Regulation of the enzymes concerned in the degradation of aromatic compounds in pseudomonas putida

Pseudomonas putida metabolises aromatic carboxylic acids by convergent pathways. The regulons for the synthesis of the enzymes involved are controlled by induction and repression by intermediates of the pathways. The results of previous studies in this system suggest that the organism should exhibit a selective enzymic response to mixed aromatic substrates. It has been the aim of this study to predict the enzymic response to mixtures of mandelate, benzoate and parahydroxybenzoate and then to check these predictions experimentally.
The affinities of these three inducer substrates for their regulons have been determined by measuring the initial rates of enzyme synthesis over a range of inducer concentrations in cultures growing on glucose. Glucose itself was shown to have very little catabolite repressor effect in this system. From the results, an induction constant, defined as the inducer concentration for half-maximum rate of induction, was estimated for each substrate. Those for the benzoate and mandelate regulons were similar (c. 0.5 mM) and much higher than that for parahydroxybenzoate hydroxylase (andlt; 0.01 mM).
By measuring the repressor effect or each aromatic substrate on the induction by one of the other substrates, repression constants (Krep) were also obtained that are a quantitative measure of the affinity of the repressor for that regulon. Mandelate had no repressor activity and the benzoate regulon was not repressed by parahydroxybenzoate. The mandelate regulon was severely repressed by p-OH benzoate (Krep = 0.02 mM) while the effect of benzoate on the parahydroxybenzoate regulon was much less pronounced (Krep = 1.0 mM) . Benzoate had a transient,severe effect on the mandelate regulon and, at high concentrations, a moderate permanent form of repression was observed.
The enzymic reponse of the organism following a changeover from growth on a single aromatic carbon source to growth in the presence of an additional aromatic carbon source was predicted by comparing the induction and repression constants for the substrates concerned. Batch cultures were used to provide an indication of the steady state enzymic reponse, which was obtained using a nitrogen-limited chemostat operated at high growth rates. In general, the predictions were experimentally validated.
Benzoate oxidase was always fully induced in mixtures containing benzoate, while the mandelate enzymes were almost completely repressed in the presence of parahydroxybenzoate. The mandelate regulon was only partially induced in mixtures containing mandelate and benzoate as was the parahydroxybenzoate regulon in cells grown on parahydroxybenzoate and benzoate. The extent to which the enzymes for the less preferred carbon source were induced was affected by the relative concentrations of the substrates.
The results indicate that comparison of induction and repression constants can be used to predict the enzymic behaviour of this organism towards mixed aromatic substrates and it is suggested that this may form the basis of a general method for predicting enzymic adaptation to mixed substrates.
Relative substrate utilisation can only be inferred from the induction and repression constants if complete repression of an enzyme system is predicted. Further data are necessary whenever the predictions indicate that more than one enzyme system will be induced. Thus, in this study, it was predicted, and experimentally verified in the chemostat, that mandelate would not be used by cells growing in the presence of mandelate + p-OH benzoate. When induction of two enzyme systems we predicted, the relative utilisation of the substrates was qualitatively related to the enzymic content of the cells.
In contrast to other systems where the reaction to mixed substrates seems to be directed towards the selection of the substrate capable of supporting the fastest growth rate, Pseudomonas putida selects, not the best growth substrate (mandelate), but the substrate requiring the synthesis of fewer enzymes for its metabolism (benzoate). Factors in the environment acting as possible selective agents are discussed.
In the course of this study, it was also found that the synthesis of the mandelate enzymes wan delayed when low external inducer concentrations were used. At high concentrations, enzyme synthesis rapidly followed the addition of inducer. A pre-induction or maintenance effect was also observed in which cells induced at saturating concentrations of mandelate were able to maintain a maximum rate of induction when transferred to sub-saturating inducer concentrations. Induced cells were able to accumulate mandelate from low external concentrations. These results are discussed in terms of an inducible mandelate transport system.
The severe transient repression of the mandelate regulon by benzoate was greatly reduced by pre-induction with mandelate. Benzoate inhibited the accumulation of mandelate by the cells and the action of benzoate on the mandelate regulon is discussed in relation to the possible induction mechanisms of the mandelate regulon.

Advisor: Mandelstam, J