DNA isolated from Escherichia coli minicells mated with F+ cells.

Abstract

A mutant of Escherichia coli K-12 produces large numbers of small cell-like structures during growth in a variety of media. These "minicells" are enclosed by an apparently normal cell wall and membrane and contain protein and RNA. They do not contain detectable amounts of DNA. ' We have asked if minicells can act as recipients of DNA during conjugation and, if so, what the properties of the transferred DNA are. This report establishes that minicells do conjugate with F+ donors and acquire DNA. Properties of the transferred DNA are discussed.Matsubara3 suggested that the F episome exists in zygotes as a linear duplex with a molecular weight of 1.0 X 108 daltons. Freifelder and Freifelder2 showed that, after replication in recipient cells, the F episome exists as a double-stranded circle with a molecular weight of 4.4 X 107 daltons. DNA isolated from minicells mated with F+ donors may represent stages in the conversion of transferred episome to vegetative episome.Materials and Methods.-(a) Materials: DNA was isolated from E. coli P678-54, the minicell producing mutant, and X15 cells by the Marmur procedure.4 Egg-white lysozyme was purchased from Sigma Chemical Company (St. Louis, Mo.). Pronase was purchased from Calbiochem (Los Angeles, Calif.). Before use, a solution of 20 mg/ml pronase was incubated at 370C for 60 min to destroy contaminating DNase. DNase was purchased from Worthington Biochemical (New York, N. Y.).E. coli exonuclease I was a gift from Drs. Paul Sadowski and Jerard Hurwitz. The preparation used in the experiments described in this paper has a specific activity of 1700 units/mg protein and contains 10 mg/mI.5 (b) Organisms and culture methods: X15 is a nonlysogenic, F + prototroph;6 P678-54 is a minicell-producing F-strain;' and X696 is an F + minicell producer which is a recombinant of X15 and P678-54. The growth-and-mating medium was L-broth.7 When bacteria were labeled with H3-thymidine, 5 jic/ml of thymidine methyl-H3 (6 c/mmole, Schwarz BioResearch, Inc., Orangeburg, N. Y.) and 100 jig/ml of adenosine8 were added to the medium. To label cells with C"4-thymidine, 1 /Ac/ml thymidine-2-C"4 (51.2 mc/mmole, Schwarz BioResearch) and 200 jig/ml adenosine were added to the medium. All incubations were at 37°C.(c) Mating procedure: With 5 ml of a stationary phase P678-54 culture, 500 ml of L-broth were inoculated and incubated on a rotary shaker for about 3 hr. The culture was then centrifuged at 4000 rpm for 10 min in the GSA rotor of an RC-2B refrigerated centrifuge (Ivan Sorvall, Inc., Norwalk, Conn.). The minicells were harvested from the supernatant fluid by centrifugation at 8000 rpm in the GSA rotor for 10 min and resuspended in medium. This procedure removes more than 99% of the normal-sized cells. Log phase X15 cells were inoculated into 50 ml of medium containing H3-or C"4-thymidine at a level of 107 cells/ml. After 2-hr incubation in standing cultures, the cells were harvested by centrifugation and resuspended in nonlabeled medium. Mating mixtures contained about 5 X 109 F+ cells and 1010 ...

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

“…For example, the highly conserved T-region border sequences are the functional equivalents of the oriT, acting as recognition and cleavage sites for the site-specific nicking complexes (35,51,55). Furthermore, both transfer processes involve singlestranded DNA transfer intermediates (1,12), and both require cell-to-cell contact (28,39). Results from heterologous transfer experiments strengthen the idea that T-DNA transfer is a modified conjugal process.…”

The ATPase Activity of the DNA Transporter TrwB Is Modulated by Protein TrwA

“…As it happened in the presence of TrwA, both linear and circular supercoiled dsDNA stimulated TrwB⌬N70 ATPase activity. Although the ATPase rates in the presence of dsDNA are high in vitro, the in vivo role for TrwB acting on dsDNA remains unclear, since it is generally accepted that ssDNA is the translocated substrate during conjugation (31,32). This capability to work with both ssDNA and dsDNA has also been described for several hexameric helicases, such as DnaB or T7 phage gp4 helicase.…”

Immunization with non-replicating E. coli minicells delivering both protein antigen and DNA protects mice from lethal challenge with lymphocytic choriomeningitis virus

“…), they do not contain a chromosome, eliminating the possibility of pathogenic reversion in this approach. The ability of minicells to contribute to horizontal gene transfer to other pathogenic strains of bacteria is minimal as it has been well documented that bacterial mating into minicells is possible while the opposite is not [11,39,40].…”