The proportion of Phase II colonies of Shigella sonnei appearing on a medium based on a papain digest of ox heart-muscle was compared with the proportion appearing on nutrient medium and a statistically significant difference noted.
A chemically defined medium was then developed for the cultivation of the organism and the mutation Phase I – Phase II studied using single cell cultures. A mutation rate of the order of 2 x 10-3 was found.
The parent and mutant were then studies in an effort to establish the basis of the trypaflavine agglutination of Phase II cells. It was shown that the surface antigens, which are chemically complex, differ in properties such as electrophoretic mobility and serological specificity and that the properties of the whole cells in this regard can be related to those of the surface antigens. In addition, the parent and mutant were shown to share at least one antigen which is not a surface antigen. The electrophoretic behaviour of trypaflavine-treated suspensions showed that the stability Phase II cells in suspension in aqueous medium depends largely on the ζ-potential at the cell surface while that of Phase I does not. Trypaflavine, by abolishing the ζ-potential of Phase II cells therefore causes their agglutination.
The amino-acid metabolism of Shigella sonei was then examined in order to study the enhanced growth of the Phase II mutant in papain digest medium. The organism was found to utilise selectively the amino acids serine, alanine, glutamic acid and arginine from acid-hydrolysed casein medium. These acids were found also to produce best growth when a full range of amino-acids was tested singly for the ability to act as sole nitrogen source. When cultures were made in chemically defined medium, valine was shown to accumulate. Isoleucine in concentrations up to 500 µM (65 µg./ml.) was shown to be inhibitory to all strains of Shigella sonnei tested, the sensitivity of Phase I strains being generally higher than that of Phase II. Isoleucine inhibition was found to be reversed be valine, inhibition of Phase II cells being more readily reversed than that of Phase I. Subsequent study of the media used showed that these aspects of the amino-acid metabolism of the organism are of significance in determining the selection of one or other of the Phases during cultivation.
The amino-acid composition of a papain-digest of ox heart-muscle was determined by quantitative paper chromatography. It contained 1435 µM (188 µg./ml/) isoleucine and 1504 µM (176 µg./ml.) valine, that is an isoleucine valine ratio of 0.95. Difco nutrient broth was shown to contain 608 µM (79 µg./ml.) isoleucine and 1300 µM (152 µg./ml.) valine, that is a molar ratio of 0.46.
Kinetic studies in which the cell counts and the proportion of Phase I and Phase II cells in culture, both in liquid and on solid medium were determined, indicated that the isoleucine : valine ratio was the major factor in determining the preponderance of either parent or mutant. High ratios favoured the mutant, low ratios, the parent. Thus the enhanced growth of Phase II cells in papain digest medium was ascribed to the high isoleucine : valine ratio of 0.95.
The biochemical basis of isoleucine inhibition and its reversal by valine was studies by means of artificially produced, nutritionally exacting mutants. A series of experiments is described which indicate that isoleucine exerts inhibition by giving rise within the cells to α-keto-β-methyl-valeric acid (“ketoisoleucine”) which acts as a competitive inhibitor of the conversion of “ketovaline” to valine.