coli lac promoter. The lipA gene was removed from the resulting plasmid
pBBLCAH by BamHI digestion and recircularization, yielding pBBLCH, which harbours a synthetic lipC/lipH operon under the transcriptional control of Plac and PT7. M9-minimal broth medium contained, per litre, 4 g glucose; 0.25 g MgSO4; 0.02 g CaCl2; 7 g Na2HPO4; 0.3 g KH2PO4; 0.5 g NaCl; 1 g NH4Cl; and 0.3% w/v agar (Oxoid). Swim plates were inoculated from overnight cultures of bacteria grown on LB plates (1.5% w/v agar) at 37 °C with a sterile toothpick. Plates were then incubated at 37 °C for 24 h. The M9-minimal broth medium described above was used with NH4Cl replaced by 0.05% w/v glutamate and solidified by the addition of 0.5% w/v agar (Oxoid). Plates were briefly dried before use, inoculated and incubated at 37 °C for 36 h. LB medium contained, per litre, 10 g see more tryptone; 5 g yeast extract; 10 g NaCl; and 1.5% w/v agar (Oxoid). Plates were inoculated with a sharp toothpick stabbed
to the bottom of a Petri dish. The zone of motility at the agar/Petri dish interface was measured after incubation for 48 h at 37 °C. Bacteria were grown on swarming agar plates as described above and cells were removed from the edge of growing colonies. For thin sections, bacteria were prefixed in 3% glutaraldehyde Akt inhibitor in vivo in cacodylate buffer (200 mM), followed by osmium tetroxide 1% in cacodylate buffer and uranylacetate 3% in water. Samples were dehydrated in acetone and embedded in glycidether (EPon 812). Sections were stained with leadcitrate 4% and examined electromicroscopically with an EM 10 (Zeiss, Germany) at an acceleration voltage of 80 kV. For negative stain preparations, bacteria were mounted on copper grids and incubated for 40 s in uranylacetate 3%. Pseudomonas aeruginosa strains used in biofilm experiments were fluorescently tagged with gfp at an intergenic neutral chromosomal locus in a miniTn7 construct. Biofilms were grown in a modified NB medium [80 mg L−1
Lab Lemco broth (Oxoid); 1 mM MgCl2; 0.1 mM CaCl2; 50 mM NaCl; 6 g L−1 Na2HPO4·2H2O; 3 g L−1 KH2PO4; and nonchelated trace metals] on glass cover slips in aminophylline flow chambers at 30 °C as described previously (Klausen et al., 2003). The flow chambers were inoculated by injecting 350 μL of an overnight culture diluted to an OD600 nm of 0.001 into individual flow channels with dimensions of 1 × 4 × 40 mm. After inoculation, flow channels were left without flow for 1 h. Then, medium flow was started using a Watson Marlow 205S peristaltic pump to produce a flow rate of 3 mL h−1. Biofilm development was documented with an LSM 510 confocal laser scanning microscope (Zeiss) using a × 63/1.4 objective. Simulated three-dimensional images and sections were generated using the imaris software package (Bitplane). The biomass, surface coverage and roughness of two independent biofilms per strain were analysed using the comstat software.