scholarly journals Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Frances Tran ◽  
James Q. Boedicker
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Extracellular Vesicles ◽  
Copy Number ◽  
Genetic Material ◽  
Plasmid Copy Number ◽  
Gene Exchange ◽  
Origin Of Replication ◽  
Plasmid Copy ◽  
The Impact

ABSTRACTHorizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles.IMPORTANCEExtracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer.

scholarly journals Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization

2007 ◽  
Vol 75 (10) ◽  
pp. 4804-4816 ◽  
Author(s):  
Yongkai Mo ◽  
Natalie M. Quanquin ◽  
William H. Vecino ◽  
Uma Devi Ranganathan ◽  
Lydia Tesfa ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Mycobacterium Smegmatis ◽  
Copy Number ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
J774 Cells

ABSTRACT Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120h E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120h E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

scholarly journals A Plasmid System with Tunable Copy Number

2021 ◽  
Author(s):  
Miles V Rouches ◽  
Yasu Xu ◽  
Louis Cortes ◽  
Guillaume Lambert
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Metabolic Effects ◽  
Massively Parallel ◽  
Simple Relationship ◽  
Plasmid Copy ◽  
Control Of Gene Expression ◽  
Recombinant Gene Expression ◽  
The Impact

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.

scholarly journals Horizontal Gene Transfer and Endophytes: An Implication for the Acquisition of Novel Traits

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 305 ◽  
Author(s):  
Pragya Tiwari ◽  
Hanhong Bae
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genetic Material ◽  
Biological Species ◽  
Economic Importance ◽  
Comprehensive Analysis ◽  
Current Perspective ◽  
Novel Traits ◽  
The Impact ◽  
Insight Into

Horizontal gene transfer (HGT), an important evolutionary mechanism observed in prokaryotes, is the transmission of genetic material across phylogenetically distant species. In recent years, the availability of complete genomes has facilitated the comprehensive analysis of HGT and highlighted its emerging role in the adaptation and evolution of eukaryotes. Endophytes represent an ecologically favored association, which highlights its beneficial attributes to the environment, in agriculture and in healthcare. The HGT phenomenon in endophytes, which features an important biological mechanism for their evolutionary adaptation within the host plant and simultaneously confers “novel traits” to the associated microbes, is not yet completely understood. With a focus on the emerging implications of HGT events in the evolution of biological species, the present review discusses the occurrence of HGT in endophytes and its socio-economic importance in the current perspective. To our knowledge, this review is the first report that provides a comprehensive insight into the impact of HGT in the adaptation and evolution of endophytes.

scholarly journals Horizontal gene transfer in 44 early diverging fungi favors short, metabolic, extracellular proteins from associated bacteria

2021 ◽  
Author(s):  
Michał Ciach ◽  
Julia Pawłowska ◽  
Anna Muszewska
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Phylogenetic Trees ◽  
Large Scale ◽  
Genetic Material ◽  
Small Sample ◽  
Extracellular Proteins ◽  
Gene Exchange ◽  
Statistical Sample

AbstractNumerous studies have been devoted to individual cases of horizontally acquired genes in fungi. It has been shown that such genes expand their metabolic capabilities and contribute to their adaptations as parasites or symbionts. Some studies have provided a more extensive characterization of the horizontal gene transfer (HGT) in Dikarya. However, in the early diverging fungi (EDF), the overall influence of HGT on the ecological adaptation and evolution is largely unknown. In order to fill this gap, we have designed a computational pipeline to obtain a sample of over 600 phylogenetic trees with evidence for recent to moderately old HGT across multiple EDF genomes ranging from Chytridiomycota and Blastocladiomycota to Mucoromycota. Our pipeline is designed to obtain a small sample of reliable HGT events with a possibly minimal number of false detections that distort the overall statistical patterns. We show that transfer rates differ greatly between closely related species and strains, but the ancestrally aquatic fungi are generally more likely to acquire foreign genetic material than terrestrial ones. A close ecological relationship with another organism is a predisposing condition, but does not always result in an extensive gene exchange, with some fungal lineages showing a preference for HGT from loosely associated soil bacteria.ImportanceAlthough it is now recognized that horizontal gene exchange is a factor influencing the adaptation and evolution of eukaryotic organisms, the so far described cases in early diverging fungi (EDF) are fragmentary, and a large-scale comprehensive study is lacking. We have designed a methodology to obtain a reliable, statistical sample of inter-kingdom xenologs across the tree of life of EDF to give a preliminary characterization of their general properties and patterns. We study how different fungal lineages vary in terms of the number of xenologs, what are their ecological associations, and the molecular properties of proteins encoded by the acquired genes. Our results help to better understand to what extent and in what way the incorporation of foreign genetic material shaped the present biodiversity of fungi.

scholarly journals Changing Plasmid Copy Number by Mutation of the pMB1 Origin of Replication (LB209)

2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Brandon Grieshaber ◽  
Erich Baker ◽  
Malcolm Campbell ◽  
Todd Eckdahl ◽  
Jeffrey Poet ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Origin Of Replication ◽  
Plasmid Copy

scholarly journals Horizontal Gene Transfer Involving Chloroplasts

2021 ◽  
Vol 22 (9) ◽  
pp. 4484
Author(s):  
Ewa Filip ◽  
Lidia Skuza
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Driving Force ◽  
Genetic Material ◽  
Adaptive Significance ◽  
Chloroplast Genomes ◽  
Organelle Genomes ◽  
Number Of Genes ◽  
Cellular Compartments

Horizontal gene transfer (HGT)- is defined as the acquisition of genetic material from another organism. However, recent findings indicate a possible role of HGT in the acquisition of traits with adaptive significance, suggesting that HGT is an important driving force in the evolution of eukaryotes as well as prokaryotes. It has been noted that, in eukaryotes, HGT is more prevalent than originally thought. Mitochondria and chloroplasts lost a large number of genes after their respective endosymbiotic events occurred. Even after this major content loss, organelle genomes still continue to lose their own genes. Many of these are subsequently acquired by intracellular gene transfer from the original plastid. The aim of our review was to elucidate the role of chloroplasts in the transfer of genes. This review also explores gene transfer involving mitochondrial and nuclear genomes, though recent studies indicate that chloroplast genomes are far more active in HGT as compared to these other two DNA-containing cellular compartments.

scholarly journals Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

2018 ◽  
Vol 36 (3) ◽  
pp. 472-486 ◽  
Author(s):  
Judith Ilhan ◽  
Anne Kupczok ◽  
Christian Woehle ◽  
Tanita Wein ◽  
Nils F Hülter ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals RNA polyadenylation and its consequences in prokaryotes

2018 ◽  
Vol 373 (1762) ◽  
pp. 20180166 ◽  
Author(s):  
Eliane Hajnsdorf ◽  
Vladimir R. Kaberdin
Keyword(s):  
Gene Expression ◽  
State Level ◽  
Rna Degradation ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Protein Coding ◽  
Mrna Metabolism ◽  
Isolation And Characterization ◽  
Polymerase I ◽  
The Impact

Post-transcriptional addition of poly(A) tails to the 3′ end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin–antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

scholarly journals Involvement of plastid, mitochondrial and nuclear genomes in plant-to-plant horizontal gene transfer

2014 ◽  
Vol 83 (4) ◽  
pp. 317-323 ◽  
Author(s):  
Maria Virginia Sanchez-Puerta
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Plant Mitochondria ◽  
Genetic Material ◽  
Single Copy ◽  
Convincing Evidence ◽  
Cell Contact ◽  
Plant Mtdna ◽  
Nuclear Genomes ◽  
Foreign Genes

This review focuses on plant-to-plant horizontal gene transfer (HGT) involving the three DNA-containing cellular compartments. It highlights the great incidence of HGT in the mitochondrial genome (mtDNA) of angiosperms, the increasing number of examples in plant nuclear genomes, and the lack of any convincing evidence for HGT in the well-studied plastid genome of land plants. Most of the foreign mitochondrial genes are non-functional, generally found as pseudogenes in the recipient plant mtDNA that maintains its functional native genes. The few exceptions involve chimeric HGT, in which foreign and native copies recombine leading to a functional and single copy of the gene. Maintenance of foreign genes in plant mitochondria is probably the result of genetic drift, but a possible evolutionary advantage may be conferred through the generation of genetic diversity by gene conversion between native and foreign copies. Conversely, a few cases of nuclear HGT in plants involve functional transfers of novel genes that resulted in adaptive evolution. Direct cell-to-cell contact between plants (e.g. host-parasite relationships or natural grafting) facilitate the exchange of genetic material, in which HGT has been reported for both nuclear and mitochondrial genomes, and in the form of genomic DNA, instead of RNA. A thorough review of the literature indicates that HGT in mitochondrial and nuclear genomes of angiosperms is much more frequent than previously expected and that the evolutionary impact and mechanisms underlying plant-to-plant HGT remain to be uncovered.

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