- Use a suitable statistical software package (seeNote 9) to draw
a standard curve of AIP concentration relative to maximum
RLU/OD 600 , Fig.3b. - Extrapolate the AIP concentration for each sample using the
linear section of the AIP standard curve, Fig.3a, b. Multiply
the concentration 20-fold to account for the dilution of super-
natant (5%) in the reporter assay. This should give an estimate
of the AIP concentration at a given time point during growth,
allowing comparisons to be made between different strains and
mutants and growth conditions.
4 Notes
- The reporter strain ROJ48 described in this protocol is suitable
for detecting AIPs from allagrgroups when combined with the
correct AgrC(X)A plasmid. Reporter strain and plasmids for all
agrgroups are available from our laboratory on request. Exam-
ples of strains which belong to eachagrgroup can therefore be
used as positive or negative controls in reporter assays:
Group 1: 8325, USA300, SH1000.
Group 2: Mu50, Mu3.
Group 3: MN8, MW2.
Group 4: RN4850, H560. - We routinely purchase synthetic AIPs from Cambridge
Research Biochemicals. - To determine whether a particularS.aureusstrain can produce
AIP or to identify theagrgroup to which a given strain belongs
to, supernatant prepared from an overnight culture is adequate
Fig. 3(a) P3 lux expression as an indicator of AIP production byS. aureusover time, peak values can be used
in combination with the calibration curve shown inbto extrapolate AIP concentration in the tested sample. (b)
AIP calibration curve.Dashed red linesindicate linear section that can be used to estimate AIP concentration
94 Ewan J. Murray and Paul Williams