A newly isolated strain of Escher ichia coli 1K12 regularly produces a large number of unusually small anucleate cells during the logarithmic phase of growth. These small cells do not divide. They may be isolated from the normal, rod-shaped cells by density gradient centrifugation and have properties that may make them useful in a variety of biological studies. In this report we communicate information regarding some of the basic properties of these miiinicells. A preliminary report of this work has been made.1 Materials and Methods. (1) Organism)is and culture methods: E. coli K12 P678 was obtained from Dr. F. Jacob of the Institut Pasteur, Paris, approximately six years ago and has been maintained on nutrient agar slants. The minicell-producing strain, P678-54, was derived from P678 after treatment of a log-phase nutrient-broth culture with triethylenemelamine (0.5 mg/ml). Both organisms are F-strains that require threonine and leucine. They are unable to utilize lactose, galactose, xylose, maltose, and mannitol as sole carbon sources.For most experiments, these organisms were cultivated in a nutrient broth prepared as described previously.2 In some experiments, appropriately supplemented synthetic medium was used.3(2) Separation of miiinicells: Cultures of P678-54 were centrifuged for 20 minutes at 10,000 X g. The pellet from 1 liter of culture medium was resuspended in approximately 20 ml of 0.067 11 potassium phosphate buffer, pH 6.8.Both minicells and normal cells were separated on sucrose gradients by centrifugation for 45 minutes at 2000 rpm (1000 X g) in a no. 253 swinging bucket rotor in an International model IPRII centrifuge. A 2.0-ml sample of the cell suspension was layered over a 40-ml linear gradient ranging from 5 to 20 per cent sucrose buffered with 0.067 Jf phosphate at pH 6.8S. The sample zones were withdrawn from the gradient, pelleted by centrifugation at 15,000 X g for 15 minutes, and resuspended in the phosphate buffer. All separations were done at approximately 40C.(3) RNA, DNA, and protein analysis: Samples of 1010 cells or 1011 to 1012 minicells were extracted in 5 ml of cold 10 per cent trichloroacetic acid for 30 minutes. After centrifugation the precipitate was resuspended in 3-5 ml of a per cent TCA and hydrolyzed at 100°for 30 minutes. The supernatant was collected after centrifugation, and the pellet digested in 2 ml of 1 N NaOH for 30 nmin. DNA was determined on the supernatant by Burton's modification of the diphenylamine reaction,4 RNA on a 5-10X dilution of the supernatant by the orcinol reaction,5 and protein on a 10-20X dilution of pellet digest by the Lowry method.6 (4) Respiration and enzyme induction: Oxygen consumption was determined in a Warburg apparatus at 370C;7 f-galactosidase was induced by methyl-fl-D-thiogalactopyranoside (TAILG) and assayed by measuring the rate of hydrolysis of 0-nitrophenol f-D-galactoside at 28S0C.8Results.(1) Isolation of mutant, general properties, and morphology: The 321

ADl.ER, HOWARD I. (Oak lRidge National Labotatory, Oak Itidge, Tenn.), AND ALICE A. HARDI-CGREE. Analysis of a genetic locus controlling cell division and sensitivity to radiation in Escherichia (o0i. J. Bacteriol. 87:720-726. 1964.-Radiation senisitivity in Eseherichia (oli is under control of

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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 ...

Changes in colony morphology were associated with the degeneration of solvent-producing strains of Clostridium acetobutylicum. The most efficient solvent-producing strains gave rise exclusively to colonies with dense centers containing large numbers of spores. Many outgrowths of various morphologies developed from the perimeter of such colonies after several days of incubation. The most degenerate cultures did not produce solvents and gave rise to large diffuse colonies that did not contain spores. These diffuse colonies did not produce outgrowths. Intermediate colony types were also observed. These could be derived from liquid cultures that were relatively poor solvent producers or from the outgrowths of colonies of efficient solvent-producing strains. Some of these intermediate types produced spores but did so less frequently than the high-solventproducing strains. The spores of the intermediate types could not be distinguished from those of the most efficient solvent producers on the basis of heat sensitivity. The relationship observed between colony morphology and solvent production provides a method for predicting the solvent-producing potential of C. acetobutylicum cultures.

A mutant strain of Escherichia coli K-12 produced amorphous cells when grown in a variety of media. The lon-allele, known to increase the radiation sensitivity of the cytokinesis mechanism, was introduced into the mutant by means of conjugation. Cells of this recombinant strain grew, after exposure to radiation, into giant amorphous cells, approximately 500 to 1,000 times the volume of a normal E. coli cell. These giant cells are analogous to the filaments formed after the irradiation of lonrod-shaped cells.

Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.