Quorum Sensing

extracellular enzymes, dispersal of biofilm and other determinants

involved in ecological competence [7, 8].

Here, we describe methods used to study the involvement of

DSF-dependent QS system in virulence of plant pathogenic bacte-

ria. These include detection and measurement of DSF, movement

in planta, colonization, and aggregate formation. The plant patho-

genic bacteriaXanthomonas hortorumpv.pelargonii(Xhp)[9]is

used as an example of theXanthomonasspp. or other genera of the

Xanthomonadaceae family.

2 Materials

2.1 Bacterial Strains
and Growth Media

1. The DSF biosensor strain used in this study isXanthomonas
   campestrispv.campestris8523 (rifampicin resistant), which is a
   mutant inrpfF[10]. It carries the plasmid pKLN55 (spectino-
   mycin resistance gene) harboring the DSF-inducibleeng::gfp
   reporter gene.
   2.Xanthomonas hortorumpv.pelargonii305 (Xhp305) (rifampi-
   cin resistant) wild type and isogenic mutants inrpfFandrpfC
   genes with kanamycin resistance (seeNote 1).


2. Luria-Bertani (LB) broth: 10 g/l NaCl, 10 g/l tryptone, 5 g/l
   yeast extract.


3. LB agar plates: LB broth plus 15 g/l agar.


4. Antibiotics: rifampicin (100μg/ml), spectinomycin (100μg/
   ml), kanamycin (50 μg/ml), chloramphenicol (10μg/ml).
   Antibiotics are added to the autoclaved and cooled growth
   media.


5. Peptone Glycerol (PG) solution for storage of cultures: glyc-
   erol (40 ml), peptone (2 g), and K 2 HPO 4 (0.15 g) in distilled
   water (to final volume of 100 ml). Overnight LB liquid cultures
   are mixed 1:1 with sterile PG before storing at 80C.

Fig. 1A 6.5 kb DNA fragment containing the QS-related genes inXanthomonas hortorumpv.pelargonii(Xhp)

strain 305

Fig. 2Chemical structure of the QS Diffusible Signal Factor molecule

244 Shulamit Manulis-Sasson and Laura Chalupowicz