-
Microbiology (Reading, England) Jun 1997Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction...
Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction enzyme profiling. In contrast, available plasmid sequence data from within the species Chlamydia trachomatis indicate that plasmids from human isolates are highly conserved. To evaluate the nature and extent of plasmid variation, the complete nucleotide sequences were determined for novel plasmids from three diverse non-human chlamydial isolates: pCpA1 from avian Chlamydia psittaci (N352); pCpnE1 from equine Chlamydia pneumoniae (N16); and pMoPn from C. trachomatis mouse pneumonitis. Comparison of the sequence data did not identify an overall biological function for the plasmid but did reveal considerable sequence conservation (> 60%) and a remarkably consistent genomic arrangement comprising eight major ORFs and four 22 bp tandem repeats. The plasmid sequences were close to 7500 nucleotides in length (pCpA1, 7553 bp; pMoPn, 7502 bp) however the equine C. pneumoniae plasmid was smaller (7362 bp) than all other chlamydial plasmids. The reduced size of this plasmid was due to a single large deletion occurring within ORF 1; this potentially generates two smaller ORFs. The disruption of ORF 1 is the only significant variation identified amongst the chlamydial plasmids and could prove important for future vector development studies.
Topics: Amino Acid Sequence; Animals; Chlamydia; Cloning, Molecular; Conserved Sequence; Genome, Bacterial; Humans; Mice; Molecular Sequence Data; Open Reading Frames; Phylogeny; Plasmids; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Sequence Homology, Amino Acid
PubMed: 9202459
DOI: 10.1099/00221287-143-6-1847 -
MicrobiologyOpen Oct 2022Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based...
Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1-based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1-based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.
Topics: Plasmids
PubMed: 36314759
DOI: 10.1002/mbo3.1325 -
Biotechnology Progress Mar 2019It is well established that the structure of plasmid DNA is a strong function of solution ionic conditions due to changes in intramolecular electrostatic interactions...
It is well established that the structure of plasmid DNA is a strong function of solution ionic conditions due to changes in intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. Multivalent cations like spermine and spermidine play a critical role in compacting and controlling the structure of supercoiled DNA in living cells. The objective of this work was to investigate the effects of these polyamines on the ultrafiltration of plasmid DNA, including possible opportunities to use these polycations to enhance the purification of specific plasmid isoforms. Data were obtained using a wide range of spermine and spermidine concentrations to evaluate DNA transmission through Biomax polyethersulfone ultrafiltration membranes. Spermine has a very strong effect on DNA transmission, with the sieving coefficient of the supercoiled plasmid decreasing by more than an order of magnitude upon addition of only 15 μM spermine. A comparable change in DNA transmission required >300 μM of the trivalent spermidine. The polyamines were able to significantly increase the selectivity for the separation of DNA from a model protein, but they were unable to provide a significant increase in the selectivity for separating DNA isoforms under the conditions examined in this study. The results do demonstrate that both spermine and spermidine can be used to control the extent of DNA transmission/purification during ultrafiltration. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2765, 2019.
Topics: DNA; Plasmids; Polyamines; Ultrafiltration
PubMed: 30537363
DOI: 10.1002/btpr.2765 -
Biotechnology and Applied Biochemistry Jun 2003The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment... (Review)
Review
The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment of early clinical results. pDNA is a large molecule and has properties that are similar to those of the contaminating chromosomal DNA. These, combined with the low initial concentration of plasmids in the host cell, provide unique process challenges that require significant upfront design to establish robust manufacturing processes that can also comply with current Good Manufacturing Practice ('cGMP') and produce milligram-to-kilogram quantities of pDNA product. This review describes promising scaleable processes that are currently being assessed for production of therapeutic supercoiled pDNA. Fermentation strategies for improving supercoiled plasmid yield and reducing contaminant concentrations are reviewed, and downstream processes are assessed for their ability to efficiently remove cellular contaminants, separate the supercoiled form of the pDNA from its open circular and linear forms, and prepare the purified drug substance for formulation. Current strategies are presented for developing stable delivery systems, and approaches to quality assurance and quality control are discussed.
Topics: Bioreactors; Drug Delivery Systems; Drug Design; Drug Industry; Genetic Engineering; Genetic Therapy; Pharmaceutical Preparations; Pilot Projects; Plasmids
PubMed: 12683955
DOI: 10.1042/BA20030011 -
Biotechnology and Applied Biochemistry Jun 2003Clinical trials for gene-therapy products began in the 1990s. Currently, most of the 630 completed or on-going trials are in Phase I and II. Because of the safety issues... (Review)
Review
Clinical trials for gene-therapy products began in the 1990s. Currently, most of the 630 completed or on-going trials are in Phase I and II. Because of the safety issues surrounding the use of viral vectors, approx. 25% of the ongoing clinical trials use DNA plasmids to carry the corrective gene (http://www.wiley.co.uk/genmed). The purpose of this perspective (and this issue) is to highlight some of the unique technical challenges and potential solutions associated with the manufacture and expression of a plasmid-based gene-therapy product.
Topics: Drug Delivery Systems; Drug Design; Drug Industry; Genetic Engineering; Genetic Therapy; Pharmaceutical Preparations; Plasmids
PubMed: 12672266
DOI: 10.1042/BA20030033 -
Microbial Genomics Nov 2023Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized...
Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 (), long before the first report of this gene in the literature. In two cases, the gene and the locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the gene was found on a large IncHI1B/IncP-type plasmid, whereas the locus was encoded on a separate chromosomally integrated plasmidic island. The sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.
Topics: Colistin; Salmonella enterica; Lactose; Serogroup; Drug Resistance, Bacterial; Plasmids
PubMed: 38031909
DOI: 10.1099/mgen.0.001149 -
Archives of Microbiology May 2011Whole genome sequences are present-day bonanzas for taxonomists. Comparative genomics provides a promising perspective to reveal the evolutionary relationship between... (Review)
Review
Whole genome sequences are present-day bonanzas for taxonomists. Comparative genomics provides a promising perspective to reveal the evolutionary relationship between organisms, but this strategy is not applicable for extrachromosomal elements due to their high recombination frequencies. Classification of plasmids is based on their compatibility, i.e., the ability to coexist within the same cell. Compatibility testing is a laborious experimental discipline of pairwise comparisons developed for a small set of replicons. Thus, novel approaches are urgently required to deal with the exponentially increasing amount of sequence data. In this minireview, a short overview about the functional role and distribution of plasmids as well as a summary of recent strategies to classify the replicons via phylogenetic analyses is given. Our own work essentially bases on genes of the replication module, i.e., the replicase and two conserved partitioning genes and we exemplified this approach for the four different plasmid types from Alphaproteobacteria. It is suitable for a reliable classification of these replicons and allows in silico predictions about their compatibility. The development of a general classification scheme for plasmids from all microbial lineages will ensure a systematic assessment of the upcoming data flood and help to understand the distribution of extrachromosomal elements.
Topics: Alphaproteobacteria; Conjugation, Genetic; Genomics; Phylogeny; Plasmids; Replicon
PubMed: 21374058
DOI: 10.1007/s00203-011-0686-9 -
Journal of Pharmaceutical Sciences Mar 2000Nonviral, plasmid-based therapeutics are a new class of pharmaceutical agents that offer the potential to cure many diseases that are currently considered untreatable.... (Review)
Review
Nonviral, plasmid-based therapeutics are a new class of pharmaceutical agents that offer the potential to cure many diseases that are currently considered untreatable. While nonviral vectors have shown promise in clinical trials, their physical instability in liquid formulations represents a major barrier to the development of these agents as marketable products. While several different approaches have been used to improve the stability of liquid formulations, it is unclear whether aqueous suspensions can be rendered sufficiently stable to withstand the stresses associated with shipping and storage. Some studies have demonstrated the potential of frozen formulations to be stored for prolonged periods of time, however the potential for phase changes in frozen samples combined with the expense of maintaining the frozen state during shipping has stimulated an interest in developing dehydrated preparations. Although the stresses associated with dehydration are considerable, several studies have reported that sugars are capable of preserving the physical characteristics and transfection activity of nonviral vectors during acute lyophilization stress. This paper discusses the merits and drawbacks of the different approaches to preserving nonviral vectors, and identifies research areas in which more work is needed to develop stable formulations of plasmid-based therapeutics.
Topics: Animals; Drug Stability; Excipients; Humans; Plasmids
PubMed: 10707010
DOI: 10.1002/(SICI)1520-6017(200003)89:3<289::AID-JPS1>3.0.CO;2-N -
International Journal of Nanomedicine 2007We prepared stable homogeneous suspensions with layered double hydroxide (LDH) nanoparticles for in vitro gene delivery tests. The viability of HEK 293T cells in the... (Review)
Review
We prepared stable homogeneous suspensions with layered double hydroxide (LDH) nanoparticles for in vitro gene delivery tests. The viability of HEK 293T cells in the presence of LDH nanoparticles at different concentrations was investigated. This revealed 50% cell viability at 500 microg/mL of LDH nanoparticles that is much higher than 50-100 microg/mL used for the delivery tests. The supercoiled pEF-eGFP plasmid (ca. 6100 base pairs) was mixed with LDH nanoparticle suspensions for anion exchange at a weight ratio of DNA/LDH between 1:25 and 1:100. In vitro experiments show that GFP expression in HEK 293T cells starts in the first day, reaches the maximum levels by the second day and continues in the third day. The GFP expression generally increases with the increase in DNA loading in DNA-LDH nanohybrids. However, the delivery efficiency with LDH nanoparticles as the agent is low. For example, the relative efficiency is 7%-15% of that of the commercial agent FuGENE 6. Three to 6% of total cells expressed GFP in an amount detectable by the FACS cytometry 2 days after transfection at 1 microg/mL of plasmid DNA with 25 microg/mL of LDH nanomaterial. The lower delivery efficiency could be attributed to the aggregation of LDH nanoparticles caused by the long-chain plasmid DNA.
Topics: Cell Line; Drug Carriers; Genetic Vectors; Humans; Hydroxides; Kidney; Nanoparticles; Particle Size; Plasmids; Transfection
PubMed: 17722544
DOI: No ID Found -
Molecular Genetics and Genomics : MGG May 2022Plasmids are acellular propagating entities that depend on bacteria, as molecular parasites, for propagation. A 'tussle' between bacteria and plasmid ensues; bacteria...
Plasmids are acellular propagating entities that depend on bacteria, as molecular parasites, for propagation. A 'tussle' between bacteria and plasmid ensues; bacteria for riddance of the plasmid and plasmid for persistence within a live host. Plasmid-maintenance systems such as endonuclease Colicin-Like Bacteriocins (CLBs) ensure plasmid propagation within the population; (i) the plasmid-cured cells are killed by the CLBs; (ii) damaged cells lyse and release the CLBs that eliminate the competitors, and (iii) the released plasmids invade new bacteria. Surprisingly, endonuclease CLB operons occur on bacterial genomes whose significance is unknown. Here, we study genetics, eco-evolutionary drive, and physiological relevance of genomic endonuclease CLB operons. We investigated plasmidic and genomic endonuclease CLB operons using sequence analyses from an eco-evolutionary perspective. We found 1266 genomic and plasmidic endonuclease CLB operons across 30 bacterial genera. Although 51% of the genomes harbor endonuclease CLB operons, the majority of the genomic endonuclease CLB operons lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the endonuclease CLB operon protects the plasmid-cured host, eliminating the metabolic burden. We show mutual exclusivity of endonuclease CLB operons on genomes and plasmids. We propose an anti-addiction hypothesis for genomic endonuclease CLB operons. Using a stochastic hybrid agent-based model, we show that the endonuclease CLB operons on genomes confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. The conflict between bacterial genome and plasmids allows the emergence of 'genetic arms' such as CLB operons that regulate the ecological interplay of bacterial genomes and plasmids.
Topics: Bacteria; Bacteriocins; Colicins; Endonucleases; Escherichia coli; Operon; Plasmids
PubMed: 35320397
DOI: 10.1007/s00438-022-01884-4