Supplementary material and methods
Strains and culture conditions
C. violaceum wild-type strain (CV 12472) and CV026 (Smr mini-Tn5 Hgr cviI::Tn5xylE Kmr)
were kindly provided by Professor Wilbert Bitter from the Department of Molecular Cell
Biology, VU University Amsterdam, Amsterdam, The Netherlands. Bacterial stocks were
maintained in glycerol stocks frozen at -80°C and single colonies for each experiment were
obtained by plating the stocks in LB plates and culture them overnight at 37°C. Precultures were done in 5 mL of LB medium at 37°C and 200 rpm shaking incubating them
for ~16 h, and cultures for the experiments were initiated by diluting pre-cultures to
turbidity of O.D. 600 nm ~0.05 and grown under the same conditions for 15, 24, or 48 h.
Phenotype determination
For violacein determination the protocol described in (Castillo-Juárez, et al., 2013) was
followed with some modifications, briefly aliquots of 0.5 mL of each culture (at 15, 24 and
48 h) were centrifuged at 13,000 rpm for 2 min and violacein was extracted from the cell
pellets by resuspending them in 500 L of 50 mM potassium phosphate buffer pH 7.0 and
adding 1 mL of ethyl acetate, then the mixture was shaken by vortex for 2 min and
centrifuged at 13,000 rpm for 5 min. The organic phase was recovered and the
absorbance at 575 nm was read with a spectrophotometer (UV-1800, Shimadzu). In order
to demonstrate the role of QS in the production of violacein 25 M of furanone C-56 which
is a potent HSL quorum quencher (Wu, et al., 2004) or ethanol, its vehicle were added to
the wild-type strain and 20 M of C6-HSL or DMSO, its vehicle were added to the CV026
mutant, the additions were done when cultures reached turbidity of O.D. 600 nm ~1.0 (~5
h after inoculation) and violacein was determined after 24 h of culturing time.
Exoprotease
The protease activity in the supernatants (obtained by centrifugation of aliquots of 1.5 mL
of cultures at 13,000 r.p.m for 2 min) was assayed following the protocol designed for the
determination of alkaline protease from P. aeruginosa (Howe & Iglewski, 1984), using 20
mg of Hide–Remazol Brilliant Blue R SIGMA (St Louis, MO, USA), to test the effect of
the pH on the proteolytic activity it was adjusted from to 6 to 10 in the Tris 20 mM CaCl 2 1
mM buffer. For testing the elastolytic activity the protocol described in (Ohman, et al.,
1980) the elastine congo-red complex SIGMA (St Louis, MO, USA) was used. In order
to evaluate the role of QS in the production of protease 25 M of furanone C-56 or ethanol,
its vehicle were added to the wild-type strain and 20 M of C6-HSL or DMSO, its vehicle
were added to the CV026 mutant, the additions were done when cultures reached turbidity
of O.D. 600 nm ~1.0 (~5 h after inoculation) and protease was determined after 24 h of
culturing time.
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Cell aggregation, biofilm formation, and swarming motility
Aggregation was assayed as reported previously (Gonzalez Barrios, et al., 2006). Briefly,
cultures were incubated for 20 h at 37°C with shaking (250 rpm) to stationary phase and
then washed and diluted in 3 mL LB medium (to a turbidity at 600 nm, 2.5) in 14-mL sterile
tubes; after the tubes were incubated quiescently at 37°C for 15 h, the absorbance 5 mm
beneath the surface was used to determine aggregation (in order to evaluate the role of
QS in aggregation 25 M of furanone C-56 or ethanol its vehicle were added to the wildtype strain and 20 M of C6-HSL or DMSO its vehicle were added to the CV026 mutant,
the additions were done immediately before the 15 h incubation). Biofilm formation was
assayed in the wild-type strain (with or without 25 M of furanone C-56 or ethanol its
vehicle) and the CV026 mutant (with or without 20 M of C6-HSL or DMSO its vehicle),
the additions were done at the beginning of the cultures, biofilms were determined by
crystal violet staining (O'Toole, et al., 1999), they were cultured in sterile 20 mL glass
tubes with 2 mL of LB medium inoculated at turbidity of 600 nm of ~ 0.05 and cultures
were growth at 37°C with gently shaking (75 r.p.m) in order to enable aeration but avoiding
biofilm disruption, after 48 h of incubation, liquid was decanted from the tubes and used to
determine growth by measuring O.D. 600 nm, then tubes were washed twice with 5 ml of
distilled H2O and stained with 2 mL of 0.1 % crystal violet for 20 min, after staining crystal
violet was discarded, tubes were again washed twice, dried at room temperature during 30
min and the crystal violet removed with 2 mL of absolute ethanol and solubilized by
vortexing 2 minutes, after that biofilm formation was determined by recording the
absorbance of the dissolved crystal violet at 540 nm (UV-1800, Shimadzu), biofilm was
normalized by growth and experiments were performed in quadruplicate. Swarming
motility was assayed as in (Ren, et al., 2001), using in LB plates with 0.5% agar, cells
were inoculated in the middle of the plate by contact, plates were incubated at 37°C, and
the diameter of the swarming colonies was measured at 24 and 48 h.
H2O2 shock
The assay was adapted from (Zhang, et al., 2007). Briefly, strains were cultured in LB from
overnight pre-cultures, until the cultures reached turbidity at O.D. 600 nm of ~ 1.5 and then
aliquots of 1 mL were taken and stressed adding H2O2 at a final concentration of 25 mM
and the cells were exposed for 30 min at 37°C without shaking. Viable counts in LB plates
before and after the stress were done to determine the survival percentages.
Phenotypic Microarrays
These assays were performed following the specifications from the manufacturer Biolog.
Briefly, cells (C. violaceum wild-type and CV026 mutant) were resuspended in inoculation
fluid one, to a turbidity of 40%, and 100 L was inoculated into each well of the plate, and
then the plates PM1, PM2A (carbon sources), PM3B (nitrogen sources) and PM4A
2
(phosphorus and sulfur sources) were incubated for 24 h. Growth was recorded in an
Elisa plate reader (iMark Microplate Absorbance Reader, BIO-RAD) by determining the
absorbance at 600 nm.
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