Circular DNA forms of a bacterial sex factor.

Bacteria are known to possess extrachromosomal genetic elements that can be classified as plasmids or episomes. Bacterial plasmids are extrachromosomal genetic elements that exist solely in the autonomous state,1 while bacterial episomes are capable of existing either in an autonomous state or stably attached to the bacterial chromosome.2 Certain bacterial sex factors, or genetic elements that promote the transfer of the bacterial chromosome, exhibit the property of episomes (see Clark and Adelberg3). Among the episomal sex factors are certain colicinogenic factors, extrachromosomal genetic elements that determine the production of extracellular, antibiotically active proteins, termed colicins. One of these elements, colicinogenic factor VB (ColVB), has been shown in Escherichia coli to stably integrate a portion of its genome into the bacterial chromosome as an alternative to existing in an autonomous state.4' I A derivative of the ColVB factor, the FColVColBtrycys factor, has been obtained by Fredericq and shown to carry genetic determinants for bacteriophage T1 sensitivity, the production of colicins V and B, and the biosynthesis of tryptophan and cysteine.4-6 The FColVColBtrycys factor also promotes bacterial conjugation and the transfer both of itself and the bacterial chromosome to female cells and, therefore, is a sex factor.' 7. 8 Theoretical considerations and genetic studies of the interaction of episomes with the bacterial chromosome have led to the prediction that sex factors are circular molecules."2 In the present study the sex factor FColVColBtrycys was transferred to a Proteus mirabilis strain and identified in this strain as doublestranded DNA with a buoyant density of 1.710 gm/cm3 in a cesium chloride gradient. The physical-chemical properties and the appearance in electron micrographs of the isolated FColVColBtrycys factor demonstrate that this sex factor is a circular DNA molecule. Materials and Methods.-Bacterial strains: The Proteus mirabilis strain used in these studies was the nicotinic acid-requiring Proteus strain AC 2505 (from A. B. Pardee). A tryptophanand thymine-requiring mutant of this strain was isolated by treatment with N-methyl-N'-nitroN-nitrosoguanidinel3 and trimethoprim,14 respectively. This mutant Proteus strain was made colicinogenic for the FColVColBtrycys factor by crossing with an E. coli strain, YS57, carrying this sex factor. The YS57 FColVColBtrycys strain was obtained from C. Yanofsky. The FColVColBtrycys factor was isolated by P. Fredericq and shown to carry at least the following sequence of genes: F,cysB,tryA,tryB,tryD,tryC,Tl,ColV,ColB.6 Colicins V and B produced by the Proteus FColVColBtrycys strain showed an antibiotic specificity range that was indistinguishable from that of the colicin produced by E. coli strains carrying this factor. The sex factor FColVColBtrycys is relatively unstable in the Proteus strain. Spontaneously occurring noncolicinogenic variants of the Proteus FColVColBtrycys strain were obtained at a frequency of 20% by plating on nutrient agar plates a culture growing logarithmically in the presence of tryptophan. To counterselect noncolicinogenic tryptophan-requiring variants formed by the spontaneous loss of the FColVColBtrycys factor, cultures of the colicinogenic Proteus strain were grown in a medium lacking tryptophan. Reagents: Lysozyme, diisopropylfluorophosphate-treated (DFP-treated) trypsin, and ribonuclease (3X crystallized) were purchased from Worthington Biochemicals. Salmon sperm