Normally these two strains will cross-feed each other (positive
control for AHL detection) such that strain CVO26 will produce
violacein in the presence of 31532. Using the first pattern (Fig.2b),
the test material is closest to strain CVO26 and so pigmentation
inhibition results could be interpreted as transcription inhibition
(luxR-effect). In the case of the second pattern (Fig.2c), the acyl-
HSL-producing strain, 31532, is closest to the test material and so
a pigmentation inhibition effect could be interpreted as interfer-
ence with C6-HSL production (luxI-effect) [57].
A schematic of a plate-based protocol for a high-throughput
bioassay is shown in Fig.2d. Here, the bioassay strain is shown as a
solid line, and the test organisms (indicated by numbers) can
be spotted near the bioassay strain. Due to possible interactions
between test organisms during the high-throughput assays,
we recommend that positive results be confirmed using the
plate-based assay (Subheading 3.2.1) or overlay assay
(Subheading3.2.2).3.2.4 Thin-Layer
Chromatography (TLC)
Detection of AHLs
- The resuspended sample and standards are added to a C18
TLC plate (seeNote 2). Apply 1 or 2μl dot-wise, approx.
2 cm apart, along a line which is 2 cm from the bottom of the
plate. (If necessary, use a pencil for labeling the TLC plate as ink
will dissolve in the organic phase). - For standards, use C6 and 3-o-C8 standards (cognate AHLs of
C. violaceumandA. tumefaciens, respectively), 5μM. - Line chromatography chamber with white paper towel or filter
paper. Add methanol/water (60:40, vol/vol), 200 ml. Let
towels saturate with the solvent. This prevents uneven solvent
front migration. - Place TLC plate into the chamber and cover with glass lid.
Develop chromatogram until the solvent front is approximately
15 cm from the starting line. - Remove TLC plate and let solvent evaporate in the fume hood.
Then place labeling tape (upright orientation) around the edge
of the plate to form a wall and prevent spills from the indicator
bacteria-agar mixture. - Overlay dried plate with indicator bacterium, as follows:
(a) From a fresh 5 ml overnight culture, inoculate 50 ml
media and grow to late exponential phase. It is important
thatsteps 6bandcbe done quickly asA. tumefaciensis
sensitive to elevated temperatures.
(b) Add this 50 ml culture to 100 ml media plus 1.12 g
melted agar plus 10 mg X-gal (500 μl of 20 mg/ml
stock in dimethylformamide), tempered at 45C.
(c) Prewarm TLC plate by putting on heater (low setting) for
approximately 5 min (allows bacteria-agar mixture to
18 Starla G. Thornhill and Robert J.C. McLean