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).In 1958 Lederberg observed that conjugal matings between two F + strains were less efficient than matings between an F+ strain and a strain that did not harbor an F fertility factor. Currently it is accepted that transmissible plasmids or episomes usually are transferred less efficiently to strains that carry similar or related elements, suggesting that a determinant linked to the sex factor confers on the cell immunity against conjugal infection by an isogenic or closely related sex factor. Novick (23) has suggested that this immunity is the result of two distinct effects, "entry exclusion" and "plasmid incompatibility."Incompatibility is considered to be operative after deoxyribonucleic acid (DNA) enters the recipient cell and is evidenced by the segregation of the newly transferred plasmid out of the population. It has been postulated that the failure of the incoming plasmid to replicate normally and its subsequent loss reflect a lack of available attachment sites (16). The occupa-tion of a maintenance site(s) by the resident plasmid presumably blocks the attachment of a similar or isogenic plasmid. However, the maintenance site model does not explain all cases of incompatibility (see Novick [23] for detailed discussion).On the other hand, it has been suggested that entry exclusion imposes a barrier to the physical transfer of DNA betwee...

A minicell-producing strain of Salmonella typhimurium contains two cryptic plasmids. One has a molecular weight of 2.6 x 106 to 2.8 x 106, is present in multiple copies per cell, and segregates into minicells. The other has a molecular weight of 130 x 106, is present in few copies per cell, and probably does not segregate into minicells. A variety of bacterial strains have been shown to harbor circular plasmid deoxyribonucleic acid (DNA) of unknown function (cryptic DNA). Cryptic DNAs, having molecular weights as indicated in parenthesis, have been obtained from Escherichia coli 15(1.5 x 106, 63 x 106, and 104 x 106) (7, 12), Salmonella pullorum (1.47 x 106, and 55 x 106) (15, 18), Salmonella typhimurium LT2(62 x 106) (10), Shigella paradysenteriae (3.4 x 106 and 1.0 x 106) (13), Shigella dysenteriae (106, 1.3 x 106, 2.6 x 106, 3.8 x 106, 20 x 106 and 24 x 106) (21), Micrococcus lysodeikticus (0.88 x 106) (16), and Bacillus megaterium (1.96 x 106 to 58.8 x 106) (4). We report here on two cryptic circular DNA species present in a S. typhimurium strain (X-1313), which is an abnormal cell division mutant isolated by W. L. Tankersley (Master's thesis, University of Tennessee, 1970), that buds off minicells, similar to those formed by the Escherichia coli K-12 strain described by Adler et al. (1). Minicells, which are easily separated from the cells that produce them, lack chromosomal DNA but may contain extrachromosomal DNA (20) and therefore are useful for the isolation and characterization of such extrachromosomal elements. Overnight growth of the Salmonella minicellproducing strain X1313 in the presence of tritiated thymidine ([3H]dThd) produced minicells containing about eight times more acidinsoluble radioactivity than minicells from the

Pseudomembranous colitis, a severe diarrheal disease, has been linked to the administration of antibiotics and to two toxins produced by Clostridium difficile. Eighty-two strains of C. difficile isolated from humans and hamsters were assayed for the presence of plasmid DNA. Agarose gel electrophoresis of Sarkosyl-lysed cells indicated that 18% of the strains contained from one to four plasmids. The plasmid DNA in these strains ranged in molecular weight from 2.7 x 106 to 60 x