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

Abstract

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 of an anaerobic degradation specialist.

Nanomolar Responsiveness of an Anaerobic Degradation Specialist to Alkylphenol Pollutants

“…A recent molecular genetic study demonstrated that the predicted 54 dependent one-component system EtpR controls the expression of the gene cluster for the anaerobic degradation of p-ethylphenol and p-hydroxyacetophenone (18). Furthermore, a subsequent physiological study revealed an in vivo response threshold of A. aromaticum EbN1 for p-hydroxyacetophenone in the range of 1 to 10 nM (19). p-Cresol is assumed to enter cells of A. aromaticum EbN1 via passive diffusion.…”

“…Furthermore, gene expression for this degradation pathway is mediated by the 54 -dependent transcriptional activator EtpR (18). The response threshold for p-ethylphenol was determined with an experimental setup and effector concentration range analogous to those described above for p-cresol, targeting three selected p-ethylphenol catabolic genes, acsA (encoding acetoacetyl-CoA synthetase), hped [encoding 1-(4-hydroxyphenyl)ethanol dehydrogenase], and pchF (herein renamed emhF; encoding ethylphenol-methylhydroxylase) for transcript profiling as previously used for p-hydroxyacetophenone (19). Highly reproducible growth curves with sampling time points for each of the eight p-ethylphenol concentrations tested and a negative control (no addition of p-ethylphenol) are provided in Fig.…”

“…The genes selected for targeted transcript analysis ( Fig. 4B) are located at the beginning (acsA), in the middle (hped), and at the end (emhF) of the large, 16.4-kb p-ethylphenol catabolic gene cluster (17,19). The onset of detectable transcript level increase agreed very well with the earlier findings for p-hydroxyacetophenone (19), albeit with 2-fold-higher levels for the latter (acsA, 18-versus 40-fold after 5 min; hped, 10-versus 26-fold after 30 min; and emhF, 7.5-versus 14-fold after 60 min).…”

“…4B) are located at the beginning (acsA), in the middle (hped), and at the end (emhF) of the large, 16.4-kb p-ethylphenol catabolic gene cluster (17,19). The onset of detectable transcript level increase agreed very well with the earlier findings for p-hydroxyacetophenone (19), albeit with 2-fold-higher levels for the latter (acsA, 18-versus 40-fold after 5 min; hped, 10-versus 26-fold after 30 min; and emhF, 7.5-versus 14-fold after 60 min). Furthermore, the highest relative transcript levels were in 2 of 3 cases less pronounced with p-ethylphenol than with p-hydroxyacetophenone (acsA, 156-versus 250-fold after 120 min; hped, 64-versus 46-fold after 120 min; and emhF, 12-fold after 120 min versus 14-fold after 60 min) (19).…”

Microbe-Driven Genotoxicity in Gastrointestinal Carcinogenesis

“…Specifically, it has been shown to interact with MUC2 in the intestine, which causes upregulation of several of its virulence genes [ 176 ]. The autotransporter protein CapC also plays a role in promoting epithelial adhesion and invasion [ 177 ]. While it establishes its niche, Campylobacter effectively disrupts tight junctions [ 178 ].…”