2.3 Culture
Preparation for DNA
Microarray
1.Bacterial strain:P. aeruginosa(PAO1-ATCC) obtained from
the Pseudomonas genetic stock center (www.pseudomonas.
med.ecu.edu, strain PAO0001).- AB minimal growth medium (seeSubheading2.1,items 4
and 5 ). - Bacto™Casamino Acid.
- RNAlater (Ambion) is used to stabilize and protect RNA.
3 Methods
We have developed a simple assay [5], which is able to detect the
inhibition of either thelasor therhlencoded QS systems inP.
aeruginosa. The monitor systems are constructed by fusing an
unstable version of green fluorescence protein (GFP) [23] to the
QS controlledlasBandrhlApromoters in a wild-type background
ofP. aeruginosa. These monitors switch on expression of GFP in
QS dependent manner in batch cultures ofP. aeruginosa, typical in
late exponential or early stationary phases of growth. Hence,
administration of a QSI compound to the growth medium will
result in reduced expression of green fluorescence compared with
the untreated batch culture. However, compounds inhibiting or
reducing growth of the monitor strains will also affect the fluores-
cence. Therefore, in order to omit scoring false positives, growth
should be measured simultaneously. We calculate the specific activ-
ity of GFP expression as change in GFP expression per time unit
divided by change in OD 450 per unit time. Reduction in specific
GFP expression and unaffected growth rate indicates the presence
of a functional QSI compound. These screens are based on tran-
scription of only two QS regulated genes. There is a minimum of
170 QS controlled genes. Consequently, only DNA microarray or
“deep sequencing” technologies give the opportunity to monitor
changes in transcription of the entire bacterial genome and thereby
gain a more specific knowledge about the target specificity. Fur-
thermore, it is important to test the efficacy of a possible QSI in
regard to bacteria living in a biofilm. The in vitro continuous-
culture flow cell system [24] makes it possible to follow and inves-
tigate biofilm development receiving fresh nutrients continually.
The continuous-culture biofilm flow cell system is perfect for visual
inspection of formation, disruption, and killing of biofilms using
CSLM. The biofilm is monitored using either GFP-tagged bacterial
cells or Syto9 as a stain. The killing of the bacteria is monitored
using PI which will stain the DNA of cells with impaired mem-
brane, i.e., dead cells.In vitro Determination of Quorum Sensing Inhibition 279