Quorum Sensing

2.2 Reporter Strain
and Plasmids
(See Note 1)

1. Strain ROJ48: RN4220Δagr::ErmBpΔagr-lux(integrated as
   a single crossover); tetracycline (tetR) and erythromycin (ermR)
   resistant [15].


2. Plasmids required to generate bio-reporters foragrgroups 1,
   2, 3, and 4 are available upon request from this laboratory:
   pAgrC1agrA, pAgrC2agrA, pAgrC3agrA, and pAgrC4agrA,
   all chloramphenicol (cmlR) resistant [15].

2.3 AIP Sample
Preparation

1. 1 ml of culture supernatant to be tested.


2. 0.22μm Sterile filter with syringe attachment.


3. 1 ml Sterile syringes.


4. Sterilized 1.5 ml microfuge tubes.

2.4 Reporter Assay
for Detection and
Quantification of AIPs
1–4 from Cell Free
Culture Supernatants

1. The appropriateagrgroup reporter.


2. Microtiter plate: Sterile, 96 well, black, 200μl well volume,
   transparent flat bottom.


3. AIP1–4: To be used as a standard (synthesized to>95% purity)
   dissolved 1 mM in dimethyl sulfoxide (DMSO) (seeNote 2).
   Filtered culture supernatant of a strain with a knownagrgroup
   can also be used as a positive control if synthetic AIP is unavail-
   able (seeNote 1).


4. Plate reader: Multi-mode plate reader capable of optical density
   (OD 600 ) and luminescence [relative light unit (RLU)
   detection].


5. Sample: Filtered supernatants to be tested.

3 Methods

3.1 Collection of
S. aureusCell-Free
Culture Supernatants
for Assays of AIP
Production
and Kinetics

1. Streak theS.aureusstrain(s) to be tested for AIP production,
   and include strains of knownagrgroups as their supernatants
   will act as positive and negative controls if synthetic AIP(s) are
   unavailable (seeNote 1foragrgroups of common lab strains),
   onto a BHI agar plate using standard microbiological sterile
   technique. Incubate agar plates at 37C overnight.


2. Inoculate a single colony of the strain to be tested, as well as
   appropriate agr group control strains if synthetic AIP is
   unavailable, into 5 ml BHI broth. Incubate at 37C overnight
   with shaking, 200 rpm.


3. Remove a 1 ml sample from each overnight culture, pellet by
   centrifugation (14,000
   gfor 1 min), and discard the super-
   natant. Wash the cell pellets three times by successive rounds of
   centrifugation and suspension in fresh sterile medium (1 ml)
   (seeNote 3).

92 Ewan J. Murray and Paul Williams