aeruginosa and S. aureus grown in a flow-chamber system. We demonstrated how adaptive mutations in regulator genes of P. aeruginosa affect interactions between P. aeruginosa and S. aureus in co-culture biofilms. Pseudomonas aeruginosa
wild-type PAO1 (Holloway & Morgan, 1986), P. aeruginosa mucA mutant (Hentzer et al., 2001), Selleckchem Alpelisib P. aeruginosa rpoN mutant (Webb et al., 2003), P. aeruginosa pilA mutant (Klausen et al., 2003b), P. aeruginosa pilH mutant (Barken et al., 2008), P. aeruginosa pqsA mutant (D’Argenio et al., 2002), S. aureus MN8 (Yarwood et al., 2004), S. aureus ISP479 (Toledo-Arana et al., 2005) and S. aureus 15981 (Toledo-Arana et al., 2005) were kindly provided by the cited authors and used in the present study. The pDA2 plasmid (An et al., 2006) was used check details to complement the pilA mutant. Fluorescence-tagged strains were constructed by the insertion of a mini-Tn7-eGFP-Gmr cassette as described (Koch et al., 2001; Klausen et al., 2003b). Escherichia coli strains MT102 and DH5α were used for standard DNA manipulations. Luria–Bertani medium (Bertani, 1951) was used to cultivate E. coli strains. A modified FAB medium (Qin et al., 2007) supplemented with 0.3 mM glucose and 3% of Tryptic Soy Broth (TSB, BD Diagnostics) was used for biofilm cultivation. Selective media were supplemented with ampicillin (100 mg L−1), gentamicin (60 mg L−1) or carbenicillin
(200 mg L−1). Biofilms were grown in flow chambers
with individual channel dimensions of 1 × 4 × 40 mm at 37 °C. The flow system was assembled and prepared as described previously (Sternberg & Tolker-Nielsen, 2006). Overnight cultures of P. aeruginosa and S. aureus were diluted to an OD600 nm of 0.001. The flow chambers were inoculated by injecting 350 μL of monospecies diluted cultures or P. aeruginosa–S. aureus 1 : 1 mixed-species diluted cultures into each flow channel with a small syringe. After inoculation, flow channels were left without flow for 1 h, after which medium flow (0.2 mm s−1) was started using a Watson Marlow 205S peristaltic pump. For DNase I treatment, biofilm medium was supplemented with 20 μg mL−1 bovine DNase I (Sigma) from the beginning of cultivation. All microscopic observations and image acquisitions were performed using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Protirelin Germany) equipped with detectors and filter sets for monitoring of green and red fluorescence from general nucleic acid staining SYTO 9 (Invitrogen) and gram-positive specific staining hexidium iodide (Invitrogen) (Mason et al., 1998), respectively. BacLite Live/Dead viability stain (Molecular Probes, Eugene, OR) was used to visualize dead and live cells in co-culture biofilms. Images were obtained using a × 40/1.3 objective. Simulated three-dimensional images and sections were generated using the imaris software package (Bitplane AG, Zürich, Switzerland).