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Plasmid Jan 2021Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification...
Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification and evolutionary rescue. Despite the relevance of plasmids in bacterial evolutionary dynamics, evaluating the population-level consequences of randomly segregating and replicating plasmids in individual cells remains a challenging problem, both in theory and experimentally. In recent years, technological advances in fluorescence microscopy and microfluidics have allowed studying temporal changes in gene expression by quantifying the fluorescent intensity of individual cells under controlled environmental conditions. In this paper, we will describe the manufacture, experimental setup, and data analysis pipeline of different microfluidic systems that can be used to study plasmid dynamics, both in single-cells and in populations. To illustrate the benefits and limitations of microfluidics to study multicopy plasmid dynamics, we will use an experimental model system consisting on Escherichia coli K12 carrying non-conjugative, multicopy plasmids (19 copies per cell, in average) encoding different fluorescent markers and β-lactam resistance genes. First, we will use an image-based flow cytometer to estimate changes in the allele distribution of a heterogeneous population under different selection regimes. Then we will use a mothermachine microfluidic device to obtain time-series of fluorescent intensity of individual cells to argue that plasmid segregation and replication dynamics are inherently stochastic processes. Finally, using a microchemostat, we track thousands of cells in time to reconstruct bacterial lineages and evaluate the allele frequency distributions that emerge in response to a range of selective pressures.
Topics: Bacteria; Computational Biology; Microfluidics; Plasmids; beta-Lactam Resistance
PubMed: 32535165
DOI: 10.1016/j.plasmid.2020.102517 -
Biochemistry and Molecular Biology... Nov 2019This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed...
This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one-week period includes the protocols for plasmid pVAX1-LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1-LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1-LacZ plasmid. Lastly, they perform the purification of pVAX1-LacZ supercoiled isoform by L-histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638-643, 2019.
Topics: Biochemistry; Biotechnology; Curriculum; DNA, Bacterial; Escherichia coli; Humans; Laboratories; Plasmids; Students; Universities
PubMed: 31390150
DOI: 10.1002/bmb.21290 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jan 2023Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during... (Review)
Review
Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of . The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene , and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene , which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of . Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.
Topics: Plasmids; Proteins; Bacteria; Recombinases; Genes, Bacterial; Anti-Bacterial Agents
PubMed: 36738199
DOI: 10.13345/j.cjb.220343 -
MSphere Feb 2022Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop...
Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop antimicrobial resistance (AMR). Several aspects of the conjugative process have long been known, namely, those related to the proteins that participate in the establishment of cell-to-cell contact and to the enzymatic processes associated with the processing of plasmid DNA and its transfer to the recipient cell. In this work, we describe the roles of newly identified proteins that influence the conjugation of several plasmids. Genes encoding high-molecular-weight bacterial proteins that contain one or several immunoglobulin-like domains (Big) are located in the transfer regions of several plasmids that usually harbor AMR determinants. These Big proteins are exported to the external medium and target two extracellular organelles: the flagella and conjugative pili. The plasmid gene-encoded Big proteins facilitate conjugation by reducing cell motility and facilitating cell-to-cell contact by binding both to the flagella and to the conjugative pilus. They use the same export machinery as that used by the conjugative pilus components. In the examples characterized in this paper, these proteins influence conjugation at environmental temperatures (i.e., 25°C). This suggests that they may play relevant roles in the dissemination of plasmids in natural environments. Taking into account that they interact with outer surface organelles, they could be targeted to control the dissemination of different bacterial plasmids carrying AMR determinants. Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid. Our paper identifies novel plasmid gene-encoded high-molecular-weight proteins that contain an immunoglobulin-like domain and are required for plasmid transmission. They are encoded by genes on different groups of plasmids. These proteins are exported outside the cell. They bind to extracellular cell appendages such as the flagella and conjugative pili. Expression of these proteins reduces cell motility and increases the ability of the bacterial cells to transfer the plasmid. These proteins could be targeted with specific antibodies to combat infections caused by AMR microorganisms that harbor these plasmids.
Topics: Anti-Infective Agents; Bacteria; Conjugation, Genetic; Gene Transfer, Horizontal; Immunoglobulin Domains; Plasmids
PubMed: 34986320
DOI: 10.1128/msphere.00978-21 -
Current Opinion in Microbiology Aug 2022Plasmids are a major driver of horizontal gene transfer in prokaryotes, allowing the sharing of ecologically important accessory traits between distantly related... (Review)
Review
Plasmids are a major driver of horizontal gene transfer in prokaryotes, allowing the sharing of ecologically important accessory traits between distantly related bacterial taxa. Within microbial communities, interspecies transfer of conjugative plasmids can rapidly drive the generation genomic innovation and diversification. Recent studies are starting to shed light on how the microbial community context, that is, the bacterial diversity together with interspecies interactions that occur within a community, can alter the dynamics of conjugative plasmid transfer and persistence. Here, I summarise the latest research exploring how community ecology can both facilitate and impose barriers to the spread of conjugative plasmids within complex microbial communities. Ultimately, the fate of plasmids within communities is unlikely to be determined by any one individual host, rather it will depend on the interacting factors imposed by the community in which it is embedded.
Topics: Bacteria; Conjugation, Genetic; Gene Transfer, Horizontal; Microbiota; Plasmids
PubMed: 35504055
DOI: 10.1016/j.mib.2022.102152 -
Scientific Reports Nov 2022Plasmids facilitate horizontal gene transfer, which enables the diversification of pathogens into new anatomical and environmental niches, implying that plasmid-encoded...
Plasmids facilitate horizontal gene transfer, which enables the diversification of pathogens into new anatomical and environmental niches, implying that plasmid-encoded genes can cooperate well with chromosomal genes. We hypothesise that such mobile genes are functionally different to chromosomal ones due to this ability to encode proteins performing non-essential functions like antimicrobial resistance and traverse distinct host cells. The effect of plasmid-driven gene gain on protein-protein interaction network topology is an important question in this area. Moreover, the extent to which these chromosomally- and plasmid-encoded proteins interact with proteins from their own groups compared to the levels with the other group remains unclear. Here, we examined the incidence and protein-protein interactions of all known plasmid-encoded proteins across representative specimens from most bacteria using all available plasmids. We found that plasmid-encoded genes constitute ~ 0.65% of the total number of genes per bacterial sample, and that plasmid genes are preferentially associated with different species but had limited taxonomical power beyond this. Surprisingly, plasmid-encoded proteins had both more protein-protein interactions compared to chromosomal proteins, countering the hypothesis that genes with higher mobility rates should have fewer protein-level interactions. Nonetheless, topological analysis and investigation of the protein-protein interaction networks' connectivity and change in the number of independent components demonstrated that the plasmid-encoded proteins had limited overall impact in > 96% of samples. This paper assembled extensive data on plasmid-encoded proteins, their interactions and associations with diverse bacterial specimens that is available for the community to investigate in more detail.
Topics: Protein Interaction Maps; Plasmids; Gene Transfer, Horizontal; Bacteria; Bacterial Proteins
PubMed: 36357451
DOI: 10.1038/s41598-022-20809-0 -
Methods in Molecular Biology (Clifton,... 2021A plasmid preparation is a method used to extract and purify plasmid DNA. Methods developed to purify plasmid DNA from bacteria generally involve harvesting and alkaline...
A plasmid preparation is a method used to extract and purify plasmid DNA. Methods developed to purify plasmid DNA from bacteria generally involve harvesting and alkaline lysis of the bacteria, precipitation of chromosomal DNA and protein, followed by purification of the plasmid DNA. Here, we describe the mini-preparation of plasmid DNA by a rapid small-scale method, adapted for Listeria monocytogenes. The quality of plasmid DNA isolated using this method is sufficient for analytical purposes but may be upscaled for further downstream analysis. Electrophoretic separation of the resultant lysate allows conclusions to be made on the presence, number, copy number, and size of the plasmids in the analyzed bacterial strains.
Topics: DNA, Bacterial; Electrophoresis, Agar Gel; Humans; Listeria monocytogenes; Listeriosis; Plasmids
PubMed: 32975773
DOI: 10.1007/978-1-0716-0982-8_12 -
Methods in Molecular Biology (Clifton,... 2018Phage Mu is the paradigm of a growing family of bacteriophages that infect a wide range of bacterial species and replicate their genome by replicative transposition....
Phage Mu is the paradigm of a growing family of bacteriophages that infect a wide range of bacterial species and replicate their genome by replicative transposition. This molecular process, which is used by other mobile genetic elements to move within genomes, involves the profound rearrangement of the host genome [chromosome(s) and plasmid(s)] and can be exploited for the genetic analysis of the host bacteria and the in vivo cloning of host genes. In this chapter we review Mu-derived constructs that optimize the phage as a series of genetic tools that could inspire the development of similarly efficient tools from other transposable phages for a large spectrum of bacteria.
Topics: Bacteriophage mu; DNA Transposable Elements; Gene Library; Genetic Techniques; Physical Chromosome Mapping; Plasmids; Replicon
PubMed: 29134601
DOI: 10.1007/978-1-4939-7343-9_19 -
Nature Communications Nov 2020Conjugative plasmids can mediate the spread and maintenance of diverse traits and functions in microbial communities. This role depends on the plasmid's ability to...
Conjugative plasmids can mediate the spread and maintenance of diverse traits and functions in microbial communities. This role depends on the plasmid's ability to persist in a population. However, for a community consisting of multiple populations transferring multiple plasmids, the conditions underlying plasmid persistence are poorly understood. Here, we describe a plasmid-centric framework that makes it computationally feasible to analyze gene flow in complex communities. Using this framework, we derive the 'persistence potential': a general, heuristic metric that predicts the persistence and abundance of any plasmids. We validate the metric with engineered microbial consortia transferring mobilizable plasmids and with quantitative data available in the literature. We believe that our framework and the resulting metric will facilitate a quantitative understanding of natural microbial communities and the engineering of microbial consortia.
Topics: Computational Biology; Conjugation, Genetic; Escherichia coli; Gene Flow; Microbiota; Models, Theoretical; Plasmids
PubMed: 33149119
DOI: 10.1038/s41467-020-19368-7 -
Microbiology Spectrum Apr 2022Plasmids are extrachromosomal genetic elements, some of which disperse horizontally between different strains and species of bacteria. They are a major factor in the...
Plasmids are extrachromosomal genetic elements, some of which disperse horizontally between different strains and species of bacteria. They are a major factor in the dissemination of virulence factors and antibiotic resistance. Understanding the ecology of plasmids has a notable anthropocentric value, and therefore, the interactions between bacterial hosts and individual plasmids have been studied in detail. However, bacterial systems often carry multiple genetically distinct plasmids, but dynamics within these multiplasmid communities have remained unstudied. Here, we set to investigate the survival of 11 mobilizable or conjugative plasmids under five different conditions where the hosts had a differing ecological status in comparison to other bacteria in the system. The key incentive was to determine whether plasmid dynamics are reproducible and whether there are tradeoffs in plasmid fitness that stem from the ecological situation of their initial hosts. Growth rates and maximum population densities increased in all communities and treatments over the 42-day evolution experiment, although plasmid contents at the end varied notably. Large multiresistance-conferring plasmids were unfit when the community also contained smaller plasmids with fewer resistance genes. This suggests that restraining the use of a few antibiotics can make bacterial communities sensitive to others. In general, the presence or absence of antibiotic selection and plasmid-free hosts (of various fitnesses) has a notable influence on which plasmids survive. These tradeoffs in different settings can help explain, for example, why some resistance plasmids have an advantage during a rapid proliferation of antibiotic-sensitive pathogens whereas others dominate in alternative situations. Conjugative and mobilizable plasmids are ubiquitous in bacterial systems. Several different plasmids can compete within a single bacterial community. We here show that the ecological setting of the host bacteria has a notable effect on the survival of individual plasmids. Selection for opportunistic genes such as antibiotic resistance genes and the presence of plasmid-free hosts can determine which plasmids survive in the system. Host bacteria appear to adapt specifically to a situation where there are multiple plasmids present instead of alleviating the plasmid-associated fitness costs of individual plasmids. Plasmids providing antibiotic resistance survived under all conditions even if there was a constant migration of higher-fitness plasmid-free hosts and no selection via antibiotics. This study is one of the first to observe the behavior of multiple genetically different plasmids as a part of a single system.
Topics: Adaptation, Physiological; Anti-Bacterial Agents; Bacteria; Plasmids
PubMed: 35416702
DOI: 10.1128/spectrum.00133-22