Adaptations to a Loss-of-Function Mutation in the Betaproteobacterium Aromatoleum aromaticum: Recruitment of Alternative Enzymes for Anaerobic Phenylalanine Degradation.

Abstract

Anaerobic phenylalanine (Phe) degradation in the betaproteobacterium Aromatoleum aromaticum involves transamination and decarboxylation to phenylacetaldehyde, followed by oxidation to phenylacetate. The latter reaction is catalyzed simultaneously by two enzymes, a highly specific phenylacetaldehyde dehydrogenase (PDH) and a rather unspecific tungsten-dependent aldehyde oxidoreductase (AOR). Attempting to establish increased synthesis of AOR, we constructed a mutant lacking the gene for PDH. This mutant still grew on phenylalanine, exhibiting increased AOR activities on medium containing tungstate. In the absence of tungstate, the mutant showed initially severe growth deficiency, but it resumed growth on Phe after longer incubation times. Moreover, the growth rates of the mutant increased during several reinoculation cycles on either tungstate-proficient or -deficient media, reaching the same values as recorded in wild-type strains. We confirmed AOR as the major alternative enzyme serving Phe degradation under tungstate-supplied conditions and identified and characterized the alternative NAD-dependent aldehyde dehydrogenase AldB taking over the function under tungstate-deficient conditions. Sequence analysis of the respective genes from adapted cultures under either growth condition revealed a mutation in the upstream region of the aor operon and a mutation within the coding region of aldB, which are likely involved in the observed adaptation of the deletion mutant to regain fast growth on Phe.IMPORTANCE The betaproteobacterium Aromatoleum aromaticum degrades many aromatic compounds under denitrifying conditions. One of the steps of phenylalanine degradation is catalyzed by two simultaneously induced enzymes, a NAD(P)-dependent phenylacetaldehyde dehydrogenase and a W-containing aldehyde oxidoreductase. We report here that the latter fully complements a constructed deletion mutant lacking the gene for phenylacetaldehyde dehydrogenase and is overproduced after several reinoculations. Moreover, an alternative NAD-dependent dehydrogenase is recruited to resume growth in tungstate-free medium, which does not allow the production of aldehyde oxidoreductase. This alternative enzyme is overproduced and seems to have acquired a point mutation in the active center. Our research illustrates the flexibility of environmentally important bacteria in adapting their metabolic pathways to new challenges within only a few generations.