Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

AbstractFlagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially restored using the tightly regulated rhamnose promoter, but wild-type motility was achieved with the T5 promoter, which, although leaky, allowed titration of induction strength. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

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

Journal of Bacteriology ◽

10.1128/jb.01422-08 ◽

2009 ◽

Vol 191 (9) ◽

pp. 3041-3049 ◽

Cited By ~ 37

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.

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Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Molecular Genetics and Genomics ◽

10.1007/s00438-002-0723-0 ◽

2002 ◽

Vol 268 (1) ◽

pp. 62-69 ◽

Cited By ~ 6

Author(s):

N. Sarkar ◽

G.-j. Cao ◽

C. Jain

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Multicopy Suppressors

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

Journal of Bacteriology ◽

10.1128/jb.181.9.2683-2688.1999 ◽

1999 ◽

Vol 181 (9) ◽

pp. 2683-2688 ◽

Cited By ~ 29

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.

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