scholarly journals Chlamydia trachomatis intra-bacterial and total plasmid copy number in clinical urogenital samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. A. M. C. Dirks ◽  
K. Janssen ◽  
C. J. P. A. Hoebe ◽  
T. H. B. Geelen ◽  
M. Lucchesi ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diagnostic Value ◽  
Plasmid Copy Number ◽  
Extracellular Dna ◽  
Chromosomal Dna ◽  
Plasmid Copy ◽  
Clinical Sensitivity ◽  
Copy Numbers ◽  
Vaginal Swabs ◽  
And Gender

AbstractChlamydia trachomatis (CT) increases its plasmid numbers when stressed, as occurs in clinical trachoma samples. Most CT tests target the plasmid to increase the test sensitivity, but some only target the chromosome. We investigated clinical urogenital samples for total plasmid copy numbers to assess its diagnostic value and intra-bacterial plasmid copy numbers to assess its natural variation. Both plasmid and chromosome copies were quantified using qPCR, and the plasmid:chromosome ratio (PCr) calculated in two cohorts: (1) 383 urogenital samples for the total PCR (tPCr), and (2) 42 vaginal swabs, with one half treated with propium-monoazide (PMA) to prevent the quantification of extracellular DNA and the other half untreated to allow for both tPCr and intra-bacterial PCr (iPCr) quantification. Mann–Whitney U tests compared PCr between samples, in relation to age and gender. Cohort 1: tPCr varied greatly (1–677, median 16). Median tPCr was significantly higher in urines than vaginal swabs (32 vs. 11, p < 0.001). Cohort 2: iPCr was more stable than tPCr (range 0.1–3 vs. 1–11). To conclude, tPCr in urogenital samples was much more variable than previously described. Transport time and temperature influences DNA degradation, impacting chromosomal DNA more than plasmids and urine more than vaginal samples. Data supports a plasmid target in CT screening assays to increase clinical sensitivity.

scholarly journals Determination of Plasmid Copy Number Reveals the Total Plasmid DNA Amount Is Greater than the Chromosomal DNA Amount in Bacillus thuringiensis YBT-1520

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e16025 ◽  
Author(s):  
Chunying Zhong ◽  
Donghai Peng ◽  
Weixing Ye ◽  
Lujun Chai ◽  
Junliang Qi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Plasmid Dna ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Chromosomal Dna ◽  
Dna Amount ◽  
Plasmid Copy

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 Plasmid Copy Number and Disease Severity in Naturally Occurring Ocular Chlamydia trachomatis Infection

2013 ◽  
Vol 52 (1) ◽  
pp. 324-327 ◽  
Author(s):  
A. R. Last ◽  
C. h. Roberts ◽  
E. Cassama ◽  
M. Nabicassa ◽  
S. Molina-Gonzalez ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Disease Severity ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Chlamydia Trachomatis Infection ◽  
Naturally Occurring

The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 893-903 ◽  
Author(s):  
Mark A. Pickett ◽  
J. Sylvia Everson ◽  
Patrick J. Pead ◽  
Ian N. Clarke
Keyword(s):  
Chlamydia Trachomatis ◽  
Confidence Interval ◽  
Copy Number ◽  
Chlamydophila Pneumoniae ◽  
Plasmid Copy Number ◽  
Developmental Cycle ◽  
Plasmid Curing ◽  
Plasmid Copy ◽  
Rep Proteins ◽  
Curing Agents

A 7·5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1–L3), ten trachoma isolates (serovars A–C) and nine urogenital isolates (serovars D–J). The results indicated an average plasmid copy number of 4·0±0·8 (mean±95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7·6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7·4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1·3±0·2 plasmids per chromosome (mean±95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number.

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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