Specific activities were determined by a modified Miller method [41]. Briefly, cells were harvested during different growth stages and resuspended in Z-Buffer to an OD600 of 0.5-0.7. Samples were prepared in triplicates by adding 100 μL of cell suspension to 900 μL Z-buffer with 0.27% (v/v) β -mercaptoethanol, 50 μL chloroform and 100 μL 0.1% SDS and vortexing for 10 seconds. After equilibration at 28°C for 10 minutes, the reaction was started by addition of 0.2 mL o-nitrophenyl-D-galactoside (ONPG) [4 mg * mL-1 ] and incubating the samples at 28°C. The reactions selleck were stopped with 0.5 mL Na2 CO3 [1M] when samples developed a yellowish color. Samples were centrifuged for 5 minutes at 13,000 rpm and OD420 was

recorded. Specific activities were expressed as Miller Units and calculated as follows: 1 Miller Unit = 1000 *

(OD420 )/(t * V * OD600 ), where t = time V= volume OD= optical density Biofilm cultivation Biofilms were grown at 30°C in three-channel flow cells as decribed previously [12]. Briefly, LB overnight cultures of the relevant S. oneidensis MR-1 strains were diluted 1/100 in LB and grown to early stationary phase. Then the optical density at 600 nm was adjusted to 0.01 in 4M MM or LM without carbon source. 1 mL of the OD600 = 0.01 cell suspension was injected into each flow channel while the medium flow was stopped. The flow Stattic manufacturer cells were inverted (glass slide facing bottom) and incubated for 40 min at 30°C. After incubation flow cells were reverted and medium was pumped through the flow cell at a constant velocity of 0.3 mm/s per channel by a Watson-Marlow Bredel (Cornwall, United Kingdom) 205S peristaltic pump. Biofilm studies were carried out in triplicate in at least two independent experiments. Biofilm image acquisition and processing Microscopic visualization of biofilms was performed using an upright Leica TCS SP2 AOBS confocal laser scanning microscope (CLSM; Leica Microsystems, Dapagliflozin Wetzlar, Germany) using the following objectives: HCX PL APO 63X/1.2 W CORR CS and HC PL FLUORTAR 20X/0.5. For three-dimensional reconstruction

of biofilm images, CLSM images were processed with the IMARIS software package (Bitplane AG, Zuerich, Switzerland) and Adobe Photoshop. Flow cytometry 24 h old LM grown biofilm of S. oneidensis MR-1 wild type and mutant cells carrying a P mxd ::gfp reporter construct were harvested from the flow chamber, passed 50 times through a 25 gauge needle to learn more suspend any cell aggregates and fixed in 2% paraformaldehyde. Flow cytometry data were obtained using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Samples were analysed using the 488 nm excitation from an argon-ion LASER at 15 mW. Detector voltages were set at defined values [800 V for the fluorescence channel (FL1) and both the FL1 and forward scatter channel amp gain were set to logarithmic scale] prior to the experimental analysis in which samples were run in succession on the same day.