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

The denitrifying betaproteobacterium " " EbN1 regulates the capacity to anaerobically degrade -ethylphenol (via -hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ -dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with -hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which -hydroxyacetophenone was added at various concentrations (from 500 μM down to 0.1 nM). Depletion profiles of this aromatic substrate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration of -hydroxyacetophenone, depletion commenced after less than 5 min and suggested a response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degradation and efflux relevant genes (e.g., , encoding a subunit of predicted -ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM. The most pronounced transcriptional response was observed, as expected, for genes located at the beginning of the two operon-like structures, related to catabolism (i.e., ) and potential efflux (i.e., ). Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their concentrations and their bioavailabilities varying over several orders of magnitude. Even though degradation pathways and underlying regulatory systems have long been studied with aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little is known about the effector concentration-dependent responsiveness. EbN1 is a model organism for the anaerobic degradation of aromatic compounds with the architecture of the catabolic network and its substrate-specific regulation having been intensively studied by means of differential proteogenomics. The present study aims at unraveling the minimal concentration of an aromatic growth substrate ( -hydroxyacetophenone here) required to initiate gene expression for its degradation pathway and to learn in principle about the lower limit of catabolic responsiveness