title: Direct quantification of N-(3-oxo-hexanoyl)-L-homoserine lactone in culture supernatant using a whole-cell bioreporter.
abstract: The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) plays a significant role in the quorum-sensing system of the marine bacterium Vibrio fischeri. Upon forming a transcriptional activation complex with LuxR, 3-oxo-C6-HSL induces transcription of the luxICDABEG operon, leading to the increased production of both the 3-oxo-C6-HSL synthase (LuxI) and the bioluminescent proteins. In order to quantitatively analyze this regulatory mechanism, a novel approach was developed to measure 3-oxo-C6-HSL concentrations in V. fischeri cell culture supernatant. A bioluminescent strain of Escherichia coli that responds to 3-oxo-C6-HSL was used as a bioreporter. Although a linear response of the bioreporter to exogenously added synthetic 3-oxo-C6-HSL was found over several orders of magnitude, we show that bioreporter performance was dramatically impacted by variations in the supernatants using samples from a V. fischeri LuxI- strain. However, when maintained in the same supernatant background, the normalized peak bioluminescence maintained a linear response to 3-oxo-C6-HSL concentrations. Therefore, a standard additions technique was developed in which a known concentration of 3-oxo-C6-HSL was added to supernatant samples from wild-type V. fischeri cultures, and the incremental increase of the normalized peak bioluminescence relative to the untreated sample was determined. The concentration of 3-oxo-C6-HSL in the supernatant of the unknown sample was then quantified from the slope of the response between the normalized bioluminescent peaks with and without the addition of 3-oxo-C6-HSL. Advantages of this method are that it is rapid, does not require concentration or extraction, uses a small sample volume (ca. 2 ml), and accounts for effects caused by the composition of the supernatant. Furthermore, the findings can be broadly applicable to other bioreporter systems involving variable background conditions.
authors.affiliation: Center for Environmental Biotechnology, Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 37996-2010, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Yan
authors.forename: Michael S
authors.initials: MS
authors.lastname: Allen
authors.forename: Michael L
authors.initials: ML
authors.lastname: Simpson
authors.forename: Gary S
authors.initials: GS
authors.lastname: Sayler
authors.forename: Chris D
authors.initials: CD
authors.lastname: Cox
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000032:analysis / Q000502:physiology; D048908:Aliivibrio fischeri / Q000737:chemistry / Q000254:growth & development / Q000502:physiology*; D001681:Biological Assay / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D049449:Luminescence; D053038:Quorum Sensing / Q000502:physiology*
chemical_list: C030654:N-(3-oxohexanoyl)-3-aminodihydro-2(3H)-furanone; D015107:4-Butyrolactone