NOVEL NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY METHODS DEMONSTRATE PREFERENTIAL CARBON SOURCE UTILIZATION BY ACINETOBACTER-CALCOACETICUS

Novel nuclear magnetic resonance spectroscopy techniques, designated m etabolic observation, were used to study aromatic compound degradation by the soil bacterium Acinetobacter calcoaceticus. Bacteria which had been rendered spectroscopically invisible by growth with deuterated ( H-2) medium were used to inoculate cultures in which natural-abundance H-1 hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise H-2 medium. Samples taken during the incubation of th ese cultures were analyzed by proton nuclear magnetic resonance spectr oscopy, and proton signals were correlated with the corresponding arom atic compounds or their metabolic descendants, This approach allowed t he identification and quantitation of metabolites which accumulated du ring growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about w hich relatively little is known. A. calcoaceticus initiates aromatic c ompound dissimilation by forming catechol or protocatechuate from a va riety of substrates. Degradation proceeds via the beta-ketoadipate pat hway, comprising two discrete branches that convert catechol or protoc atechuate to tricarboxylic acid cycle intermediates, As shown below, w hen provided with several carbon sources simultaneously, all degraded via the beta-ketoadipate pathway, A. calcoaceticus preferentially degr aded specific compounds. For example, benzoate, degraded via the catec hol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To d etermine if this preference were governed by metabolites unique to cat echol degradation, pathway mutants were constructed, Studies of these mutants indicated that the product of catechol ring cleavage, cis,cis- muconate, inhibited the utilization of p-hydroxybenzoate in the presen ce of benzoate. The accumulation of high levels of cis,cis-muconate al so appeared to be toxic to the cells.