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Current Opinion in Microbiology Aug 2017Conjugative plasmids are the keystone of horizontal gene transfer. Metagenomic research and clinical understanding of plasmid transmission beg for a taxonomical approach... (Review)
Review
Conjugative plasmids are the keystone of horizontal gene transfer. Metagenomic research and clinical understanding of plasmid transmission beg for a taxonomical approach to conjugative plasmid classification. Up to now, a meaningful classification was difficult to achieve for lack of appropriate analytical tools. The advent of the genomic era revolutionized the landscape, offering a plethora of plasmid sequences as well as bioinformatic analytical tools. Given the need and the opportunity, in view of the available evidence, a taxonomy of conjugative plasmids is proposed in the hope that it will leverage plasmid studies.
Topics: Conjugation, Genetic; Gene Transfer, Horizontal; Plasmids
PubMed: 28586714
DOI: 10.1016/j.mib.2017.05.005 -
Infection and Immunity Mar 2013Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role...
Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.
Topics: Animals; Bacterial Proteins; Cell Line; Chlamydia trachomatis; Chromosomes, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Mice; Plasmids; Protein Array Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Virulence
PubMed: 23319558
DOI: 10.1128/IAI.01305-12 -
Mycopathologia Jun 2005Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe, which has been sporadically recorded in Italy. A collection...
Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe, which has been sporadically recorded in Italy. A collection of 26 Diaporthe helianthi isolates deriving from different geographic origins was analysed in order to determine the presence of extra-chromosomal genetic determinants and their molecular diversity. Extra-chromosomal bands in total genomic DNAs were identified in every French and the Yugoslavian isolate and in only one Italian isolate, while no Romanian and Argentinean isolates resulted to host any plasmids. When tested for their chemicophysical nature, they were recognised as linear plasmids sized about 2.3 Kb. A more detailed analysis was performed on a plasmid purified from a French isolate (plasmid F). Its intracellular localisation resulted as mitochondrial. Plasmid F was also exploited as a probe in Southern hybridisation experiments, in which it recognised only plasmids present in the genomes of French and Yugoslavian isolates (countries were the disease has a heavy incidence) indicating a strong correlation to geographic origin. An RFLP hybridisation analysis performed on genomic DNAs revealed a homogeneous restriction pattern in all French and Yugoslavian isolates, suggesting molecular homology among plasmids present in those isolates.
Topics: Ascomycota; Blotting, Southern; DNA, Fungal; DNA, Mitochondrial; Electrophoresis, Agar Gel; Europe; Helianthus; Plant Diseases; Plasmids; Polymorphism, Restriction Fragment Length
PubMed: 15983747
DOI: 10.1007/s11046-005-1327-0 -
Analytical Biochemistry May 2003We have developed a LabChip-based plasmid assay that runs on the Agilent 2100 Bioanalyzer. The assay determines the sizes and relative concentrations of the multiple... (Comparative Study)
Comparative Study
We have developed a LabChip-based plasmid assay that runs on the Agilent 2100 Bioanalyzer. The assay determines the sizes and relative concentrations of the multiple forms of plasmid samples. Twelve samples can be analyzed on each chip in an automated run lasting approximately 30min. By using a supercoiled DNA sizing standard of 2-16kb, the size of the analyzed plasmid can be determined. The resulting MW has a relative standard deviation (CV) <5% and error <5%. Plasmids from 2-8kb can be separated with resolution better than 1kb. Topological isoforms in a plasmid sample can also be separated. However, due to differential staining, the heterogeneity of plasmid samples can only be measured if the signal of each isomer peak can be calibrated with pure standards for every isomer form. For a typical plasmid preparation which predominately is in the supercoiled form, the normalized corrected peak area for the supercoiled form correlates with the plasmid concentration in a broad range of 1-100ng/microl. The measurement is semiquantitative with a CV lower than 20%. A number of applications of this assay on a Labchip will be shown.
Topics: Chemistry Techniques, Analytical; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Superhelical; Electrophoresis; Molecular Weight; Plasmids; Reproducibility of Results; Software; Substrate Specificity; Time Factors
PubMed: 12694731
DOI: 10.1016/s0003-2697(03)00037-x -
Advanced Drug Delivery Reviews Apr 2005While a vast array of liposomes, peptides, and molecular conjugates have been evaluated for nonviral gene transfer, the entity containing the actual gene itself is... (Review)
Review
While a vast array of liposomes, peptides, and molecular conjugates have been evaluated for nonviral gene transfer, the entity containing the actual gene itself is almost always a plasmid. The layout of most plasmid DNA (pDNA) vectors is usually quite simple, consisting of a promoter, transgene, polyadenylation signal, and a backbone that permits propagation of the plasmid in bacteria. Additional sequence elements and modifications can be incorporated to influence the stability of gene expression and retention of the pDNA molecule in a given tissue. This review describes the different choices that can be made when designing a pDNA vector for transient, sustained, or regulated expression. The choice of promoter is a major determinant governing the kinetics of expression, but other factors, such as CpG content and the topological form of the pDNA are also influential. Vectors can also be designed to respond to the local environment of a given cell or tissue, or engineered to respond to a small molecule drug.
Topics: Animals; Gene Expression Regulation; Humans; Plasmids; Transgenes
PubMed: 15757760
DOI: 10.1016/j.addr.2004.12.009 -
Biotechnology and Bioengineering Sep 2011Application of plasmid DNA as pharmaceutical to be used in gene therapy and vaccination has been investigated intensively in recent years. To be able to provide...
Application of plasmid DNA as pharmaceutical to be used in gene therapy and vaccination has been investigated intensively in recent years. To be able to provide sufficient material that is in accordance with quality of pharmaceutical grade it is mandatory to gain comprehensive process knowledge which is even requested by regulatory agencies. Regarding plasmid DNA production the specific growth rate has been identified as one of the key parameters. The reduction of specific growth rate results in an increase of plasmid DNA formation. However, quantitative explanations that allow for efficient process development and design are still missing. The presented study proposes a model that clearly demonstrates the relationship between specific growth rate and plasmid formation due to identification of the specific plasmid production rate as relevant key parameter. In addition the model is proved to serve as a useful tool in process development and design.
Topics: Biotechnology; Cell Culture Techniques; DNA; Escherichia coli; Genetic Therapy; Models, Biological; Plasmids; Temperature; Vaccination
PubMed: 21437880
DOI: 10.1002/bit.23138 -
Environmental Microbiology Dec 2019Although plasmids play an important role in biological evolution, the number of plasmid families well-characterized in terms of geographical distribution and evolution...
Although plasmids play an important role in biological evolution, the number of plasmid families well-characterized in terms of geographical distribution and evolution remains limited, especially in archaea. Here, we describe the first systematic study of an archaeal plasmid family, the pT26-2 plasmid family. The in-depth analysis of the distribution, biogeography and host-plasmid co-evolution patterns of 26 integrated and 3 extrachromosomal plasmids of this plasmid family shows that they are widespread in Thermococcales and Methanococcales isolated from around the globe but are restricted to these two orders. All members of the family share seven core genes but employ different integration and replication strategies. Phylogenetic analysis of the core genes and CRISPR spacer distribution suggests that plasmids of the pT26-2 family evolved with their hosts independently in Thermococcales and Methanococcales, despite these hosts exhibiting similar geographic distribution. Remarkably, core genes are conserved even in integrated plasmids that have lost replication genes and/or replication origins suggesting that they may be beneficial for their hosts. We hypothesize that the core proteins encode for a novel type of DNA/protein transfer mechanism, explaining the widespread oceanic distribution of the pT26-2 plasmid family.
Topics: Archaea; Evolution, Molecular; Phylogeny; Plasmids
PubMed: 31503394
DOI: 10.1111/1462-2920.14800 -
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 -
BMC Bioinformatics Apr 2018In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological...
BACKGROUND
In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological conditions. Such intrinsically disordered proteins (IDPs) and intrinsically disordered (protein) regions (IDRs) are involved in essential cell processes through two basic mechanisms: the entropic chain mechanism which is responsible for rapid fluctuations among many alternative conformations, and molecular recognition via short recognition elements that bind to other molecules. IDPs possess a high adaptive potential and there is special interest in investigating their involvement in organism evolution.
RESULTS
We analyzed 2554 Bacterial and 139 Archaeal proteomes, with a total of 8,455,194 proteins for disorder content and its implications for adaptation of organisms, using three disorder predictors and three measures. Along with other findings, we revealed that for all three predictors and all three measures (1) Bacteria exhibit significantly more disorder than Archaea; (2) plasmid-encoded proteins contain considerably more IDRs than proteins encoded on chromosomes (or whole genomes) in both prokaryote superkingdoms; (3) plasmid proteins are significantly more disordered than chromosomal proteins only in the group of proteins with no COG category assigned; (4) antitoxin proteins in comparison to other proteins, are the most disordered (almost double) in both Bacterial and Archaeal proteomes; (5) plasmidal proteins are more disordered than chromosomal proteins in Bacterial antitoxins and toxin-unclassified proteins, but have almost the same disorder content in toxin proteins.
CONCLUSION
Our results suggest that while disorder content depends on genome and proteome characteristics, it is more influenced by functional engagements than by gene location (on chromosome or plasmid).
Topics: Archaea; Archaeal Proteins; Bacteria; Bacterial Proteins; Chromosomes, Archaeal; Chromosomes, Bacterial; Intrinsically Disordered Proteins; Plasmids; Proteome; Toxins, Biological
PubMed: 29699482
DOI: 10.1186/s12859-018-2158-6 -
MBio Aug 2021Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence...
Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (Tra) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the Tra T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of or , which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of , , , or . Conversely, translocation of PsiB or SSB, but not PsiA, through the Tra T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or "mating" channels, can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.
Topics: Bacterial Proteins; Conjugation, Genetic; DNA, Bacterial; Escherichia coli; F Factor; Plasmids; SOS Response, Genetics; Translocation, Genetic; Type IV Secretion Systems
PubMed: 34253063
DOI: 10.1128/mBio.01629-21