Quorum Sensing

2 Materials

2.1 Bacterial Cell
Cultures

1. Bacterial strain: The methods presented here use cultures of the
   Gram-negative bacteriumP. aeruginosa, strain PA14 [7].


2. Tryptic soy broth (TSB) medium.


3. 18
   150 mm borosilicate glass tubes.


4. Roller drum for culture tubes.

2.2 LC/MS Analysis 1. Triple-quadrupole mass spectrometer (MS) equipped with a
Z-spray interface.

2. Nitrogen is used for drying and argon is used as collision gas in
   multiple reaction monitoring (MRM) mode.


3. HPLC equipped with a 4.6
   150 or 3.0
   100 mm C8 or C18
   column: the MS is connected to the HPLC through a Tsplitter.
   The third output of the splitter is fitted with a tube of internal
   diameter and length such as only 10% of the initial flow goes to
   the electrospray probe.


4. Solvent A: Distilled water containing 1% ACS-grade acetic acid.


5. Solvent B: HPLC-grade acetonitrile (or 2-propanol), contain-
   ing 1% ACS-grade acetic acid.

2.3 Internal
Standards

1. 5,6,7,8-Tetradeutero-4-hydroxy-2-heptylquinoline (HHQ-
   d 4 ) and 5,6,7,8-tetradeutero-3,4-dihydroxy-2-heptylquinoline
   (PQS-d 4 ) are synthesized as described in [8].


2. Standard stock solution: 10 mg/l HHQ-d 4 and 20 mg/l PQS-
   d 4 are prepared in HPLC-grade methanol and kept at 20 C.

3 Methods

Mass spectrometric analysis of the samples is performed under

positive electrospray ionization conditions, a process that adds a

proton to the analytes, thus producing pseudomolecular ions which

correspond to the mass of the neutral molecule with the addition of

one proton.

The methods presented here mostly deal with the quantifica-

tion of 4-hydroxy-2-heptylquinoline (HHQ), 3,4-dihydroxy-2-

heptylquinoline (Pseudomonas quinolone signal; PQS), and 4-

hydroxy-2-alkylquinolineN-oxide (HQNO), each the best studied

HAQ congener of its family [3], and of C 4 -HSL and 3-oxo-C 12 -

HSL as the most representative AHLs ofP. aeruginosa(Fig.1).

Within each family, the various congeners only differ by the length

of their alkyl side chain. These molecules can thus be analyzed by

the same methods by taking into account the mass difference due

to the successive addition (or subtraction) of one methylene

50 Franc ̧ois Le ́pine et al.