Derbyshire, Paul (1986) Plasmid maintenance in Escherichia coli K-12. PhD thesis, University of Warwick.

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Abstract

This study investigated the maintenance of several plasmid derivatives of pBR322 and ColEl present within Escherichia coli K-12, with a view to establishing a strategy that would ensure the stable maintenance of plasmid pBR322 and the identification of a postulated partitioning function on plasmid ColEl.

Two strategies were adopted. The first of these used selective nitrogen-limited chemostat culture of a glutamate-dependent strain of E.coli, carrying a pBR322 derivative expressing glutamate dehydrogenase. Using this approach, it was found that the plasmid conferred a reproductive advantage and persisted, during nitrogen limitation, for several generations beyond that of pBR322 under glucose- or phosphate- limited conditions.

The second strategy used nonselective chemostat culture of a strain of E.coli carrying derivatives of pBR322 encoding stability functions from either plasmid pSClOl, the partitioning region par, or plasmid R1, the cell division/plasmid inheritance coupling region parB. Although these plasmids persisted for many generations beyond that of pBR322 under similar chemostat culture conditions, no conclusions could be made with respect to the ability of these functions to ensure stable plasmid maintenance, since the copy numbers of the respective plasmids were several-fold greater than that of pBR322, a factor that in itself would contribute to the segregational stability of the plasmids. Plasmid- free segregants which did arise were found not to be isogenic with the host strain. These mutants exhibited an increased resistance to U.V.-light irradiation, a mucoid colony phenotype, an altered cell division cycle, giving rise to minicell, filament and Y-shaped cellular morphologies, an enhanced ability to form tandemly repeated plasmid multimers and an altered sensitivity to the DNA gyrase specific antibiotics novobiocin and nalidixic acid.
A study of plasmid configuration in Ion and Ion sul strains of E.coli, which share similar phenotypic characteristics with the above mutants, revealed that strains which carry a sul or azi mutation express an enhanced capacity for plasmid multimerization.

Finally, no conclusive evidence could be obtained to indicate the presence of a partitioning function on plasmid ColEl.

This study concludes by postulating several factors which may affect the maintenance of plasmids in E.coli K-12.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Plasmids, Escherichia coli, Continuous culture (Microbiology)
Official Date:	July 1986
Dates:	Date Event July 1986 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Mann, Nicholas H. ; Primrose, S. B. ; Robinson, Colin, 1958-
Format of File:	pdf
Extent:	432 leaves : illustrations, charts
Language:	eng

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