Glutamine in the production of tetanus toxin.

A strain of Cbstridium tetani under investigation in this laboratory produces high titers of exotoxin when cultivated on a medium containing trypsin-digested protein and high concentrations of iron salts (1). Toxin production fails when acidor alkali-hydrolyzed protein (casein) is substituted for the enzyme digest, and it has been shown (2) that at least two acid-labile substances or groups of substances are required in order to obtain suitable yields. In addition to these, certain amino acids (tryptophan, tyrosine, phenylalanine, and cystine) are known to be required and others will undoubtedly prove necessary as the simplification of the medium proceeds. This phase of the investigation has been hampered by the fact that certain free amino acids exert an inhibitory effect on toxin production. This is particularly true of synthetic serine, in which instance the inhibition has been shown (3) to arise from the presence of the D form of the compound. It has now been possible to show that glutamine is involved in growth and toxin formation. Glutamic acid, while it favors growth, has led to low yields of toxin. Although there appears to be relatively little free dicarboxylic amino acid in the tryptic digest of casein used in our experiments, both glutamic acid and aspartic acid are readily recognized after acid hydrolysis of almost every peptide-containing fraction which has been prepared from the material, by whatever method of separation. Presumably some of these peptides contain glutamine rather than glutamic acid, and in a form which is both relatively heat-stable and available to the tetanus organism. Much time may be required to isolate, characterize, and test individually such peptides; nor can suitably quantitative comparisons be made until other unknown factors can be supplied to the medium in purified form. It consequently seems desirable to report at this time the general fact that glutamine is involved, leaving for subsequent reports a further quantitative elaboration of the observation. Glutamine has, of course, been shown to act as a growth essential or stimulant for a number of bacterial species (4, 5), although its functions in microbial growth are still largely matters of speculation (6, 7). There is also a possibility that glutamine enters into the strepogenin molecule