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Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay

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Abstract
Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe3+ within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 mu M. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of > 120 with a standard deviation of similar to 30% in a reliable and reproducible way.
Keywords
AL-2, quorum sensing, SALMONELLA-TYPHIMURIUM, CELL-DENSITY, IRON CONTROL, BACTERIA, BORON, LUXS, autoinducer-2, QUORUM-SENSING SIGNAL, bioassay, FISCHERI LUMINESCENCE SYSTEM, PSEUDOMONAS-AERUGINOSA, luminescence, Vibrio harveyi

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MLA
Vilchez Vargas, Ramiro, André Lemme, Verena Thiel, et al. “Analysing Traces of Autoinducer-2 Requires Standardization of the Vibrio Harveyi Bioassay.” ANALYTICAL AND BIOANALYTICAL CHEMISTRY 387.2 (2007): 489–496. Print.
APA
Vilchez Vargas, R., Lemme, A., Thiel, V., Schulz, S., Sztajer, H., & Wagner-Döbler, I. (2007). Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 387(2), 489–496.
Chicago author-date
Vilchez Vargas, Ramiro, André Lemme, Verena Thiel, Stefan Schulz, Helena Sztajer, and Irene Wagner-Döbler. 2007. “Analysing Traces of Autoinducer-2 Requires Standardization of the Vibrio Harveyi Bioassay.” Analytical and Bioanalytical Chemistry 387 (2): 489–496.
Chicago author-date (all authors)
Vilchez Vargas, Ramiro, André Lemme, Verena Thiel, Stefan Schulz, Helena Sztajer, and Irene Wagner-Döbler. 2007. “Analysing Traces of Autoinducer-2 Requires Standardization of the Vibrio Harveyi Bioassay.” Analytical and Bioanalytical Chemistry 387 (2): 489–496.
Vancouver
1.
Vilchez Vargas R, Lemme A, Thiel V, Schulz S, Sztajer H, Wagner-Döbler I. Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. 2007;387(2):489–96.
IEEE
[1]
R. Vilchez Vargas, A. Lemme, V. Thiel, S. Schulz, H. Sztajer, and I. Wagner-Döbler, “Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay,” ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 387, no. 2, pp. 489–496, 2007.
@article{4421193,
  abstract     = {{Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe3+ within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 mu M. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of > 120 with a standard deviation of similar to 30% in a reliable and reproducible way.}},
  author       = {{Vilchez Vargas, Ramiro and Lemme, André and Thiel, Verena and Schulz, Stefan and Sztajer, Helena and Wagner-Döbler, Irene}},
  issn         = {{1618-2642}},
  journal      = {{ANALYTICAL AND BIOANALYTICAL CHEMISTRY}},
  keywords     = {{AL-2,quorum sensing,SALMONELLA-TYPHIMURIUM,CELL-DENSITY,IRON CONTROL,BACTERIA,BORON,LUXS,autoinducer-2,QUORUM-SENSING SIGNAL,bioassay,FISCHERI LUMINESCENCE SYSTEM,PSEUDOMONAS-AERUGINOSA,luminescence,Vibrio harveyi}},
  language     = {{eng}},
  number       = {{2}},
  pages        = {{489--496}},
  title        = {{Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay}},
  url          = {{http://dx.doi.org/10.1007/s00216-006-0824-4}},
  volume       = {{387}},
  year         = {{2007}},
}

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