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Abstract:
The beta-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 mu M
(detection limit) to 250 mu M 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.
