Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus

Abstract

Novel nuclear magnetic resonance spectroscopy techniques, designated metabolic 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 ( 2 H) medium were used to inoculate cultures in which natural-abundance 1 H hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise 2 H medium. Samples taken during the incubation of these cultures were analyzed by proton nuclear magnetic resonance spectroscopy, and proton signals were correlated with the corresponding aromatic compounds or their metabolic descendants. This approach allowed the identification and quantitation of metabolites which accumulated during growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about which relatively little is known. A. calcoaceticus initiates aromatic compound dissimilation by forming catechol or protocatechuate from a variety of substrates. Degradation proceeds via the ␤-ketoadipate pathway, comprising two discrete branches that convert catechol or protocatechuate to tricarboxylic acid cycle intermediates. As shown below, when provided with several carbon sources simultaneously, all degraded via the ␤-ketoadipate pathway, A. calcoaceticus preferentially degraded specific compounds. For example, benzoate, degraded via the catechol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To determine if this preference were governed by metabolites unique to catechol 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 presence of benzoate. The accumulation of high levels of cis,cis-muconate also appeared to be toxic to the cells.Despite extensive research on bacterial hydrocarbon metabolism, little is known about how the presence of multiple carbon sources influences the degradation of an individual compound. In these studies, a new nuclear magnetic resonance (NMR) spectroscopy technique was used to monitor the catabolism of aromatic compound mixtures by the soil bacterium Acinetobacter calcoaceticus. This technique, designated metabolic observation NMR (MO-NMR), is based on the principle that NMR spectrometers will not detect deuterated ( 2 H) compounds when tuned to proton ( 1 H) frequencies (11,14,20,32). As described in this report, A. calcoaceticus was grown in fully deuterated medium, thereby rendering the bacterial cultures spectroscopically invisible. Mixtures of 1 H aromatic compounds were added to the deuterated cultures as sources of carbon and energy, and samples were taken at specific times. NMR spectra of the samples were used to monitor the metabolic fates of the 1 H compounds over time; these spectra provided information about the identity and the accumulation of meta...

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

“…Benzoate is only one of many aromatic compounds degraded via the catechol branch of the ␤-ketoadipate pathway (37). Tight transcriptional control is needed to regulate benzoate degradation in environments with multiple growth substrates to prevent the build up of toxic metabolites, such as muconate (38). Thus, the synergistic response of BenM to two pathway metabolites, muconate and benzoate, provides an elegant mechanism for fine tuning transcription in a complex catabolic pathway.…”

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

“…In contrast, there is no evidence of the catechol branch of the pathway in the Roseobacter group isolates (4; www.tigr.org) or in other pca-containing ␣-proteobacteria, including A. tumefaciens C58, S. meliloti 1021, M. loti MAFF303099, or C. crescentus CB15, based on genome sequence analyses. Organisms possessing both branches of the pathway may require additional regulatory mechanisms for dictating a preferential hierarchy in substrate uptake when presented with the mixture of aromatic compounds found in natural environments (1,15,29). This crossregulation between the two branches of the pathway might serve to prevent mismatched interactions between the structurally similar enzymes and substrates of the pathway (3).…”

Benzoate Catabolite Repression of the Phthalate Degradation Pathway in Rhodococcus sp. Strain DK17

“…One interesting observation was the preferred metabolism of benzoate over 4-hydroxybenzoate (4-HBA) in some gram-negative soil bacteria, such as Acinetobacter and Pseudomonas spp. (8,21).…”