3 Methods
The GFP from the jellyfishAequorea victoriawas chosen as the
reporter since it only requires a trace amount of oxygen to mature
and thereby no external compounds needs to be added in order to
detect green fluorescence [22]. However, since the GFP is
extremely stable (derived fromgfpmut3[23]) differentgfpgenes
with varying half-lives ranging from 40 min to a few hours was
constructed [15]. One of these variants is the unstable versiongfp
(ASV). The construct of a live bacterial AHL sensor using theluxR
gene fromV. fischerias the quorum sensor acts in the presence of
exogenous AHLs by LuxR, and positively affects the expression of
theluxIpromoter (luxR-PluxI), which in turn induces the expres-
sion of thegfp-reporter [14]. The sensitivity of thisgfp-reporter
enables the visualization of AHLs produced by P. aeruginosa
in vivo. In practice this means that no signal should be observed
in mice infected with alginate beads containing either theE. coli
monitor strain or aP. aeruginosastrain alone. However, when a
mixture of the two strains are used to inoculated the lungs of mice,
one should be able to detect a GFP signal produced by theE. coli
monitor strain in response to AHLs produced byP. aeruginosa.
Other AHL monitor strains have been constructed to study the
ability of anti-pathogenic drugs to block QS. One example is theP.
aeruginosastrain constitutively expressing a red fluorescent protein,
which is advantageous for easy identification of the bacteria in the
tissue samples. Moreover the same strain contains alasR-PlasBgfp
(ASV) reporter fusion in order to detect an activated QS system. In
practice this means that the strain always fluorescence red, and
when QS is activated it will produce a GFP signal in addition.
When QS is blocked by the presence of exogenous QSI com-
pounds, the GFP signal will disappear. For further detailssee[13].
The different monitor stains can also be used in in vitro continuous-
culture flow cell experiments (seedetails in Chapter21) where both
the AHL production from different strains and QSIs can be
investigated.
In the present method section the protocol for in vivo detec-
tion of AHLs produced byP. aeruginosais described. When isolat-
ing lungs for ex vivo inspection, lungs should also be isolated, at the
same time, for quantitative bacteriology to verify that bacteria are
present and that the monitor strain is functional.3.1 Preparation
of Bacteria in Alginate
Beads
- Plate bacteria from a freezer stock onto a blue agar plate, which
are selective for Gram-negative bacilli [24], and incubate the
plate at 37C overnight. Use your choice ofP. aeruginosa
strain and theE. colimonitor strain JB525-gfp(ASV). - Use one bacterial colony from the plate to inoculate an over-
night culture in 100 ml LB liquid medium in a 250 ml conical
ImagingN-Acyl Homoserine Lactone Quorum Sensing In Vivo 207