github.com/phac-nml/mob-suite). We updated the MOB-suite database from 12 091 to 23 671 complete sequences, representing 17 779 unique plasmids. With advances in new algorithms for rapidly calculating average nucleotide identity (ANI), we compared clustering characteristics using two different distance measures - Mash and ANI - and three clustering algorithms on the unique set of plasmids. The plasmid nomenclature is designed to group highly similar plasmids together that are unlikely to have multiple representatives within a single cell. Based on our results, we determined that clusters generated using Mash and complete-linkage clustering at a Mash distance of 0.06 resulted in highly homogeneous clusters while maintaining cluster size. The taxonomic distribution of plasmid biomarker sequences for replication and relaxase typing, in combination with MOB-suite whole-sequence-based clusters have been examined in detail for all high-quality publicly available plasmid sequences. We have incorporated prediction of plasmid replication host range into the MOB-suite based on observed distributions of these sequence features in combination with known plasmid hosts from the literature. Host range is reported as the highest taxonomic rank that covers all of the plasmids which share replicon or relaxase biomarkers or belong to the same MOB-suite cluster code. Reporting host range based on these criteria allows for comparisons of host range between studies and provides information for plasmid surveillance.
Topics: Bacteria; Conjugation, Genetic; Databases, Genetic; Host Specificity; Humans; Molecular Typing; Plasmids
PubMed: 32969786
DOI: 10.1099/mgen.0.000435 -
Plasmid May 2017
Topics: Bacteria; Plasmids
PubMed: 28109681
DOI: 10.1016/j.plasmid.2017.01.002