On the metabolism of tryptophan by Agrobacterium tumefaciens

Abstract

In connection with investigations on the selective inhibition of tumorous growth in plants, an analysis of the formation of indoleacetic acid (IAA) from tryptophan (Tryp) by the Crown-gall-inducing organism Agrobacterium tumefaciens was carried out by chromatography of extracts from the culture medium. Tryp is generally recognized as the principal precursor of IAA, oxidative deamination or transamination presumably leading to indolepyruvic acid (IPyA) as an intermediate. Its occurrence in biological media had not been proved conclusively, however. Synthetic IPyA appeared to be an extremely labile substance under the conditions of chromatography (especially with alkaline solvents), decomposing into at least seven compounds, two of which were identified as IAA and indolealdehyde (IAld) and it seems probable that indoleglycollic acid was present. Analysis by ultraviolet spectrography pointed to an enol-form for crystalline IPyA, while in solution a complex, pH-dependent tautomeric equilibrium, together with decomposition reactions were found. Most of the spots on the chromatograms of the culture media turned out to be artefacts, which are due to spontaneous breakdown of Tryp (by the extraction procedure) and IpyA (during chromatography). The appearance of the highly specific chromatographic pattern of IPyA decomposition provided definite proof for the occurrence of IPyA in the media. Indolelactic acid (ILA) and tryptophol (Tol) were identified as products of physiological origin. Under anaerobic conditions they are the main products, IPyA very probably being their immediate precursor. By varying the duration of the incubation it was shown that during the first 23 h IAA is formed mainly, IPyA, ILA and Tol accumulating in the next 15 h. By using Tryp labelled with 14C in different positions and by radioautography, conclusive evidence was obtained for the indole character of the IPyA-decomposition products. In a trial to characterize IPyA in the culture medium by reaction with 2,4-dinitrophenylhydrazine, a yellow compound was formed, identical with a similar reaction product from synthetic IPyA, which could not, however, be identified as the 2,4-dinitrophenylhydrazone of IPyA. Pure crystalline indolealdehyde was isolated from the medium.