| Description |
The development of a neonatal mouse model system for the detection of exfoliative toxin (ET) made it possible to score for the exfoliative toxin marker in genetic studies. As a result of initial studies, it was demonstrated that the genes controlling the production of ET were not associated with a temperate phage, but were probably located on plasmid DNA. The objectives of this study were: (i) to demonstrate the extrachromosomal location for the genes controlling ET production by phage group II staphylococci, (ii) to determine if genetic markers other than the one for ET synthesis occupied the ET plasmid, (iii) to isolate the ET plasmid, and (iv) to characterize the physical properties of the ET plasmid. The ability of two staphylococcal strains to produce ET was lost at a high frequency after growth at 44 C or in the presence of ethidium bromide (EB) or sodium dodecyl sulfate (SDS). The ability of EB to eliminate ET production was investigated further. Using the optimum innoculum size and concentration of EB, the rate of appearance of Tox~ variants was determined. The early appearance, rapid doubling time, and lack of a selective growth advantage of Tox~ variants in EB suggested that the loss of genes for ET synthesis was due to the elimination of a plasmid. The coordinate loss of genetic markers on the same plasmid should occur at a high frequency. The ability of strain UT 0007 to produce penicillinase, coagulase, and /3 hemolysin could not be associated with the loss of ET production. However, the ability of strain UT 0007 to produce a bacteriocin and ET were co-eliminated at a high frequency after growth in EB or at high temperatures. These data suggested that the genes for bacteriocin synthesis were on the ET plasmid. In order to further demonstrate the extrachromosomal location of the genes for ET production, two substrains were isolated after growth of UT 0007 in the presence of EB. Substrain UT 0101 produced both ET and bacteriocin, while substrain UT 0100 produced neither product. Extrachromosomal DNA was isolated by cesium chloride-EB buoyant density centrifugation of SD3-sodium chloride lysates prepared from substrain UT 0101 (Tox+ , Bac+), while no extrachromosomal DNA was found in the cured substrain UT 0100 (Tox-, Bac-). Two species of extrachromosomal DNA were isolated from substrain UT 0101 with sedimentation values of 563 and 383. The 38S species was identified as the open circular (0G) form of the 563 covalently closed circular (CGC) form. The molecular 7 weight of the 563 ET plasmid was calculated to be 2.9 x 10' daltons. Therefore, a single plasmid was isolated from substrain UT 0101 (Tox+, Bac+) and was identified as the ET plasmid. The 56S ET plasmid isolated by Brij detergent cocktail lysis of substrain UT 0101 was rapidly converted to the 383 OC form by treatment with agents known to denature proteins (pronase, SDS, and alkali) or storage at 4 C or -70 C# These data suggested that the ET plasmid was a DNA-protein relaxation complex. Finally, the complete conversion of the 56S CCC DNA to the 38S OC form by treatment with alkali suggested that all of the plasmid DNA existed as the DNA-protein relaxation complex. |
