Anaerobic Degradation of p -Ethylphenol by “ Aromatoleum aromaticum ” Strain EbN1: Pathway, Regulation, and Involved Proteins

Abstract

The denitrifying "Aromatoleum aromaticum" strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure (ϳ15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol-and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.Alkylphenols, such as p-ethylphenol, are present in coal tars and crude oils (2, 45). Besides the accidental release of fuelderived alkylphenols to the environment, phenolic compounds are also prominent constituents of petrochemical wastewaters arising from spent caustic and coal gasification. Alkylphenol concentrations in these effluents (mainly phenol, cresols, and ethylphenols) range from 100 to 68,000 mg liter Ϫ1 , depending on the source (2). Moreover, p-ethylphenol can also be plant derived; e.g., native olive oils contain up to 52 mg p-ethylphenol per kg, and the content can reach 470 mg kg Ϫ1 during storage (5). p-Ethylphenol can also be formed from p-coumaric acid, a major component of cereal cell walls, by several yeast and Lactobacillus species (11,50). Due to their cytotoxicity and relatively high water solubility (www.epa.gov/safewater/mcl .html), alkylphenols are of environmental concern.Anaerobic degradation of aromatic compounds requires reactions independent of molecular oxygen. They are fundamentally different from the oxygenase-catalyzed reactions employed under oxic conditions (for an overview, see references 14, 17, and 18). A variety of anaerobic aromatic compounddegrading bacteria were newly isolated during the last decades (for an overview, see references 46 and 51). Most of them are denitrifiers belonging to the "Aromatoleum"/Azoarcus/Thauera cluster within Betaproteobacteria (R. Rabus et al., unpublished data). "Aromatoleum aromaticum" strain EbN1, a metabolically versatile representative of this group, was originall...

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…The chromosome of strain EbN1 harbors two additional gene clusters, which display high sequence similarity to the gene cluster comprising the ebA1926 to ebA1936 cluster, and they exhibit the same gene order: (i) the cluster comprising the ebA326 to ebA335 cluster, which is likely involved in anaerobic p-ethylphenol and p-hydroxyacetophenone metabolism (see above), and (ii) the cluster comprising ebA5762 to ebA5768, which is located near to the catabolic genes involved in a putative phenolic compounds degradation pathway (see above). Interestingly, these three paralogous gene clusters are specifically expressed in the presence of different aromatic compounds, which correlate well with the genomic content in each case (379). The products of the ebA326 to ebA335, ebA1926 to ebA1936, and ebA5762 to ebA5768 genes were specifically formed during anaerobic growth on p-ethylphenol (379), toluene (203), and phenol/p-cresol (378), respectively.…”

“…Thus, 7.2 kb upstream of the bbs cluster, there are five genes (ebA1926 to ebA1936) organized in an operon-like structure, two of which (ebA1932 and ebA1936) encode proteins that are highly induced when the cells were grown in toluene and whose putative promoter region shows significant similarity to that of the bbs and bss operons, agreeing well with its toluene-specific induction (203). Within this operon-like structure, the ebA1926 gene may encode a universal stress-like protein, and the ebA1928-ebA1932-ebA1936 genes may encode a putative toluene-specific RND-type efflux pump involved in coping with toxic toluene concentrations (203,379). Interestingly, ebA1932 and ebA1936 are highly similar to the products of orf1 and orf2 from T. aromatica K172.…”

“…b Data taken from data reported previously by Matsunaga et al (236) and from the M. magneticum AMB-1 genome project at the NCBI. c Data taken from data reported previously by Rabus et al (293), Rabus (290), and Wöhlbrand et al (379) and from the Azoarcus sp. strain EbN1 genome project at the NCBI.…”

“…7). Nevertheless, hydrolytic cleavage of the carboxylation product without prior CoA activation cannot be excluded (379). All the predicted enzymes involved in anaerobic p-ethylphenol degradation are encoded in a large operon-like structure on the chromosome of Azoarcus sp.…”

“…Both operon-like structures harbor the Ϫ12/Ϫ24 consensus sequence of 54 -dependent promoters located around 100 bp upstream of the translational start of the acsA and ebA335 genes, suggesting the transcriptional control of the corresponding promoters by the ebA324 regulator. The participation of ebA327-ebA332-ebA335 in a so-far-uncharacterized solventspecific efflux system analogous to the RND (resistance nodulation cell division)-type ArcA-ArcB-TolC efflux pump that is known to be involved in solvent tolerance in several gramnegative bacteria has been proposed (379). The fact that the ebA326 gene product shows domains related to the cytoplasmic universal stress protein (UspA) from E. coli is also in agreement with the potential participation of the ebA326 to ebA335 gene products in a specific solvent stress system in p-ethylphenol and p-hydroxyacetophenone metabolism (379).…”