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.

scholarly journals Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.

1989 ◽  
Vol 9 (4) ◽  
pp. 1488-1497 ◽  
Author(s):  
K W Runge ◽  
V A Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
High Copy Number ◽  
Limiting Factor ◽  
Telomeric Dna ◽  
Base Pairs ◽  
Plasmid Copy ◽  
Telomere Elongation

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.

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

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3609-3623 ◽  
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.

Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation

1989 ◽  
Vol 9 (4) ◽  
pp. 1488-1497
Author(s):  
K W Runge ◽  
V A Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
High Copy Number ◽  
Limiting Factor ◽  
Telomeric Dna ◽  
Base Pairs ◽  
Plasmid Copy ◽  
Telomere Elongation

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.

scholarly journals Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number

2021 ◽  
Vol 118 (4) ◽  
pp. e2011577118
Author(s):  
Songwei Ni ◽  
Baiyuan Li ◽  
Kaihao Tang ◽  
Jianyun Yao ◽  
Thomas K. Wood ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Negative Regulator ◽  
Conjugative Plasmid ◽  
Plasmid Replication ◽  
Plasmid Copy ◽  
Conjugative Plasmids ◽  
Low Copy Number ◽  
Replication Initiator ◽  
Postsegregational Killing

Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

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