Attempts to detect by physicochemical methods plasmid DNA in mycoplasmas of human origin before and after transformation to tetracycline resistance.

Physicochemical methods have been used to compare mycoplasma DNA capable of the genetic transformation of tetracycline resistance with DNA from tetracycline-sensitive mycoplasmas and their transformants. These mycoplasmas were isolated from human patients. The DNA extracted from Mycoplasma hominis tetr resistant to 100 microgram/mL tetracycline transforms tetracycline resistance to sensitive strains of Mycoplasma salivarium tets and Mycoplasma hominis tets but not Mycoplasma fermentans tets. Bulk DNA and DNA extracted by methods which increase the yield of circular DNA moieties were analyzed by cesium chloride and cesium chloride--ethidium bromide buoyant density ultracentrifugation and by horizontal and vertical agarose gel electrophoresis. Extrachromosomal DNA was not detected, which suggests that transformation was mediated by the recombination of chromosomal genes for tetracycline resistance and not by R factors. Moreover, no significant differences were detected in the DNA from the resistant and sensitive species or from their transformants and Mycoplasma fermentans tets which could not be transformed to resistance to 10 micrograms tetracycline/mL medium.

Preparation of competent single-cell suspensions of Mycoplasma hominis tets and Mycoplasma salivarium tets for genetic transformation to tetracycline resistance by DNA extracted from Mycoplamsa hominis tetr.

DNA extracted from Mycomplasma hominis (Sprott strain), resistant to 100 micrograms of tetracycline/ml transformed M. hominis strain H29 and Mycoplasma salivarium strain S9, which are sensitive to 2.5 and 5.0 micrograms of tetracycline/ml, respectively, to resistance. The transformants were selected on agar medium containing 10 micrograms of tetracycline/ml. Some transformants were resistant also to 20 micrograms of tetracycline/ml, a finding confirming that transformation occurred between homologous and heterologous species and that resistance is stepwise and controlled by several genetic loci. Medium containing 10 micrograms of tetracycline/ml was bacteriostatic. Prototype experiments employing mixtures of strains that were tetr and tets (tetracycline-resistant and tetracycline-sensitive, respectively) demonstrated that tetr mutants and transformants formed typical fried-egg colonies when mixtures containing not more than 10(9) mycoplasmas were spread on tetracycline agar plates. No mutants to tetracycline resistance were detected. Both M. hominis and M. salivarium were competent after treatment with MgCl2 and CaCl2, while Mycoplasma orale type 2 was inactivated. During DNA extraction different quantities of DNA formed insoluble precipitates with protein, thus preventing quantitative experiments.