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Plasmid May 2023Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid... (Review)
Review
Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs).
Topics: Plasmids; Bacteria; Anti-Bacterial Agents; Gene Transfer, Horizontal
PubMed: 37116631
DOI: 10.1016/j.plasmid.2023.102684 -
Acta Biochimica Et Biophysica Sinica Apr 2021The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established...
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.
Topics: CRISPR-Cas Systems; Escherichia coli; Gene Editing; Genome, Bacterial; Plasmids
PubMed: 33764372
DOI: 10.1093/abbs/gmab036 -
EcoSal Plus Dec 2022In the late 1950s, a number of laboratories took up the study of plasmids once the discovery was made that extrachromosomal antibiotic resistance (R) factors are the... (Review)
Review
In the late 1950s, a number of laboratories took up the study of plasmids once the discovery was made that extrachromosomal antibiotic resistance (R) factors are the responsible agents for the transmissibility of multiple antibiotic resistance among the enterobacteria. The use of incompatibility for the classification of plasmids is now widespread. It seems clear now on the basis of the limited studies to date that the number of incompatibility groups of plasmids will likely be extremely large when one includes plasmids obtained from bacteria that are normal inhabitants of poorly studied natural environments. The presence of both linear chromosomes and linear plasmids is now established for several species. One of the more fascinating developments in plasmid biology was the discovery of linear plasmids in the 1980s. A remarkable feature of the Ti plasmids of Agrobacterium tumefaciens is the presence of two DNA transfer systems. A definitive demonstration that plasmids consisted of duplex DNA came from interspecies conjugal transfer of plasmids followed by separation of plasmid DNA from chromosomal DNA by equilibrium buoyant density centrifugation. The formation of channels for DNA movement and the actual steps involved in DNA transport offer many opportunities for the discovery of proteins with novel activities and for establishing fundamentally new concepts of macromolecular interactions between DNA and specific proteins, membranes, and the peptidoglycan matrix.
Topics: Plasmids; Agrobacterium tumefaciens; Plant Tumor-Inducing Plasmids; Bacteria; DNA, Bacterial
PubMed: 35373578
DOI: 10.1128/ecosalplus.esp-0028-2021 -
Current Biology : CB Oct 2020Plasmids are genetic elements that colonize and replicate in prokaryotic cells (Box 1). They are considered a major driving force of prokaryote evolution, as they can...
Plasmids are genetic elements that colonize and replicate in prokaryotic cells (Box 1). They are considered a major driving force of prokaryote evolution, as they can migrate between populations, making them potent agents of lateral DNA transfer and microbial warfare. The importance of plasmids goes beyond microbial evolution, as they are widely used as vectors for genetic engineering in basic research (e.g., random mutagenesis) as well as applications in biotechnology (e.g., insulin production), synthetic biology, agriculture (e.g., genetic engineering of crops) and medicine (e.g., biopharmaceuticals).
Topics: Biological Evolution; Biotechnology; Genetic Vectors; Mutagenesis; Plasmids; Prokaryotic Cells
PubMed: 33022260
DOI: 10.1016/j.cub.2020.07.003 -
MSphere Apr 2022Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In...
Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In natural systems, bacteria simultaneously possess or encounter multiple MGEs, yet their combined influences on microbial communities are poorly understood. Here, we investigate interactions among MGEs in the marine bacterium Sulfitobacter pontiacus. Two related strains, CB-D and CB-A, each harbor a single prophage. These prophages share high sequence identity with one another and an integration site within the host genome, yet these strains exhibit differences in "spontaneous" prophage induction (SPI) and consequent fitness. To better understand mechanisms underlying variation in SPI between these lysogens, we closed their genomes, which revealed that in addition to harboring different prophage genotypes, CB-A lacks two of the four large, low-copy-number plasmids possessed by CB-D. To assess the relative roles of plasmid content versus prophage genotype on host physiology, a panel of derivative strains varying in MGE content were generated. Characterization of these derivatives revealed a robust link between plasmid content and SPI, regardless of prophage genotype. Strains possessing all four plasmids had undetectable phage in cell-free lysates, while strains lacking either one plasmid (pSpoCB-1) or a combination of two plasmids (pSpoCB-2 and pSpoCB-4) produced high (>10 PFU/mL) phage titers. Homologous plasmid sequences were identified in related bacteria, and plasmid and phage genes were found to be widespread in Oceans metagenomic data sets. This suggests that plasmid-dependent stabilization of prophages may be commonplace throughout the oceans. The consequences of prophage induction on the physiology of microbial populations are varied and include enhanced biofilm formation, conferral of virulence, and increased opportunity for horizontal gene transfer. These traits lead to competitive advantages for lysogenized bacteria and influence bacterial lifestyles in a variety of niches. However, biological controls of "spontaneous" prophage induction, the initiation of phage replication and phage-mediated cell lysis without an overt stressor, are not well understood. In this study, we observed a novel interaction between plasmids and prophages in the marine bacterium Sulfitobacter pontiacus. We found that loss of one or more distinct plasmids-which we show carry genes ubiquitous in the world's oceans-resulted in a marked increase in prophage induction within lysogenized strains. These results demonstrate cross talk between different mobile genetic elements and have implications for our understanding of the lysogenic-lytic switches of prophages found not only in marine environments, but throughout all ecosystems.
Topics: Bacteriophages; Ecosystem; Plasmids; Prophages; Rhodobacteraceae
PubMed: 35311569
DOI: 10.1128/msphere.00930-21 -
PloS One 2020There was an explosion in the amount of commercially available DNA in sequence repositories over the last decade. The number of such plasmids increased from 12,000 to...
There was an explosion in the amount of commercially available DNA in sequence repositories over the last decade. The number of such plasmids increased from 12,000 to over 300,000 among three of the largest repositories: iGEM, Addgene, and DNASU. A challenge in biodesign remains how to use these and other repository-based sequences effectively, correctly, and seamlessly. This work describes an approach to plasmid design where a plasmid is specified as simply a DNA sequence or list of features. The proposed software then finds the most cost-effective combination of synthetic and PCR-prepared repository fragments to build the plasmid via Gibson assembly®. It finds existing DNA sequences in both user-specified and public DNA databases: iGEM, Addgene, and DNASU. Such a software application is introduced and characterized against all post-2005 iGEM composite parts and all Addgene vectors submitted in 2018 and found to reduce costs by 34% versus a purely synthetic plasmid design approach. The described software will improve current plasmid assembly workflows by shortening design times, improving build quality, and reducing costs.
Topics: DNA; Databases, Genetic; Genetic Vectors; Plasmids; Software
PubMed: 31917791
DOI: 10.1371/journal.pone.0223935 -
Clinical and Translational Science May 2021VM202 is a plasmid DNA encoding two isoforms of hepatocyte growth factor (HGF). A previous phase II study in subjects with painful diabetic peripheral neuropathy (DPN)... (Randomized Controlled Trial)
Randomized Controlled Trial
VM202 is a plasmid DNA encoding two isoforms of hepatocyte growth factor (HGF). A previous phase II study in subjects with painful diabetic peripheral neuropathy (DPN) showed significant reductions in pain. A phase III study was conducted to evaluate the safety and efficacy of VM202 in DPN. The trial was conducted in two parts, one for 9 months (DPN 3-1) with 500 subjects (VM202: 336 subjects; and placebo: 164) and a preplanned subset of 101 subjects (VM202: 65 subjects; and placebo: 36) with a noninterventional extension to 12 months (DPN 3-1b). VM202 or placebo was administered to calf muscles on days 0 and 14, and on days 90 and 104. The primary end point in DPN 3-1 was change from baseline in the mean 24-h Numerical Rating Scale (NRS) pain score. In DPN 3-1b, the primary end point was safety, whereas the secondary efficacy end point was change in the mean pain score. VM202 was well-tolerated in both studies without significant adverse events. VM202 failed to meet its efficacy end points in DPN 3-1. In DPN 3-1b, however, VM202 showed significant and clinically meaningful pain reduction versus placebo. Pain reduction in DPN 3-1b was even greater in subjects not receiving gabapentin or pregabalin, confirming an observation noted in the phase II study. In DPN 3-1b, symptomatic relief was maintained for 8 months after the last injection suggesting that VM202 treatment might change disease progression. Despite the perplexing discrepancy between the two studies, the safety and long-lasting pain-relieving effects of VM202 observed in DPN 3-1b warrant another rigorous phase III study. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Current therapies for painful diabetic peripheral neuropathy (DPN) are palliative and do not target the underlying mechanisms. Moreover, symptomatic relief is often limited with existing neuropathic pain drugs. Thus, there is a great medical need for safer and effective treatments for DPN. WHAT QUESTION DID THIS STUDY ADDRESS? Can nonviral gene delivery of hepatocyte growth factor reduce pain in patients with DPN and potentially modify progression of the disorder? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Nonviral gene therapy can be used safely and practically to treat DPN. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? As the first gene medicine to enter advanced clinical trials for the treatment of DPN, this study provides the proof of concept of an entirely new potential approach to the disorder.
Topics: Aged; Diabetic Neuropathies; Double-Blind Method; Female; Genetic Therapy; Hepatocyte Growth Factor; Humans; Injections, Intramuscular; Male; Middle Aged; Neuralgia; Pain Measurement; Placebos; Plasmids; Treatment Outcome
PubMed: 33465273
DOI: 10.1111/cts.12977 -
Nature Communications Apr 2023Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that...
Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids.
Topics: Bacteriophages; Plasmids; Lysogeny; Virus Activation; Mutation
PubMed: 37041135
DOI: 10.1038/s41467-023-37512-x -
Philosophical Transactions of the Royal... Jan 2022Conjugative plasmids play an important role in bacterial evolution by transferring niche-adaptive traits between lineages, thus driving adaptation and genome... (Review)
Review
Conjugative plasmids play an important role in bacterial evolution by transferring niche-adaptive traits between lineages, thus driving adaptation and genome diversification. It is increasingly clear, however, that in addition to this evolutionary role, plasmids also manipulate the expression of a broad range of bacterial phenotypes. In this review, we argue that the effects that plasmids have on the expression of bacterial phenotypes may often represent plasmid adaptations, rather than mere deleterious side effects. We begin by summarizing findings from untargeted omics analyses, which give a picture of the global effects of plasmid acquisition on host cells. Thereafter, because many plasmids are capable of both vertical and horizontal transmission, we distinguish plasmid-mediated phenotypic effects into two main classes based upon their potential fitness benefit to plasmids: (i) those that promote the competitiveness of the host cell in a given niche and thereby increase plasmid vertical transmission, and (ii) those that promote plasmid conjugation and thereby increase plasmid horizontal transmission. Far from being mere vehicles for gene exchange, we propose that plasmids often act as sophisticated genetic parasites capable of manipulating their bacterial hosts for their own benefit. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.
Topics: Adaptation, Physiological; Bacteria; Conjugation, Genetic; Gene Transfer, Horizontal; Phenotype; Plasmids
PubMed: 34839708
DOI: 10.1098/rstb.2020.0461 -
Nucleic Acids Research Jul 2021Engineered plasmids are widely used in the biological sciences. Since many plasmids contain DNA sequences that have been reused and remixed by researchers for decades,...
Engineered plasmids are widely used in the biological sciences. Since many plasmids contain DNA sequences that have been reused and remixed by researchers for decades, annotation of their functional elements is often incomplete. Missing information about the presence, location, or precise identity of a plasmid feature can lead to unintended consequences or failed experiments. Many engineered plasmids contain sequences-such as recombinant DNA from all domains of life, wholly synthetic DNA sequences, and engineered gene expression elements-that are not predicted by microbial genome annotation pipelines. Existing plasmid annotation tools have limited feature libraries and do not detect incomplete fragments of features that are present in many plasmids for historical reasons and may impact their newly designed functions. We created the open source pLannotate web server so users can quickly and comprehensively annotate plasmid features. pLannotate is powered by large databases of genetic parts and proteins. It employs a filtering algorithm to display only the most relevant feature matches and also reports feature fragments. Finally, pLannotate displays a graphical map of the annotated plasmid, explains the provenance of each feature prediction, and allows results to be downloaded in a variety of formats. The webserver for pLannotate is accessible at: http://plannotate.barricklab.org/.
Topics: Amino Acyl-tRNA Synthetases; Bioengineering; Databases, Genetic; Dependovirus; Internet; Molecular Sequence Annotation; Plasmids; Software
PubMed: 34019636
DOI: 10.1093/nar/gkab374