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Analysis of the protein synthesized by Escherichia coliminicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R−minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.

R factor proteins are synthesized in R factor-containing Escherichia coliminicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.

Penicillin has been used to purify large quantities of minicells.

Salmonella enteritidis14C-endotoxin was recovered predominantly from the nuclear and mitochondrial subcellular fractions of livers and spleens of mice and rats, 3.5 hr and 3 days after intravenous administration. Of the recovered radioactivity, 10 to 20% was present in the liver mitochondrial fraction as high-molecular-weight, biologically active material, suggesting the presence of intact endotoxin. Autoradiographic studies demonstrated nuclear and cytoplasmic labeling in the liver and at least nuclear label in spleen cells. The resistance of rats, as compared to mice, to the induction of amyloidosis does not appear to be based on a difference in subcellular localization of endotoxin within the reticuloendothelial system.

Levy, StuartB. (Institut du Radium, Paris, France). Isologous interference with ultraviolet and X-ray irradiated bacteriophage T2. J. Bacteriol. 87:1330–1338. 1964.—Qualitative and quantitative analysis of the interference capacity of an irradiated T2bacteriophage was made with ultraviolet and X-ray irradiation. Two different effects were found to explain the total interference picture in the ultraviolet-irradiated system: exclusion and depression. Exclusion is the absolute inhibition of infectious phage growth in the bacterial host. Depression is the diminution of burst size in instances where the infectious phage has not been excluded. Both effects were seen when the infectious phage was added after the addition of ultraviolet-irradiated phage. Doses between 1,600 and 2,200 ergs/mm2(survivals, ca. 10−7) showed the greatest exclusion effect (70%). Exclusion was lost between 6,500 and 7,500 ergs/mm2. The depression effect was highest (90%) at lower doses (survivals, ca. 10−6), falling off as the dose range went above 1,600 ergs/mm2or survivals of 10−7. Depression was lost at 3,000 ergs/mm2. X-ray irradiation (both direct and indirect) to survivals less than 10−2showed no interference capacity in the phage irradiated. Indirect X-ray irradiation to survivals between 5 and 10% showed 50% exclusion, but no depression.

Both fi+and fi−type R factors function as intact replicative units when segregated into Escherichia coliminicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R+minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R+minicells in contrast to R−minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.