Differences in Response of a Virulent Strain of the Tubercle Bacillus and Its Avirulent Variant to Metabolites and Their Genetic Significance

Abstract

When colonies of virulent tubercle bacilli were maintained on solid egg media, Steenken (1935) found that eventually lysis occurred and new colonies of bacteria resistant to the lytic process appeared which proved to be avirulent. The phenomenon was observed in several human strains and in bovine strains as well (Steenken, 1938). Similar results were obtained when colonies from single cell cultures were grown to the point of lysis, indicating that the resistant colonies were not merely the result of selection of pre-existing resistant cells (Steenken, 1938). The distinguishing characteristics of the virulent and avirulent strains were maintained indefinitely on subculture, indicating that the virulent and avirulent strains differed genetically. Because of its long history (Steenken and Gardner, 1946) the strain H37Rv and its avirulent variant H37Ra were selected for investigation of the possible metabolic and genetic differences between the two strains. The results to be reported here show that the virulent and avirulent forms differ in their response to 16 different metabolites. The frequency of appearance of avirulent colonies (Steenken, 1938) suggests a single gene mutation as the basis for the difference between H37Ra and H37Rv. However, difficulty is encountered in reconciling the finding of a multiplicity of differences in response to metabolites in the two strains, with a single gene mutation, if the hypothesis that each enzyme is controlled by a single gene is accepted. Methods. In a liquid medium, the H37 strain of tubercle bacillus will synthesize all the materials it requires for growth from glucose as the sole carbon source and ammonium chloride as the source of nitrogen (Schaefer, Marshak, and Burkhart, 1949). The medium was NH4Cl-1.0 g, Na2HPO4-12H20-6.5 g, KH2PO4-1.0 g, MgSO4 7H20-0.02 g, CaCl2-0.0005 g, and FeCl3-6H20-0.0005 g dissolved in 1 liter of distilled water that also contained sorbitan monooleate ("tween 80") in a concentration of 0.02 per cent. After being autoclaved, sterile 50 per cent glucose was added to a final concentration of 0.1 per cent and 5 per cent sterile bovine serum albumin2 to a concentration of 0.01 per cent. Cultures of tubercle bacilli in 6 ml of Dubos medium (Davis and Dubos, 1947) grown in slanted screw-cap tubes, 2 cm by 13 cm, for 9 to 10 days were centrifuged, and the cells were washed twice in the foregoing medium and then resus

Studies on the Metabolism of Mycobacterium Tuberculosis Vi

“…The results of these studies indicated a decreased metabolic efficiency of the avirulent mammalian strains tested since these were unable to utilize some as yet undefined substance in bovine serum which markedly stimulated the growth of the H37Rv strain. Differences in the utilization of amino acids (Dubos, 1949;Marshak, 1951), purines and pyrimidines (Marshak, 1951), and of organic acids (Dubos, 1950) also have been reported.…”