Quorum Sensing

5. Syto 9 and PI are light sensitive and have to be covered with
   aluminum foil. Remember to also cover the flow cells with
   aluminum foil during the 15 min of staining.


6. It is possible to add PI to the medium from the beginning of
   the experiment or when the media is changed to contain anti-
   biotics and QSI. If this procedure is used, the final concentra-
   tion of PI in the media has to be 0.0015 mM. Remember to
   cover every media containing parts with aluminum foil.


7. The flask has to be warmed in a 37C incubator before use.


8. It is important that the growth of the treated and untreated
   culture is similar and they reach the decided density
   (OD 600 ¼2.0) at the same time.


9. Detailed description of RNA isolation for transcriptomic anal-
   ysis can be found in Chapter14 of this book.

Acknowledgments

We acknowledge the contributions made by Drs. Morten Hentzer,

Hong Wu, Thomas Bovbjerg Rasmussen, Jens Bo Andersen, and

Allan Bech Christensen with regard to the initial developments of

the model systems. We are greateful for the support we have

received over the years from the Lundbeck foundation, the Danish

Strategical research Council, the German CF organization,

Kirsten & Freddy foundation, the Villum foundation and Novo-

seeds to MG.

References

1. Lewenza S, Conway B, Greenberg EP, Sokol
   PA (1999) Quorum sensing inBurkholderia
   cepacia: identification of the LuxRI homologs
   CepRI. J Bacteriol 181:748–756


2. Bjarnsholt T, Jensen PO, Burmolle M, Hentzer
   M, Haagensen JA, Hougen HP et al (2005)
   Pseudomonas aeruginosatolerance to tobramy-
   cin, hydrogen peroxide and polymorphonu-
   clear leukocytes is quorum-sensing
   dependent. Microbiology 151:373–383


3. Costerton JW, Stewart PS, Greenberg EP
   (1999) Bacterial biofilms: a common cause of
   persistent infections. Science 284:1318–1322


4. de Kievit TR, Iglewski BH (2000) Bacterial
   quorum sensing in pathogenic relationships.
   Infect Immun 68:4839–4849


5. Hentzer M, Riedel K, Rasmussen TB, Heydorn
   A, Andersen JB, Parsek MR et al (2002) Inhibi-
   tion of quorum sensing inPseudomonas aerugi-
   nosabiofilm bacteria by a halogenated furanone
   compound. Microbiology 148:87–102
   6. Hentzer M, Wu H, Andersen JB, Riedel K,
   Rasmussen TB, Bagge N et al (2003) Attenua-
   tion ofPseudomonas aeruginosavirulence by
   quorum sensing inhibitors. EMBO J
   22:3803–3815
   7. Wu H, Song Z, Hentzer M, Andersen JB,
   Molin S, Givskov M, Høiby N et al (2004)
   Synthetic furanones inhibit quorum-sensing
   and enhance bacterial clearance inPseudomonas
   aeruginosalung infection in mice. J Antimicrob
   Chemother 53:1054–1061
   8. Givskov M, de Nys R, Manefield M, Gram L,
   Maximilien R, Eberl L et al (1996) Eukaryotic
   interference with homoserine lactone-
   mediated prokaryotic signalling. J Bacteriol
   178:6618–6622
   9. Rasmussen TB, Skindersoe ME, Bjarnsholt T,
   Phipps RK, Christensen KB, Jensen PO et al
   (2005) Identity and effects of quorum-sensing
   inhibitors produced by Penicilliumspecies.
   Microbiology 151:1325–1340

284 Tim Holm Jakobsen et al.