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

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.

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Cloning and functional characterization of Komagataella phaffii centromeres by a color-based plasmid stability assay

10.1101/433417 ◽

2018 ◽

Author(s):

Luiza Cesca Piva ◽

Janice Lisboa De Marco ◽

Lidia Maria Pepe de Moraes ◽

Viviane Castelo Branco Reis ◽

Fernando Araripe Gonçalves Torres

Keyword(s):

Copy Number ◽

Plasmid Stability ◽

Functional Characterization ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Komagataella Phaffii ◽

High Transformation ◽

Colony Color ◽

Stable Vectors

AbstractThe yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing K. phaffii centromeres and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

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Genetic dissection of centromere function.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3156 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3156-3166 ◽

Cited By ~ 10

Author(s):

I G Schulman ◽

K Bloom

Keyword(s):

Chromosome Segregation ◽

Copy Number ◽

Artificial Chromosome ◽

Plasmid Copy Number ◽

Genetic Dissection ◽

Negative Effects ◽

Plasmid Copy ◽

Complex Cells ◽

Copy Number Control

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.

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An unusually long-lived antisense RNA in plasmid copy number control: in vivo RNAs encoded by the streptococcal plasmid pIP501

Journal of Molecular Biology ◽

10.1006/jmbi.1996.0023 ◽

1996 ◽

Vol 255 (2) ◽

pp. 275-288 ◽

Cited By ~ 27

Author(s):

Sabine Brantl ◽

E. Gerhart H. Wagner

Keyword(s):

Antisense Rna ◽

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Copy Number Control

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A statistical framework for determination of minimal plasmid copy number required for transgene expression in mammalian cells

Bioelectrochemistry ◽

10.1016/j.bioelechem.2020.107731 ◽

2020 ◽

pp. 107731

Author(s):

Liangli Wang ◽

Chun-Chi Chang ◽

Justin Sylvers ◽

Fan Yuan

Keyword(s):

Copy Number ◽

Mammalian Cells ◽

Transgene Expression ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Statistical Framework ◽

Expression In Mammalian Cells

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Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

Journal of Bacteriology ◽

10.1128/jb.00430-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 4

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.

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Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum

Microbiology ◽

10.1099/mic.0.043745-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3609-3623 ◽

Cited By ~ 11

Author(s):

Naoko Okibe ◽

Nobuaki Suzuki ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Corynebacterium Glutamicum ◽

Antisense Rna ◽

Copy Number ◽

Shuttle Vector ◽

Fold Increase ◽

Plasmid Copy Number ◽

Plasmid Replication ◽

Plasmid Copy ◽

Rna Molecules ◽

Target Mrnas

pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem–loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.

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