title: A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon.
abstract: The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.
authors: Laboratory of Microbial Biochemistry, Department of Microbiology, Philipps-Universität Marburg Marburg, Germany.
authors: Enrico
authors: E
authors: Muhr
authors: Laboratory of Cellular Microbiology, Department of Microbiology, Philipps-Universität Marburg Marburg, Germany.
authors: Oliver
authors: O
authors: Leicht
authors: LOEWE Center for Synthetic MicrobiologyMarburg, Germany; Max Planck Institute for Terrestrial MicrobiologyMarburg, Germany.
authors: Silvia
authors: S
authors: González Sierra
authors: Laboratory of Cellular Microbiology, Department of Microbiology, Philipps-Universität MarburgMarburg, Germany; LOEWE Center for Synthetic MicrobiologyMarburg, Germany; Max Planck Institute for Terrestrial MicrobiologyMarburg, Germany.
authors: Martin
authors: M
authors: Thanbichler
authors: Laboratory of Microbial Biochemistry, Department of Microbiology, Philipps-Universität MarburgMarburg, Germany; LOEWE Center for Synthetic MicrobiologyMarburg, Germany.
authors: Johann
authors: J
authors: Heider
keywords: Aromatoleum aromaticum; acetophenone; bioreporter; flow cytometry; fluorescence microscopy; immunoblot; mCherry