Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Abstract

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by Fparents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by Fparents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', RI, or Col+ parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 106 minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB (ts) Donors by Minicells

“…All strains were grown in a mineral salts solution (12) supplemented with 0.5% Casamino Acids (Difco), 0.5% glucose, and 2 pg of thiamine hydrochloride per ml. The minicell-producing strains were diluted 1:100 into fresh medium and incubated overnight (approximately 9 h) in a rotory shaker at 35 C. The minicells were isolated from their parent cells by sucrose gradient centrifugation as described by Roozen et al (37). The purified minicell preparation was suspended in ice-cold growth medium to an optical density of 0.4 at 620 nm in a Spectronic 20 (Bausch and Lomb) spectrophotometer and kept in an ice bath until needed for a mating.…”

“…The terminated mating mixtures were pelleted by centrifugation at 10,000 rpm in a Sorvall SS34 rotor at 4 C for 10 min. After suspension in 2 ml of BSG, the donor cells and recipient minicells were separated by sucrose gradient centrifugation (37). Both the minicells (which form a band in the upper part of the gradient) and the cells (which form a pellet at the bottom of the tube) were collected, diluted with cold BSG, sedimented in the SS34 rotor as above, suspended in 2 ml of BSG, and purified by a second sucrose gradient sedimentation.…”

Bacteriophage-coded specific enzyme synthesis in minicells of Bacillus subtilis

“…Filtering 15 ml of a clarified solution of crude cell lysate through 0.45/~m membrane filters (Millipore Corp., Bedford, MA) provided adequate phage stocks for induction of phage-coded enzyme synthesis. Isolation of free minicells from the producing culture was achieved following established methods of sucrose gradient centrifugation [1,2]. The final minicell preparation contained approx.…”

Preferential synthesis, localisation and solubilisation of a precursor form of Bacillus subtilis levansucrase in an Escherichia coli minicell system

“…The minicell-producing strain (E. coli AR1062, a restrictionless derivative of P678-54 [11]) carrying the plasmid pLS51 [7] coding for levansucrase synthesis was grown in LB medium containing ampicillin (150/xg/ml) by incubation overnight at 37°C. Minicells were purified according to Roozen et al [12] using a glycerol gradient.…”