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

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

Streptococcus mutans Inhibits Candida albicans Hyphal Formation by the Fatty Acid Signaling Molecule trans‐2‐Decenoic Acid (SDSF)

“…harveyi BB170 was grown in autoinducer bioassay (AB) medium at 30 8C according to the method of Greenberg et al [62] and modified by Vilchez et al [63] The sensor strains E. coli MT102 pSB403, P. putida F117 pKR-C12, and E. coli MT102 pJB A132 were grown overnight in LB medium at 30 8C (P. putida) or 378C (E. coli). Chromobacterium violaceum CV026 was grown in 0.5 % yeast extract and 1 % tryptone (Difco) at 30 8C.…”

“…The V. harveyi bioassay was carried out as described previously [62,63] by using white microtiter plates (Nunc Ltd., Langenselbold, Germany). The solvent was evaporated from the test sample (20 mL) with a stream of N 2 gas before addition of 100 mL of the working dilution of V. harveyi (BB170, BB886, or BB120 depending on the experiment).…”

Directed assembly of a bacterial quorum

“…Hence, the E. coli CT104 cells constructed precisely report the AI-2 level in culture media; they serve as robust sensors of AI-2. Cellbased sensors have been widely used for reporting the activity of autoinducers, including AI-2, in fluids both from natural environments and from laboratory samples (Mok et al, 2003;Taga, 2005;Gantner et al, 2006;Vilchez et al, 2007;Dulla and Lindow, 2008;Zhu and Pei, 2008). Perhaps the most widely used is the Vibrio harveyi BB170 assay of Surette and Bassler (1998).…”

Identification, Quantification, and Determination of the Absolute Configuration of the Bacterial Quorum‐Sensing Signal Autoinducer‐2 by Gas Chromatography–Mass Spectrometry

“…LuxS cleaves the intermediate S-ribosylhomocysteine into homocysteine and (S)-4,5-dihydroxy-2,3-pentanedione [(S)-DPD, 1], the metabolic precursor of AI-2. [4] Through the processes of cyclization (2,3), hydration (4,5,10,11), and borate ester formation if enough borate is present in solution (6)(7)(8)(9), [5] DPD exists as an equilibrium mixture of several compounds (Scheme 1), some of which function as signaling molecules.…”