THE BACTERIAL OXIDATION OF AROMATIC COMPOUNDS V. C14

B. P. Sleeper
1951 Journal of Bacteriology  
There is good evidence (Stanier, 1950) that the bacterial oxidation of benzoic acid proceeds via catechol, but the biochemical mechanism of catechol formation is completely obscure at the present time. Since all likely aromatic intermediates, including phenol, the monohydroxybenzoic acids, 2,3-and 3 ,4-dihydroxybenzoic acid, and salicylaldehyde have been excluded either on the basis of unutilizability or through the analysis of adaptive patterns , it seems probable that the conversion is
more » ... nied by a temporary partial dearomatization of the benzene ring. Unfortunately, the enzyme system catalyzing the reaction is inactivated during the preparation of dried cells , and hence the problem cannot yet be investigated on the enzymatic level. It appeared possible, however, that some additional information about the process could be obtained from an analysis of the metabolism of specifically-labeled benzoic acids by intact cells, which was accordingly undertaken. During the course of this work the ultimate metabolic fate of two of the carbon atoms in benzoic acid was also determined. MATERIALS AND METHODS Pseudomona fluorescerts, strain A .3.1 (Stanier, 1948) , was used in all experiments on living cells. This strain, whose metabolism of aromatic compounds is otherwise typical, accumulates unusually large amounts of catechol during the oxidation of benzoic acid under unfavorable physiological conditions, a property which was advantageous for the purposes of the present work. In order to obtain an accumulation of catechol, cells were grown on yeast extract, harvested, and resuspended at a high density in M/30 phosphate buffer (pH 7.0) to which was added an amount of benzoic acid (previously neutralized and dissolved in the same buffer mixture) sufficient to provide a final substrate concentration of M/50. The mixture was then dispensed in amounts of 50 ml into Erlenmeyer flasks of 500 ml capacity, and the flasks were closed with rubber stoppers and incubated at 30 C. The stoppers were subsequently removed only to obtain periodic aliquots for analysis. The production of catechol was followed colorimetrically by the method of Evans (1947). It had usually reached a maximum after 40 to 44 hours. At this time, the bacterial cells were removed by centrifugation, the pH of the supernatant liquid was adjusted to 8.0, and the catechol was
doi:10.1128/jb.62.5.657-662.1951 fatcat:wczcn2hd2zezzglma2lkudqw6e