Sensitive and selective phenol sensing in denitrifying Aromatoleum aromaticum EbN1 ^T

Abstract

Aromatoleum aromaticum EbN1 T anaerobically degrades phenol, p -cresol, and p -ethylphenol, each via a distinct peripheral pathway. The compound-specific regulation of each pathway is proposed to occur on the transcriptional level in the case of phenol supposedly mediated by the one-component system PheR. To confirm its predicted function, we generated an unmarked, in-frame deletion mutant (Δ pheR ). This mutant did not express the ppsA1 gene, which encodes the A1 subunit of phenol-activating phenylphosphate synthase. The expression of ppsA1 was restored by in trans complementation of pheR into the Δ pheR background. The responsiveness to phenol was studied in vivo in benzoate-limited anaerobic cultures by adding, upon benzoate depletion, single defined pulses of phenol (from 100 µM down to 0.1 nM). Time-resolved, targeted transcript profiling by qRT-PCR revealed a response threshold for ppsA1 expression of 30‒50 nM phenol. Notably, ppsA1 expression could not be induced by p -cresol or p -ethylphenol. Conversely, lack of expression was also observed for the additional target genes cmh ( p -cresol degradation) and acsA1 ( p -ethylphenol degradation) applying phenol or p -ethylphenol as well as phenol or p -cresol as stimuli. Thus, the sensory proteins PheR, PcrS, and EtpR should be highly selective for phenol, p -cresol, and p -ethylphenol, respectively. The implicated incapability of cross-stimulus binding was corroborated by comparing the predicted 3D structural models of the proteins’ sensory domains. While the ligand-binding pockets share the conserved hydroxy group-anchoring histidine and tryptophane, their distal faces in PcrS and EtpR are, compared to PheR, enlarged to accommodate the bulkier methyl ( p -cresol) and ethyl group ( p -ethylphenol), respectively. IMPORTANCE Aromatic compounds are globally abundant organic molecules with a multitude of natural and anthropogenic sources, underpinning the relevance of their biodegradation. A. aromaticum EbN1 T is a well-studied environmental betaproteobacterium specialized on the anaerobic degradation of aromatic compounds. The here studied responsiveness toward phenol in conjunction with the apparent high ligand selectivity (non-promiscuity) of its PheR sensor and those of the related p -cresol (PcrS) and p -ethylphenol (EtpR) sensors are in accord with the substrate-specificity and biochemical distinctiveness of the associated degradation pathways. Furthermore, the present findings advance our general understanding of the substrate-specific regulation of the strain’s remarkable degradation network and of the concentration thresholds below which phenolic compounds become essentially undetectable and as a consequence should escape substantial biodegradation. Furthermore, the findings may inspire biomimetic sensor designs for detecting and quantifying phenolic contaminants in wastewater or environments.