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Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability.

ABSTRACT: The ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes. Many plasmids are found in their host cell in multiple copies. This leads to an increased mutational supply of plasmid-encoded genes and genetically heterogeneous plasmid genomes. Nonetheless, the segregation of plasmid copies into daughter cells during cell division is considered to occur in the absence of selection on the plasmid alleles. We investigate the implications of random genetic drift of multicopy plasmids during cell division-termed here "segregational drift"-to plasmid evolution. Performing experimental evolution of low- and high-copy non-mobile plasmids in Escherichia coli, we find that the evolutionary rate of multicopy plasmids does not reflect the increased mutational supply expected according to their copy number. In addition, simulated evolution of multicopy plasmid alleles demonstrates that segregational drift leads to increased loss frequency and extended fixation time of plasmid mutations in comparison to haploid chromosomes. Furthermore, an examination of the experimentally evolved hosts reveals a significant impact of the plasmid type on the host chromosome evolution. Our study demonstrates that segregational drift of multicopy plasmids interferes with the retention and fixation of novel plasmid variants. Depending on the selection pressure on newly emerging variants, plasmid genomes may evolve slower than haploid chromosomes, regardless of their higher mutational supply. We suggest that plasmid copy number is an important determinant of plasmid evolvability due to the manifestation of segregational drift.

SUBMITTER: Ilhan J

PROVIDER: S-EPMC6389322 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability.

Ilhan Judith J Kupczok Anne A Woehle Christian C Wein Tanita T Hülter Nils F NF Rosenstiel Philip P Landan Giddy G Mizrahi Itzhak I Dagan Tal T

Molecular biology and evolution 20190301 3

The ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes. Many plasmids are found in their host cell in multiple copies. This leads to an increased mutational supply of plasmid-encoded genes and genetically heterogeneous plasmid genomes. Nonetheless, the segregation of plasmid copies into daughter cells during cell division is considered to occur in the absence of selection on the plasmi ...[more]

PMID: 30517696

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Project description:Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5? cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

Dataset Information

Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability.

Publications

Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability.

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