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Molecular Studies on Entry Exclusion in Escherichia coli Minicells

University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

ABSTRACT

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R⁺ donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd⁺ gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).

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• Marrero, J., Waldor, M. K. (2007). Determinants of Entry Exclusion within Eex and TraG Are Cytoplasmic. J. Bacteriol. 189: 6469-6473 [Abstract] [Full Text]