Aromatic ring cleavage by a Thiobacillus.

As part of a study on the assimilation of organic compounds by autotrophic microorganisms, enrichment cultures were set up for thiobacilli. A facultatively autotrophic Thiobacillus was isolated in pure culture and grew heterotrophically on many organic compounds including p-hydroxybenzoate (PHBz). Since degradation of aromatic compounds is carried out by a limited range of bacterial genera, the mechanism of aromatic ring cleavage was investigated in the Thiobacillus isolate. Aromatic ring cleavage was established in Hydrogenomonas facilis, but the mechanism has not been elucidated (2). Two distinct ring cleavage mechanisms have been established among the aerobic pseudomonads (1). The isolate is assigned to the genus Thiobacillus because it grows autotrophically at the expense of thiosulfate, which is oxidized to sulfate. The organism grows best at pH 8.0 to 9.0, and a complete description will be published. The physiological properties relevant to the metabolism of PHBz are discussed in this note. The organism grew on a chemically defined medium, in the absence of thiosulfate, with 5 mM PHBz as sole carbon and energy source, and cultures could be transfered between heterotrophic and autotrophic media. Replica platings were made from well-isolated colonies on autotrophic media with thiosulfate to heterotrophic media with PHBz. Both PHBz and protocatechuic acid (PCA) were oxidized by suspensions of PHBz-grown organisms, but not by suspensions of thiosulfate-grown organisms. Oxidation of PHBz and PCA was biphasic and did not go to completion. PCA was completely used up, as judged by the catechol test (3). The reaction products gave an intense violet color in the Rothera test (5), suggesting the occurence of 3-keto-adipate, a key intermediate in the "orthocleavage" mechanism. Cell-free extracts were made by sonic disruption of a suspension of cells in 100 mm tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.0), and by centrifugation for 20 min at 20,000 X g followed by 2 hr at 96,000 x g. Such extracts oxidized PCA, but were without effect on PHBz or catechol. Approximately 1 ,umole of oxygen was consumed, and 1 ,umole of carbon dioxide was produced per ,umole of PCA added (Table 1). These results are consistent with the "orthocleavage" mechanism. Since ,B-keto-adipic acid is somewhat unstable and is not readily available in a high state of purity, its formation from PCA in extracts of the Thiobacillus was confirmed by a number of indirect tests. Apart from the characteristic color reaction in the Rothera test, the chemical decarboxylation of f3-keto-adipate to levulinate by 4-amino-antipyrine at pH 4.0 (6) was used for