Abstract
Our knowledge of the metabolism of Clostridium botulinum is at present scanty. Wagner, Meyer and Dozier 1 analyzed the products of the action of Cl. botulinum on complex media but obtained little information as to the reactions by which the products had been formed. Knight, 2 summarizing the growth-requirements of Cl. botulinum, reports that the simplest medium which will support growth contains the amino-acids proline, glycine, leucine, alanine, lysine, and cystine, together probably with traces of tryptophan and of the “sporogenes growth factor.” He suggests that Cl. botulinum may obtain its energy through the “Stickland reaction,” a coupled oxido-reduction between pairs of amino-acids. Glycine and proline act as hydrogen acceptors while alanine and leucine act as hydrogen donators in the case of Cl. sporogenes (Stickland 3 ). Therefore these amino-acids were tested to determine whether or not they are attacked directly as is true for Cl. tetanomorphum with various amino-acids (Woods and Clifton 4 ) or by coupled reactions between pairs of amino-acids.
All the experiments reported below were carried out with washed suspensions of Cl. botulinum, Type B (E-44). 800 ml of plain broth plus 0.1% yeast extract were inoculated with 0.5 ml of a beef-brain culture of this organism and incubated for 20 hours at 37°C in a McIntosh and Fildes anaërobic jar. The culture was then centrifuged, the cells were washed in saline and finally suspended in phosphate buffer of pH 7.4. The suspension so prepared was immediately deaërated in vacuo. The usual Thunberg-tube method was employed for the detection of hydrogen donators and it was found that alanine and leucine were the only amino-acids of those essential for growth which reduced methylene blue readily.
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