The predicted σ54-dependent regulator EtpR is essential for expression of genes for anaerobic p-ethylphenol and p-hydroxyacetophenone degradation in “Aromatoleum aromaticum” EbN1

Abstract

BackgroundThe denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 anaerobically utilizes a multitude of aromatic compounds via specific peripheral degradation routes. Compound-specific formation of these catabolic modules is assumed to be mediated by specific transcriptional activators. In case of the recently elucidated p-ethylphenol/p-hydroxyacetophenone pathway, the highly substrate-specific regulation was implicated to involve the predicted σ54-dependent, NtrC-type regulator EbA324. The latter was suggested to control the expression of the two neighboring gene clusters encoding the catabolic enzymes as well as a corresponding putative solvent efflux system. In the present study, a molecular genetic approach was used to study the predicted function of EbA324.ResultsAn unmarked in frame ΔebA324 (here renamed as ΔetpR; p-ethylphenol regulator) deletion mutation was generated. The ΔetpR mutant was unable to grow anaerobically with either p-ethylphenol or p-hydroxyacetophenone. Growth similar to the wild type was restored in the ΔetpR mutant background by in trans expression of plasmid-born etpR. Furthermore, expression of the "p-ethylphenol" gene clusters as well as corresponding protein formation was shown to depend on the presence of both, EtpR and either p-ethylphenol or p-hydroxyacetophenone. In the wild type, the etpR gene appears to be constitutively expressed and its expression level not to be modulated upon effector presence. Comparison with the regulatory domains of known phenol- and alkylbenzene-responsive NtrC-type regulators of Pseudomonas spp. and Thauera aromatica allowed identifying >60 amino acid residues in the regulatory domain (in particular positions 149 to 192 of EtpR) that may contribute to the effector specificity viz. presumptively restricted effector spectrum of EtpR.ConclusionsThis study provides experimental evidence for the genome predicted σ54-dependent regulator EtpR (formerly EbA324) of "A. aromaticum" EbN1 to be responsive to p-ethylphenol, as well as its degradation intermediate p-hydroxyacetophenone, and to control the expression of genes involved in the anaerobic degradation of these two aromatic growth substrates. Overall, the presented results advance our understanding on the regulation of anaerobic aromatic compound catabolism, foremost based on the sensory discrimination of structurally similar substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0571-9) contains supplementary material, which is available to authorized users.

Comparative Genomic Analysis of the Regulation of Aromatic Metabolism in Betaproteobacteria

“…BH72, where AphT regulates itself and the divergent operon comprised of distant homologs of the four aforementioned genes, as well as genes encoding COG589 universal stress protein, methyl-accepting chemotaxis transducer, and regulator LapR (Figure 4 and Supplementary Table S2). These proteins are possibly involved in sensing some aromatic compounds (as it has been shown that several methyl-accepting chemotaxis proteins are induced while growing on aromatic substrates), coping with their toxic concentrations, and solvent stress response (Wöhlbrand et al, 2008; Carmona et al, 2009; Büsing et al, 2015). LapR, a close homolog of AphR, has been described as an alkylphenol metabolism regulator (Jeong et al, 2003), hence it should control transcription of the adjacent lap genes of the alkylphenol metabolism (close homologs of aph ), forming a regulatory cascade AphT–LapR, but no candidate LapR (AphR-type) binding sites have been identified upstream of the lap genes.…”

Responsiveness of Aromatoleum aromaticum EbN1 T to Lignin-Derived Phenylpropanoids

“…Complementation of pprR in trans into DpprR deletion mutant. In trans-complementation was performed as described in two previous studies (29,30) and using the primers listed in Table 1. Essentially, a 1,434-bp fragment containing the pprR gene together with its ribosomal binding site was amplified from genomic DNA of A. aromaticum EbN1 T as described above.…”

Towards the Response Threshold for p -Hydroxyacetophenone in the Denitrifying Bacterium “Aromatoleum aromaticum” EbN1

“…An accordingly substrate-specific regulation was demonstrated on the basis of targeted transcript analysis as well as differential proteomic and enzymatic profiling (16,20,21). Furthermore, generation of an unmarked ⌬etpR in-frame deletion mutation resulted in loss of anaerobic growth with p-ethylphenol and p-hydroxyacetophenone as well as respective transcript and protein formation, underpinning the essential role of EtpR (22).…”