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Gas chromatographic-mass spectrometric analysis on the distribution of alpha-ketodicarboxylic acids in various bacteria determined that alpha-ketoglutarate and alpha-ketoadipate are widely distributed in all the bacteria examined, whereas alpha-ketopimelate and alpha-ketosuberate are found only in the methanogenic archaebacteria. Labeling experiments with stable isotopes indicated that each of these acids arises from alpha-ketoglutarate by repeated alpha-ketoacid chain elongation. The final product in this series of reactions, alpha-ketosuberate, serves in the methanogenic bacteria as the biosynthetic precursor to the 7-mercaptoheptanoic acid portion of 7-mercaptoheptanoylthreonine phosphate, a methanogenic coenzyme.

The yeast Candida tropicalis HP 15 was able to utilize phenol up to concentrations of 2.5 g/l as a sole carbon and energy source. Phenol was metabolized via the beta-ketoadipate pathway by an inducible enzyme system. Besides phenol, resorcinol, quinol, hydroxyquinol, catechol, and to a lesser extend 4-chlorocatechol, protocatechuate, p-cresol, m-chlorophenol, and p-chlorophenol were metabolized by the yeast. A total of 30 aromatic compounds were tested as substrates.

The pathway of mandelate metabolism in Pseudomonas aeruginosa is composed of the following steps: l(+)-mandelate --> benzoylformate --> benzaldehyde --> benzoate. These three steps are unique to mandelate oxidation; the benzoate formed is further metabolized via the beta-ketoadipate pathway. The first enzyme, l(+)-mandelate dehydrogenase, is induced by its substrate. The second and third enzymes, benzoylformate decarboxylase and benzaldehyde dehydrogenase, are both induced by benzoylformate. The same benzaldehyde dehydrogenase, or one very similar to it, is also induced by beta-ketoadipate, an intermediate in the subsequent metabolism of benzoate. This dehydrogenase may also be induced by adipate or a metabolite of adipate. These conclusions have been drawn from the physiological and genetic properties of wild-type P. aeruginosa strains and from the study of mutants lacking the second and third enzyme activities.

Crude cell-free extracts of d-glucarate-grown cells of Bacillus megaterium converted d-glucarate to alpha-keto-beta-deoxy-d-glucarate (KDG). Charcoal-treated cell-free extracts or partially purified enzyme preparations converted KDG to an intermediate which was isolated and identified as 2,5-diketoadipate (DKA). This compound was synthesized, and the cell-free extracts of d-glucarate grown cells were found to catalyze the reduction of nicotinamide adenine dinucleotide (NAD) in its presence. In the absence of NAD, the same enzyme preparation catalyzed the decarboxylation of the DKA to alpha-ketoglutarate semialdehyde (KGS), whereas in the presence of NAD the KGS was subsequently oxidized to alpha-ketoglutarate by alpha-ketoglutarate semialdehyde dehydrogenase. Since galactarate-grown B. megaterium contains a galactarate dehydrase forming KDG, the complete pathway for the metabolism of d-glucarate or galactarate to alpha-ketoglutarate and CO(2) is now known in a gram-positive bacterium.

Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD(+))-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD(+)-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD(+)-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD(+)-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.

The regulation of the synthesis of the inducible enzymes that mediate the reactions of the beta-ketoadipate pathway in Alcaligenes eutrophus has been examined by determining the inductive responses of the wild type and of mutants derived from it to metabolites of the pathway. The system of control differs in many respects from those which operate in the genera Pseudomonas and Acinetobacter.

The isolation of several mutant strains blocked in l-lysine degradation has permitted an assessment of the physiological significance of enzymatic reactions related to lysine metabolism in Pseudomonas putida. Additional studies with intact cells involved labeling of metabolic intermediates from radioactive l- or d-lysine, and patterns of enzyme induction in both wild-type and mutant strains. These studies lead to the conclusions that from l-lysine, the obligatory pathway is via delta-aminovaleramide, delta-aminovalerate, glutaric semialdehyde, and glutarate, and that no alternative pathways from l-lysine exist in our strain. A distinct pathway from d-lysine proceeds via Delta(1)-piperideine-2-carboxylate, l-pipecolate, and Delta(1)-piperideine-6-carboxylate (alpha-aminoadipic semialdehyde). The two pathways are independent in the sense that certain mutants, unable to grow on l-lysine, grow at wild-type rates of d-lysine, utilizing the same intermediates as the wild type, as inferred from labeling studies. This finding implies that lysine racemase in our strain, while detectable in cell extracts, is not physiologically functional in intact cells at a rate that would permit growth of mutants blocked in the l-lysine pathway. Pipecolate oxidase, a d-lysine-related enzyme, is induced by d-lysine and less efficiently by l-lysine. Aminooxyacetate virtually abolishes the inducing activity of l-lysine for this enzyme, suggesting that lysine racemase, although functionally inactive for growth purposes, may still have regulatory significance in permitting cross-induction of d-lysine-related enzymes by l-lysine, and vice versa. This finding suggests a mechanism in bacteria for maintaining regulatory patterns in pathways that may have lost their capacity to support growth. In addition, enzymatic studies are reported which implicate Delta(1)-piperideine-2-carboxylate reductase as an early step in the d-lysine pathway.