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E. coli growth inhibition by a high copy number derivative of plasmid pBR322
M S Valenzuela1, E V Ikpeazu, K A Siddiqui
1Division of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208, USA. valenz44@ccvax.mmc.edu
Abstract:
We have observed that plasmid pKH47, a pBR322-derivative containing a 100bp poly(dA)-poly(dT) insertion, causes growth inhibition of host E. coli cells harboring it. In this paper we show that this inhibitory effect is due to an increased copy number property of this plasmid, which is turn leads to an over expression of the plasmid-encoded tet gene. Our work also indicates that contrary to other pleiotropic effects caused by the tet gene product, which solely depend on the expression of the 5' end of the gene, growth inhibition requires an intact tet gene. In addition we present the isolation of an E. coli mutant that is refractive to the inhibitory effect of pKH47 and shares some properties with the parental bacteria containing plasmid pKH4.
Insights
Plasmid pKH47 causes E. coli growth inhibition due to high copy number and tet gene overexpression. This effect requires an intact tet gene, unlike other pleiotropic effects.
Area of Science:
- Molecular Biology
- Bacterial Genetics
Background:
- Plasmids like pBR322 are crucial tools in molecular biology.
- The tet gene product can have various effects on host cells.
Purpose of the Study:
- To investigate the mechanism behind the growth inhibition caused by plasmid pKH47 in E. coli.
- To determine the role of the tet gene and its expression in this phenomenon.
- To isolate and characterize E. coli mutants resistant to pKH47-induced growth inhibition.
Main Methods:
- Construction and characterization of plasmid pKH47, a pBR322 derivative with a poly(dA)-poly(dT) insertion.
- Analysis of plasmid copy number and tet gene expression in E. coli.
- Genetic analysis to determine the requirement of an intact tet gene for growth inhibition.
- Isolation and phenotypic analysis of E. coli mutants resistant to pKH47.
Main Results:
- Plasmid pKH47 exhibits increased copy number in E. coli host cells.
- The increased copy number leads to overexpression of the plasmid-encoded tet gene.
- Growth inhibition is directly linked to tet gene overexpression and requires an intact tet gene.
- An E. coli mutant resistant to pKH47's inhibitory effects was isolated, sharing properties with cells harboring a related plasmid.
Conclusions:
- The growth inhibitory effect of pKH47 in E. coli is mediated by high plasmid copy number and subsequent tet gene overexpression.
- Unlike other pleiotropic effects, pKH47-induced growth inhibition necessitates a functional, intact tet gene.
- The identification of a resistant mutant provides a tool for further understanding the host-plasmid interactions and resistance mechanisms.