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.
| Original language | English |
|---|---|
| Pages (from-to) | 40-45 |
| Number of pages | 6 |
| Journal | Journal of Microbiological Methods |
| Volume | 68 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1 Jan 2007 |
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Keywords
- 3-oxo-C6-HSL
- Autoinducer
- Bioreporter
- Quorum sensing
- Vibrio fischeri
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Direct quantification of N-(3-oxo-hexanoyl)-l-homoserine lactone in culture supernatant using a whole-cell bioreporter. / Yan, Ling; Allen, Michael Shane; Simpson, Michael L.; Sayler, Gary S.; Cox, Chris D.
In: Journal of Microbiological Methods, Vol. 68, No. 1, 01.01.2007, p. 40-45.Research output: Contribution to journal › Article
TY - JOUR
T1 - Direct quantification of N-(3-oxo-hexanoyl)-l-homoserine lactone in culture supernatant using a whole-cell bioreporter
AU - Yan, Ling
AU - Allen, Michael Shane
AU - Simpson, Michael L.
AU - Sayler, Gary S.
AU - Cox, Chris D.
PY - 2007/1/1
Y1 - 2007/1/1
N2 - 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.
AB - 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.
KW - 3-oxo-C6-HSL
KW - Autoinducer
KW - Bioreporter
KW - Quorum sensing
KW - Vibrio fischeri
UR - http://www.scopus.com/inward/record.url?scp=33846014687&partnerID=8YFLogxK
U2 - 10.1016/j.mimet.2006.06.002
DO - 10.1016/j.mimet.2006.06.002
M3 - Article
C2 - 16916554
AN - SCOPUS:33846014687
VL - 68
SP - 40
EP - 45
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
SN - 0167-7012
IS - 1
ER -