3 Methods
3.1 Protocol for AHL
Detection During
Bacterial Growth
This is a general protocol for^14 C-radiolabeling of AHL signals
during bacteria growth, but it should be modified according to
the needs of the particular strain as noted below. To illustrate the
range of labeling times and^14 C-HSL production among strains, we
performed the radiolabel assay using three different bacteria (Fig.3,
seeNote 4):Pseudomonas aeruginosaPAO1 [25],Pseudomonas
chlororaphis GM17 [26], and Bradyrhizobium japonicum
USDA110 [27].- Grow your bacterium of interest in a small volume (usually
5 ml) of methionine-free medium with the appropriate growth
conditions (e.g., temperature, aerobic with shaking, anaerobic,
photosynthetic) until the culture reaches desired density,
usually late logarithmic phase or early stationary phase
(seeNote 10).
a10 20 30 40 50 60 7002000040000600000255075100CPMC43OC12methanol (%)bCPM10 20 30 40 50 60 7002000400060000255075100C4 3OHC83OHC63OHC10methanol (%)10 20 30 40 50 60 7005001000150020000255075100CPMcFractionIV methanol (%)Fig. 3HPLC profiles of^14 C-AHLs synthesized by (a)Pseudomonas aeruginosaPAO1 [25] (labeled for 30 min),
(b)Pseudomonas chlororaphisGM17 [26] (labeled for 2 h), and (c)Bradyrhizobium japonicumUSDA110 [27]
(labeled for 1 day). In all graphs thex-axis indicates the fraction numbers that were collected over a 10–100%
methanol-in-water gradient. Theleft y-axis denotes the counts per minute (CPM) of radiolabel in each fraction
(black circles) and theright y-axis indicates the methanol concentration of the HPLC run (dashed line). The
synthetic AHL compound that co-elutes with the observed radiolabel peak is abbreviated as follows: butanoyl-
HSL (C4), 3-oxo-dodecanoyl-HSL (3OC12), 3-hydroxy-hexanoyl-HSL (3OHC6), 3-hydroxy-octanoyl-HSL
(3OHC8), 3-hydroxy-decanoyl-HSL (3OHC10), and isovaleryl-HSL (IV). Radioactivity that eluted in the column
void volume (fraction 4) is presumed to be unincorporated methionine
40 Amy L. Schaefer et al.