Nanomolar Responsiveness of an Anaerobic Degradation Specialist to Alkylphenol Pollutants.

Anaerobic degradation of -cresol (4-methylphenol) by the denitrifying betaproteobacterium EbN1 is regulated with high substrate specificity, presumed to be mediated by the predicted σ-dependent two-component system PcrSR. An unmarked, in-frame Δ deletion mutant showed reduced expression of the genes (21-fold) and (8-fold) that encode the two enzymes for initial oxidation of -cresol to -hydroxybenzoate compared to their expression in the wild type. The expression of and was restored by in complementation with in the Δ background to even higher levels than in the wild type. This is likely due to ∼200-/∼30-fold more transcripts of in the complemented mutant. The responsiveness of EbN1 to -cresol was studied in benzoate-limited anaerobic cultures by the addition of -cresol at various concentrations (from 100 μM down to 0.1 nM). Time-resolved transcript profiling by quantitative reverse transcription-PCR (qRT-PCR) revealed that the lowest -cresol concentrations just affording and expression (response threshold) ranged between 1 and 10 nM, which is even more sensitive than the respective odor receptors of insects. A similar response threshold was determined for another alkylphenol, -ethylphenol, which strain EbN1 anaerobically degrades via a different route and senses by the σ-dependent one-component system EtpR. Based on these data and theoretical considerations, -cresol or -ethylphenol added as a single pulse (10 nM) requires less than a fraction of a second to reach equilibrium between intra- and extracellular space (∼20 molecules per cell), with an estimated (dissociation constant) of <100 nM alkylphenol (-cresol or -ethylphenol) for its respective sensory protein (PcrS or EtpR). Alkylphenols (like -cresol and -ethylphenol) represent bulk chemicals for industrial syntheses. Besides massive local damage events, large-scale micropollution is likewise of environmental and health concern. Next to understanding how such pollutants can be degraded by microorganisms, it is also relevant to determine the microorganisms' lower threshold of responsiveness. EbN1 is a specialist in anaerobic degradation of aromatic compounds, employing a complex and substrate-specifically regulated catabolic network. The present study aims at verifying the predicted role of the PcrSR system in sensing -cresol and at determining the threshold of responsiveness for alkylphenols. The findings have implications for the enigmatic persistence of dissolved organic matter (escape from biodegradation) and for the lower limits of aromatic compounds required for bacterial growth.