Pyocyanin was added to Congo red plates at a final concentration of 50 μM. HHQ (a gift from M. whitelely, University of Texas) and HNQ (a gift from P. Williams, University of Nottingham) were added to MOPS-buffered Congo red plates at a final concentration of 50 μM or directly to the bacterial inoculum at final concentrations of 20, this website 100 and 500 μM. The respective solvents ethyl acetate, dimethyl sulfoxide (DMSO), and methanol were used as controls. Pel’-lacZ-reporter construction and β-galactosidase measurements A 555 bp promoter region of the pel operon was amplified from the ZK strain using
the primers listed in Additional file 1: Table S1 and cloned upstream of the lacZ gene in the integration vector mini-CTX-lacZ . The resulting plasmid pRG11 was then inserted into the chromosome of the wild-type and the lasR mutant as described . As a control, the mini-CTX-lacZ parent vector was also integrated into the genome. The colonies of the ZK wild-type and the lasR mutant grown on Congo red plates at 37°C were used to measure β-galactosidase levels. A colony was cut
out on the 3rd, 4th, and the 5th day and suspended in 2 ml of 50 mM phosphate buffer, pH 7.4, in a 15 ml conical tube. Cells were lysed by sonication. The total protein was estimated by Bradford assay . The sonicated sample was centrifuged at 4°C for 30 min. The resulting supernatant was NVP-BGJ398 molecular weight used to measure β-galactosidase activity as described previously . Pellicle biofilm assay Cultures were inoculated in tryptone broth at an OD600 of 0.0025 and incubated at 22°C and 37°C without shaking . After 24, 48 and 72 h, pellicle formation was observed at the air-liquid interface. Microtiter plate biofilm assay Biofilm formation in a microtiter format was assayed as described . Overnight cultures of the wild-type and the lasR mutant grown in LB broth at 37°C were diluted 1:100 in tryptone Dimethyl sulfoxide broth. One hundred and fifty μl of the diluted culture was added to 96-well polystyrene microtiter plates (Cellstar-Greiner Bio-one) and incubated at 22°C and 37°C without shaking for 48
and 72 h. Microtiter plates were rinsed in running hot water. Adherent cells were then stained with 1% crystal violet for 20 min. The microtiter plate was again rinsed in running hot water. Ethanol was added to each dry well and the samples were allowed to stand for 20 min. Absorbance was measured at 590 nm. Flow-cell biofilm assay Biofilms were grown at 37°C in flow chambers. The system was assembled as described [33, 51]. The cultures for inoculation were prepared from mid-exponential phase (OD600 of 0.4-0.8) TSB cultures grown at 37°C. The cultures were diluted to an OD600 of 0.05 in 1:100 diluted TSB medium and injected into the flow cell. Flow was initiated after 1 h. The diluted TSB was supplied at a flow rate of 180 μl/min using a peristaltic pump (Watson Marlow 205S).