Cyanide production and degradation by chromobacterium violaceum

Chromobacterium violacewn is one of a small number of bacteria that have previously been found to produce inorganic cyanide. Glycine is known to be the substrate for cyanide formation by these organisms but the mechanism by which cyanide is formed from glycine is unknown. Methionine has been found to stimulate cyanogenesis by bacteria; again the mechanism is unknown. C.violaceum also possesses the ability to metabolise the cyanide produced, and the formation of the amino acids g-cyanoalanine, asparagine, aspartate and y-cyano-a-aminobutyric acid from cyanide by non­proliferating cells has previously been demonstrated. This study is an attempt to investigate further the conditions of cyanide production by C.violaceum, when grown on L-glutamate as carbon and nitrogen source, and the relationship between the formation of cyanide and its utilisation or degradation. Although glycine is the substrate for cyanide production the relation­ship between the glycine concentration of the growth medium, and the amount of cyanide produced was found to be more complex than a simple precursor- product relationship. At low glycine or methionine concentrations glycine was found to inhibit cyano-genesis but this inhibitory effect was overcome at high glycine or methionine concentrations. Glycine is postulated to partially repress synthesis of the cyanide producing system. Cyanide is produced during the transition from exponential growth to the stationary phase of growth, and work with an inhibitor of protein synthesis suggests that the cyanide producing system is induced during exponential growth. Increases in the ferrous ion and phosphate concentrations of the growth medium were found to stimulate cyanide production but variations in growth 605C0:@63:0 and initial pH value of the growth medium, over ranges which supported growth, had no effect on cyanide production. The conditions of cyanide production are typical of those of microbial secondary metabolites and it is suggested that cyanide is such a metabolite. C.Kiolacevm has previously been found to contain two cyanide utilising enzymes, g-cyanoalanine synthase and y-cyano-a-amincbutyric acid synthase. The presence of these two activities is confirmed, and a third cyanide utilising enzyme, rhodanese, was also detected. The three cyanide utilising enzymes are found to be induced towards the end of exponential growth in cells growing under high cyanide - (i.e. grown on glutamate supplemented with glycine and methionine) or low cyanide - (i.e. grown on glutamate alone) producing conditions. This occurs at a time when the cyanide content of the medium is increasing and cyanide is postulated to induce the synthesis of the cyanide utilising enzymes. Inclusion of glycine in the growth medium partially represses the induction of B-cyanoalanine synthase and y-Myano-a-amino butyric acid synthase, and methionine is shown to inhibit S-Myanoalanine synthase activity. B-cyanoalanine was found to accumulate in the medium of stationary phase cultures but neither y-cyano-a-amino- butyric acid nor thiocyanate could be detected, suggesting that B-cyano­alanine synthase is the major enzyme involved in further metabolism of cyanide. No evidence could be found for the conversion of B-cyanoalanine to asparagine and aspartate. Addition of chloramphenicol to low- and high- cyanide evolving cultures was found to have a marked effect on the medium cyanide levels. These observations are shown to have been caused by chlor­amphenicol blocking the induction of the cyanide utilising enzymes and the resulting inhibition of B-cyanoalanine formation from cyanide. A scheme (Fig.41) is put forward to explain the effects of glycine and methionine on the cyanide content of growing cultures of C.violaoeum.