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 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 resumed growth on Phe after longer incubation times. Moreover, the growth rates of the mutant increased during several re-inoculation 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 operon and a mutation within the coding region of , which are likely involved in the observed adaptation of the deletion mutant to regain fast growth on Phe. The betaproteobacterium 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 re-inoculations. 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.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1

“…A. aromaticum EbN1 is known to grow with the aromatic amino acids Phe and Tyr as sole substrates (16), but growth on related amines has not been tested so far. We observed that this organism also grows very well on the primary aromatic amines benzylamine (BAm) and 2-phenylethylamine (2-PEA) under nitrate-reducing conditions, reaching an optimal growth rate of 0.11 h Ϫ1 , which is equal to that observed with Phe (20). In contrast, no growth was detected with one of the secondary amines (R)-and (S)-1phenylethylamine as the substrate.…”

“…Further degradation pathways of aromatic amines. In analogy to Phe or benzyl alcohol degradation in A. aromaticum EbN1 (20,21), we expected that the aldehydes generated by the amine dehydrogenases are further oxidized by aldehyde dehydrogenases or a tungsten-containing aldehyde oxidoreductase (AOR). Cells grown with 2-PEA or Phe, but not with other substrates, indeed contained a highly specific phenylacetaldehyde dehydrogenase (PDH) using NAD or NADP as electron acceptors with a characteristic ratio of activities of 0.56, as described in reference 22 (Table 1).…”

“…We also checked the degradative activities of a deletion mutant of A. aromaticum lacking the gene for PDH (strain SR7_Δpdh), which compensates for this enzyme with a markedly increased AOR activity (20). The mutant also grew with 2-PEA reaching slightly lower growth rates than the wild type and exhibited similar specific activities of 2-PEA dehydrogenase (Table 1).…”

“…However, a derivative of strain SR7_Δpdh, which has adapted to growth on Phe in the absence of tungstate by using a mutant version of the aldB gene product as an alternative NAD-dependent phenylacetaldehyde (PAld) dehy- In the wild-type strain, growth capabilities similar to those observed for the aromatic amines were expected if the corresponding alcohols, BAlc or 2-phenylethanol (2-PE), were offered as sole substrates. Growth of A. aromaticum EbN1 with BAlc has been recognized and studied before (17,20), and the enzyme activities obtained from cultures grown with benzyl alcohol or benzaldehyde (Table 1) indicate that benzylamine shares the same downstream metabolic pathway. However, 2-PE surprisingly did not support growth under nitrate-reducing conditions, even at lowered concentrations to avoid potential toxic effects or after prolonged incubation times of up to 6 weeks.…”

“…This is confirmed by similar adaptive complementations of the degradation pathways of Phe or 2-PEA in a knockout mutant lacking PDH (strain SR7_⌬pdh). PDH activity was replaced by increased AOR activity in the presence or by another induced NAD-specific aldehyde dehydrogenase (AldB) in the absence of tungstate (20), although the observed growth rates of the adapted strains were lower than those of the wild-type cells, and the AldB-overproducing mutant did not arise spontaneously in 2-PEA-degrading cultures, as observed with Phe (20). This may be explained by a higher rate of PAld production from 2-PEA than from Phe, as indicated by significantly increased PDH activities in wild-type cells grown with 2-PEA compared to that with Phe and high PAld accumulation in failed cultures of strain SR7_⌬pdh with 2-PEA in the absence of tungstate.…”