While intracellular c-di-GMP concentrations become elevated in
biofilms via diguanylate cyclases, the levels decrease notably in
planktonic populations. Phosphodiesterases are coupled directly or
indirectly to environmental sensors, which in turn are activated by a
variety of cues. Reduced c-di-GMP levels trigger gene expression
related to the return to planktonic growth. Other mechanisms
besides alterations of c-di-GMP levels have been described includ-
ing activation of phage and other genes that can act as mechanisms
of apoptosis [48–50] of a portion of the organisms within a biofilm.
While there is considerable work related to the identification of
quorum inhibition in prevention of biofilms, there is a need for
work in identifying compounds that may trigger biofilm dispersion.
We address a microtiter plate strategy for screening and identifying
potential biofilm dispersion molecules. This conceptis adapted from
the flow-cell protocol by Karin Sauer and colleagues [48], and
representative data is shown in Fig.6.2 Materials
2.1 AHL Extraction
[35, 38]
- Centrifuge capable of 4000g, and solvent-resistant tubes.
- Ethyl acetate (containing 0.1% (v/v) acetic acid) (store in the
dark in volatile chemical storage). - Overnight broth culture (stationary phase) of organism to be
investigated. - Alternatively, if naturally occurring biofilms are to be investi-
gated, then prepare small glass containers with water—fill half
full with water, cover, and autoclave. Container size is chosen
based on environmental sample size.
Fig. 5TLC analysis of AHL extracts from several strains ofP. aeruginosa.Of
note,A. tumefaciensA136 (PCF218)(PCF372) (left) detected many more AHLs
than did theC. violaceumCV026 (right)Bioassays of Quorum and Biofilm Dispersion Signals 11