scholarly journals Quantitative characterization of random partitioning in the evolution of plasmid-encoded traits

2019 ◽  
Author(s):  
Andrew D. Halleran ◽  
Emanuel Flores-Bautista ◽  
Richard M. Murray
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Stochastic Simulations ◽  
Quantitative Characterization ◽  
Plasmid Copy ◽  
Daughter Cells ◽  
Copy Numbers ◽  
Plasmid Partitioning ◽  
Beneficial Allele

AbstractPlasmids are found across bacteria, archaea, and eukaryotes and play an important role in evolution. Plasmids exist at different copy numbers, the number of copies of the plasmid per cell, ranging from a single plasmid per cell to hundreds of plasmids per cell. This feature of a copy number greater than one can lead to a population of plasmids within a single cell that are not identical clones of one another, but rather have individual mutations that make a given plasmid unique. During cell division, this population of plasmids is partitioned into the two daughter cells, resulting in a random distribution of different plasmid variants in each daughter. In this study, we use stochastic simulations to investigate how random plasmid partitioning compares to a perfect partitioning model. Our simulation results demonstrate that random plasmid partitioning accelerates mutant allele fixation when the allele is beneficial and the selection is in an additive or recessive regime where increasing the copy number of the beneficial allele results in additional benefit for the host. This effect does not depend on the size of the benefit conferred or the mutation rate, but is magnified by increasing plasmid copy number.

scholarly journals Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

2009 ◽  
Vol 191 (9) ◽  
pp. 3041-3049 ◽  
Author(s):  
Drew S. Cunningham ◽  
Zhu Liu ◽  
Nathan Domagalski ◽  
Richard R. Koepsel ◽  
Mohammad M. Ataai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Pyruvate Kinase ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Phosphotransferase System ◽  
Plasmid Copy ◽  
Copy Numbers ◽  
High Copy Number Plasmid ◽  
Pathway Flux

ABSTRACT Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37°C. At 42°C, its plasmid content was ninefold higher than JM101 at 37°C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp uv , under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

scholarly journals DnaA Boxes Are Important Elements in Setting the Initiation Mass of Escherichia coli

1999 ◽  
Vol 181 (9) ◽  
pp. 2683-2688 ◽  
Author(s):  
Bjarke Bak Christensen ◽  
Tove Atlung ◽  
Flemming G. Hansen
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Moderate Increase ◽  
Plasmid Copy ◽  
Dnaa Protein ◽  
Copy Numbers ◽  
Dna Binding Sites ◽  
Dnaa Box

ABSTRACT The binding of DnaA protein to its DNA binding sites—DnaA boxes—in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.

scholarly journals Characterization of the Replication Region of Plasmid pLS32 from the Natto Strain of Bacillus subtilis

2005 ◽  
Vol 187 (13) ◽  
pp. 4315-4326 ◽  
Author(s):  
Teruo Tanaka ◽  
Hirofumi Ishida ◽  
Tomoko Maehara
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Tandem Repeats ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Gram Positive Bacteria ◽  
Low Copy Number ◽  
Replication Region ◽  
Translational Start

ABSTRACT Plasmid pL32 from the Natto strain of Bacillus subtilis belongs to a group of low-copy-number plasmids in gram-positive bacteria that replicate via a theta mechanism of replication. We studied the DNA region encoding the replication protein, RepN, of pLS32, and obtained the following results. Transcription of the repN gene starts 167 nucleotides upstream from the translational start site of repN. The copy number of repN-coding plasmid pHDCS2, in which the repN gene was placed downstream of the IPTG (isopropyl-1-thio-β-d-galactopyranoside)-inducible Pspac promoter, was increased 100 fold by the addition of IPTG. Histidine-tagged RepN bound to a specific region in the repN gene containing five 22-bp tandem repeats (iterons) with partial mismatches, as shown by gel retardation and foot printing analyses. Sequence alterations in the first three iterons resulted in an increase in plasmid copy number, whereas those in either the forth or fifth iteron resulted in the failure of plasmid replication. The iterons expressed various degrees of incompatibility with an incoming repN-driven replicon pSEQ243, with the first three showing the strongest incompatibility. Finally, by using a plasmid, pHDMAEC21, carrying the sequence alterations in all the five iterons in repN and thus unable to replicate but encoding intact RepN, the region necessary for replication was confined to a 96-bp sequence spanning the 3′-terminal half of the fourth iteron to an A+T-rich region located downstream of the fifth iteron. From these results, we conclude that the iterons in repN are involved in both the control of plasmid copy number and incompatibility, and we suggest that the binding of RepN to the last two iterons triggers replication by melting the A+T-rich DNA sequence.

scholarly journals Segregational drift and the interplay between plasmid copy number and evolvability

2018 ◽  
Author(s):  
Judith Ilhan ◽  
Anne Kupczok ◽  
Christian Woehle ◽  
Tanita Wein ◽  
Nils F. Hülter ◽  
...  
Keyword(s):  
Cell Division ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Fixation Time ◽  
Multicopy Plasmid ◽  
Plasmid Copy ◽  
Plasmid Evolution ◽  
Host Chromosome ◽  
Daughter Cells ◽  
Multiple Copies

AbstractThe 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.

scholarly journals Characterization of a Novel Partition System Encoded by the δ and ω Genes from the Streptococcal Plasmid pSM19035

2006 ◽  
Vol 188 (12) ◽  
pp. 4362-4372 ◽  
Author(s):  
Michał Dmowski ◽  
Izabela Sitkiewicz ◽  
Piotr Cegłowski
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Promoter Regions ◽  
Plasmid Copy ◽  
Partition System ◽  
Segregational Stability ◽  
Plasmid Stabilization ◽  
Postsegregational Killing

ABSTRACT High segregational stability of the streptococcal plasmid pSM19035 is achieved by the concerted action of systems involved in plasmid copy number control, multimer resolution, and postsegregational killing. In this study, we demonstrate the role of two genes, δ and ω, in plasmid stabilization by a partition mechanism. We show that these two genes can stabilize the native pSM19035 replicon as well as other θ- and σ-type plasmids in Bacillus subtilis. In contrast to other known partition systems, in this case the two genes are transcribed separately; however, they are coregulated by the product of the parB-like gene ω. Analysis of mutants of the parA-like gene δ showed that the Walker A ATPase motif is necessary for plasmid stabilization. The ParB-like product of the ω gene binds to three regions containing repeated WATCACW heptamers, localized in the copS (regulation of plasmid copy number), δ, and ω promoter regions. We demonstrate that all three of these regions can cause partition-mediated incompatibility. Moreover, our data suggest that each of these could play the role of a centromere-like sequence. We conclude that δ and ω constitute a novel type of plasmid stabilization system.

Cloing and characterization of an Escherichia coli gene, pcnB, affecting plasmid copy number

1989 ◽  
Vol 3 (7) ◽  
pp. 903-910 ◽  
Author(s):  
J. B. March ◽  
M. D. Colloms ◽  
D. Hart-Davis ◽  
I. R. Oliver ◽  
M. Masters
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Isolation and characterization of ColE1-derived plasmid copy-number mutant.

1978 ◽  
Vol 75 (12) ◽  
pp. 5869-5873 ◽  
Author(s):  
D. H. Gelfand ◽  
H. M. Shepard ◽  
P. H. O'Farrell ◽  
B. Polisky
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Isolation And Characterization

scholarly journals Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

2006 ◽  
Vol 73 (4) ◽  
pp. 1296-1307 ◽  
Author(s):  
Ryan Fong ◽  
Zhihao Hu ◽  
C. Richard Hutchinson ◽  
Jianqiang Huang ◽  
Stanley Cohen ◽  
...  
Keyword(s):  
Copy Number ◽  
Transfer Functions ◽  
Biosynthetic Gene Cluster ◽  
Plasmid Copy Number ◽  
High Copy Number ◽  
Plasmid Replication ◽  
Biosynthetic Genes ◽  
Plasmid Copy ◽  
Metabolite Production ◽  
Copy Numbers

ABSTRACT A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

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