Formation of 20 beta-dihydrosteroids by anaerobic bacteria.

Cortisol was metabolized to a variety of products, among them small amounts of cortol by fecal flora of humans and rats. A microorganism, Bifidobacterium adolescentis, isolated from both sources, synthesized a 20-hydroxysteroid dehydrogenase which reduced cortisol to 20 beta-dihydrocortisol. The metabolite was reduced to cortol by Clostridium paraputrificum. The 20-hydroxysteroid dehydrogenase showed a wide substrate specificity; it was independent of the 4-ene and the configuration at C3, C-11, C-17 and C-21. Cortol was resistant to any further alteration by human fecal flora, i.e. it is a metabolic end product. As expected, B. adolescentis effectively prevented 21-dehydroxylation of cortisol by Eubacterium lentum.

Side-chain cleavage of cortisol by fecal flora.

The side chain of certain C-21 steroids may be removed by an enzyme, desmolase, synthesized by intestinal bacteria. With a view to isolate these organisms we examined the conditions required for their multiplication and function. The model substrate, cortisol (11 beta, 17 alpha, 21-trihydroxy-4-pregnene-3,20-dione), was metabolized by mixed fecal flora of humans and rats to a number of C-21 and C-19 compounds. The major C-21 metabolites were 21-deoxycortisol, tetrahydro-21-deoxycortisol, and tetrahydrocortisol. The C-19 metabolites obtained were identified as 5 beta-androstane-3 alpha, 11 beta-triol and 5 xi-androstane-3 alpha, 11 beta-diol-17-one. The prevalence of converting microorganisms was approximately 10(6)/g feces in both humans and rats. Conversion required an Eh below -130 mV, and an initial pH of 7.0. Optimal yield of C-19 products occurred with a fecal dilution of 10(5), though C-19 metabolites were evident from 10(1) through 10(8) fecal dilutions. Preliminary investigation indicates that the ability of converting organisms to form colonies varied with the composition of the media and the gaseous environment.

The preparation of transforming DNA from Mycoplasma hominis strain Sprott tetr and quantitative studies of the factors affecting the genetic transformation of Mycoplasma salivarium strain S9 tets to tetracycline resistance.

DNA extracted by a standard method from Mycoplasma hominis Sprott, resistant to 100 micrograms tetracycline, permitted the quantitative genetic transformation of tetracycline-sensitive Mycoplasma salivarium to resistance. The yield was 1 microgram DNA/10(9) cells. This DNA enabled determination of the optimum conditions for making M. Salivarium competent with CaCl2 and for studying some factors affecting transformation. Mycoplasma salivarium was transformed to resistance to 10, 20, and 30 micrograms tetracycline but not to 40 micrograms. The optimum DNA concentration for transforming resistance to 10, 20, and 30 micrograms tetracycline was the same, i.e., 50 micrograms DNA/10(8) viable cells. Treatment with DNase indicated that DNA uptake took 30 min. Competition between transforming DNA and DNA from calf thymus and M. salivarium tets inhibited transformation.