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Advanced Biosystems Apr 2020The rapid dissemination of non-conjugative virulence plasmids among non-K1/K2 types of Klebsiella pneumoniae poses an unprecedented threat to human health, yet the...
The rapid dissemination of non-conjugative virulence plasmids among non-K1/K2 types of Klebsiella pneumoniae poses an unprecedented threat to human health, yet the underlying mechanisms governing dissemination of such plasmids is unclear. In this study, a novel 68 581 bp IncFIA plasmid is discovered that can be fused to a hypervirulence-encoding plasmid to form a hybrid conjugative virulence plasmid in conjugation experiments; such fusion events involve homologous recombination between a 241 bp homologous region located in each of the two plasmids. The fusion hypervirulence-encoding plasmid can be conjugated to both classic and bla -bearing carbapenem-resistant K. pneumoniae strains through conjugation, enabling such strains to acquire the ability to express the hypervirulence phenotype. Dissemination of this fusion virulence plasmid will impose an enormous burden on current efforts to control and treat infections caused by multidrug resistant and hypervirulent K. pneumoniae.
Topics: Conjugation, Genetic; Klebsiella pneumoniae; Plasmids; Virulence Factors
PubMed: 32293159
DOI: 10.1002/adbi.201900239 -
The Journal of Antimicrobial... Feb 2022To assess the genetic contexts surrounding blaNDM-1 genes carried on IncM plasmids harboured by six carbapenemase-producing Enterobacterales (CPE) isolates referred to...
OBJECTIVES
To assess the genetic contexts surrounding blaNDM-1 genes carried on IncM plasmids harboured by six carbapenemase-producing Enterobacterales (CPE) isolates referred to the UK Health Security Agency's Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit.
METHODS
Between 2014 and 2018, the AMRHAI Reference Unit undertook WGS of CPE isolates using Illumina NGS. Nanopore sequencing was used for selected isolates and publicly available plasmid references were downloaded. Analysis of incRNA, which encodes the antisense RNA regulating plasmidic repA gene expression, was performed and bioinformatics tools were used to analyse whole plasmid sequences.
RESULTS
Of 894 NDM-positive isolates of Enterobacterales, 44 NDM-1-positive isolates of five different species (Citrobacter spp., Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca) encoded the IncRNA locus of IncM2 plasmids. Long-read sequencing of six diverse isolates revealed related IncM2, NDM-1-encoding plasmids. Plasmid 'backbone' areas were conserved and contrasted with highly variable resistance regions. Sub-groupings of IncM2 plasmids encoding blaNDM-1 were detected; one sub-group occurred in five different health regions of England in every year. The diversity of NDM-1-encoding resistance gene integrons and transposons and their insertions sites in the plasmids indicated that NDM-1 has been acquired repeatedly by IncM2 variants.
CONCLUSIONS
The use of sequencing helped inform: (i) a wide geographical distribution of isolates encoding NDM-1 on emergent IncM2 plasmids; (ii) variant plasmids have acquired NDM-1 separately; and (iii) dynamic arrangements and evolution of the resistance elements in this plasmid group. The geographical and temporal distribution of IncM2 plasmids that encode NDM-1 highlights them as a public health threat that requires ongoing monitoring.
Topics: Bacterial Proteins; Drug Resistance, Bacterial; Enterobacteriaceae; Microbial Sensitivity Tests; Plasmids; beta-Lactamases
PubMed: 34993543
DOI: 10.1093/jac/dkab466 -
Philosophical Transactions of the Royal... Jan 2022Naturally occurring plasmids come in different sizes. The smallest are less than a kilobase of DNA, while the largest can be over three orders of magnitude larger....
Naturally occurring plasmids come in different sizes. The smallest are less than a kilobase of DNA, while the largest can be over three orders of magnitude larger. Historically, research has tended to focus on smaller plasmids that are usually easier to isolate, manipulate and sequence, but with improved genome assemblies made possible by long-read sequencing, there is increased appreciation that very large plasmids-known as megaplasmids-are widespread, diverse, complex, and often encode key traits in the biology of their host microorganisms. Why are megaplasmids so big? What other features come with large plasmid size that could affect bacterial ecology and evolution? Are megaplasmids 'just' big plasmids, or do they have distinct characteristics? In this perspective, we reflect on the distribution, diversity, biology, and gene content of megaplasmids, providing an overview to these large, yet often overlooked, mobile genetic elements. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.
Topics: Plasmids
PubMed: 34839707
DOI: 10.1098/rstb.2020.0472 -
Gene 1986The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of...
The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accommodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.
Topics: Base Sequence; Cloning, Molecular; DNA Replication; DNA Restriction Enzymes; DNA, Recombinant; DNA, Viral; Gene Amplification; Gene Expression Regulation; Genetic Vectors; Plasmids; R Factors; Recombinant Proteins; Selection, Genetic; Transcription, Genetic
PubMed: 3034735
DOI: 10.1016/0378-1119(86)90307-0 -
MBio Aug 2023Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth...
Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth rates of the host bacteria. Compensatory mutations are known as an effective evolutionary solution to reduce the fitness cost and improve plasmid persistence. However, whether the plasmid transmission by conjugation is sufficient to improve plasmid persistence is debated since it is an inherently costly process. Here, we experimentally evolved an unstable and costly plasmid pHNSHP24 under laboratory conditions and assessed the effects of plasmid cost and transmission on the plasmid maintenance by the plasmid population dynamics model and a plasmid invasion experiment designed to measure the plasmid's ability to invade a plasmid-free bacterial population. The persistence of pHNSHP24 improved after 36 days evolution due to the plasmid-borne mutation A51G in the 5'UTR of gene . This mutation largely increased the infectious transmission of the evolved plasmid, presumably by impairing the inhibitory effect of FinP on the expression of . We showed that increased conjugation rate of the evolved plasmid could compensate for the plasmid loss. Furthermore, we determined that the evolved high transmissibility had little effect on the -deficient ancestral plasmid, implying that high conjugation transfer is vital for maintaining the -bearing plasmid. Altogether, our findings emphasized that, besides compensatory evolution that reduces fitness costs, the evolution of infectious transmission can improve the persistence of antibiotic-resistant plasmids, indicating that inhibition of the conjugation process could be useful to combat the spread of antibiotic-resistant plasmids. IMPORTANCE Conjugative plasmids play a key role in the spread of antibiotic resistance, and they are well-adapted to the host bacteria. However, the evolutionary adaptation of plasmid-bacteria associations is not well understood. In this study, we experimentally evolved an unstable colistin resistance () plasmid under laboratory conditions and found that increased conjugation rate was crucial for the persistence of this plasmid. Interestingly, the evolved conjugation was caused by a single-base mutation, which could rescue the unstable plasmid from extinction in bacterial populations. Our findings imply that inhibition of the conjugation process could be necessary for combating the persistence of antibiotic-resistance plasmids.
Topics: Plasmids; Drug Resistance, Microbial; Bacteria; Mutation; Anti-Bacterial Agents
PubMed: 37314200
DOI: 10.1128/mbio.00442-23 -
Plasmid Jan 2009Although plasmid DNA vectors have been extensively applied in biotechnology, there is still a lack of standard plasmid vector classification. Here, we propose a...
Although plasmid DNA vectors have been extensively applied in biotechnology, there is still a lack of standard plasmid vector classification. Here, we propose a classification method for commonly used plasmid vectors. Plasmid vectors were classified into different classes based on their replication origin, selection marker and promoter information. The replication origins of plasmid vectors were classified as: prokaryotic replication origin, eukaryotic replication origin and viral replication origin. Selection markers of plasmid vectors were mainly classified as ampicillin, kanamycin, neomycin, chloramphenicol, gentamycin, tetracycline, erythromycin, streptomycin, vancomycin and spectinomycin resistance gene markers. Promoter sequences were also classified as prokaryotic, eukaryotic and viral promoters. Finally, the nomenclature of common plasmid vectors has three determinants. We believe that the classification of plasmid vectors can provide useful information for researchers employing molecular cloning procedures. A web service of the plasmid classification was established and it is available from http://www.computationalmedicalbiology.org/plasclas.aspx.
Topics: Base Sequence; Cloning, Molecular; Genetic Markers; Genetic Vectors; Plasmids; Promoter Regions, Genetic; Replication Origin
PubMed: 18840464
DOI: 10.1016/j.plasmid.2008.09.003 -
Mutation Research Jun 2006pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay...
pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most sensitive genetic toxicity assays and a useful short-term test for predicting carcinogenicity in animals. Plasmid pKM101, which is a deletion derivative of plasmid R46 (also referred to as R-Brighton after its origin of isolation in Brighton, England), has also been used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker's phenomenal insight into the genetics of Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis.
Topics: Carcinogenicity Tests; History, 20th Century; Mutagenesis; Mutagenicity Tests; Plasmids; R Factors; Salmonella
PubMed: 16716644
DOI: 10.1016/j.mrrev.2006.03.002 -
PloS One 2020Evolutionary studies have been conducted that have investigated the chromosomal variance in the genus of Chlamydia. However, no all-encompassing genus-wide comparison...
BACKGROUND
Evolutionary studies have been conducted that have investigated the chromosomal variance in the genus of Chlamydia. However, no all-encompassing genus-wide comparison has been performed on the plasmid. Therefore, there is a gap in the current knowledge on Chlamydia plasmid diversity.
AIMS
This project is aimed to investigate and establish the nature and extent of diversity across the entire genus of Chlamydia, by comparing the sequences of all currently available plasmid carrying strains.
METHODS
The PUBMED database was used to identify plasmid sequences from all available strains that met the set quality criteria for their inclusion in the study. Alignments were performed on the 51 strains that fulfilled the criteria using MEGA X software. Following that Maximum Likelihood estimation was used to construct 11 phylogenetic trees of the whole plasmid sequence, the individual 8 coding sequences, the iteron and a chromosomal gene ompA as a comparator.
RESULTS
The genus-wide plasmid phylogeny produced three distinct lineages labelled as alpha, beta and gamma. Nineteen genotypes were found in the initial whole plasmid analysis. Their distribution was allocated as six C. pecorum, two C. pneumoniae, one C. gallinacea, one C. avium, one C. caviae, one C. felis, two C. psittaci, one C. trachomatis, one C. muridarum, and two C. suis. The chromosomal comparative gene ompA supported this distribution, with the same number of primary clades with the same species distribution. However, ompA sequence comparison resulted in fewer genotypes due to a reduced amount of available sequences (33 out of 51). All results were statistically significant.
CONCLUSION
The results of this study indicate that the common bacterial ancestor of all the species had a plasmid, which has diverged over time. Moreover, it suggests that there is a strong evolutionary selection towards these species retaining their plasmids due to its high level of conservation across the genus, with the notable exception of C. pneumoniae. Furthermore, the evolutionary analysis showed that the plasmid and the chromosome have co-evolved.
Topics: Animals; Chlamydia; Chlamydia Infections; Genetic Variation; Genome, Bacterial; Genotype; Phylogeny; Plasmids; Sequence Analysis, DNA
PubMed: 32469898
DOI: 10.1371/journal.pone.0233298 -
Applied and Environmental Microbiology Feb 2011Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other...
Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during in vitro culture. The analysis of B. burgdorferi plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain B. burgdorferi B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many B. burgdorferi strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage B. burgdorferi B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease Borrelia studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease Borrelia strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.
Topics: Borrelia burgdorferi; DNA Transposable Elements; Fluorescent Dyes; High-Throughput Nucleotide Sequencing; Microspheres; Plasmids; Polymerase Chain Reaction
PubMed: 21169439
DOI: 10.1128/AEM.01877-10 -
Antimicrobial Agents and Chemotherapy Sep 2022The MOX lineage of β-lactamases includes a group of molecular class C enzymes (AmpCs) encoded by genes mobilized from the chromosomes of spp. to plasmids. MOX-9,...
The MOX lineage of β-lactamases includes a group of molecular class C enzymes (AmpCs) encoded by genes mobilized from the chromosomes of spp. to plasmids. MOX-9, previously identified as a plasmid-encoded enzyme from a Citrobacter freundii isolate, belongs to a novel sublineage of MOX enzymes, derived from the resident Aeromonas media AmpC. The gene was found to be carried on a transposon, named Tn, likely responsible for its mobilization to plasmidic context. MOX-9 was overexpressed in Escherichia coli, purified, and subjected to biochemical characterization. Kinetic analysis showed a relatively narrow-spectrum profile with strong preference for cephalosporin substrates, with some differences compared with MOX-1 and MOX-2. MOX-9 was not inhibited by clavulanate and sulbactam, while both tazobactam and avibactam acted as inhibitors in the micromolar range.
Topics: Bacterial Proteins; Cephalosporins; Clavulanic Acid; Kinetics; Plasmids; Sulbactam; Tazobactam; beta-Lactamases
PubMed: 36040170
DOI: 10.1128/aac.00595-22