Transfer of plasmid-DNA into Roseobacter strains by electroporation Electro-competent cells were prepared as described previously by Miller and Belas [2006] with slight modifications. Therefore, cells were grown in MB medium at 30°C and 200 rpm to an OD578 of 0.5. Ten ml culture was centrifuged for 15 min at 3,200

× g. Sedimented cells were washed 5 times with 10 ml cold 10% (v/v) glycerol in ultra-pure water. Then, the cell pellet was resuspended in 400 μl 10% (v/v) glycerol and 50 μl aliquots were frozen in liquid nitrogen and stored at -80°C. For electroporation, 25 – 50 ng plasmid-DNA were added to 50 μl competent cells selleck screening library in an ice cold 2 mm pulser cuvette (Bio-Rad, Munich, Germany). The mixture was treated in a Bio Rad gene pulser II with field strength of 1.5 – 3.0 kV, resistance of 200 Ω and capacitance of 25 μF. After electroporation the cells were transferred to 1 ml cold MB media and incubated overnight at room temperature with shaking at 300 rpm to allow the expression of antibiotic resistance genes. To investigate electroporation efficiency, cells were serially diluted in 1.7% (w/v) sea salt solution and plated on hMB agar plates with

the appropriate antibiotic concentration and incubated for 2 days (Phaeobacter strains and O. indolifex) or 4 days (Roseobacter strains and D. shibae) at 30°C. Subsequently, colony forming units (cfu) were determined. Conjugal transfer of plasmid-DNA from E. coli into Roseobacter strains The conjugation procedure was modificated DMXAA cell line for Roseobacter strains from the protocol of Thoma and Schobert

[2009]. The recipient Roseobacter strains were cultivated for 18 h in MB-Medium. The donor E. coli strain ST18 was grown in LB-medium supplemented with 50 μg/ml ALA (Sigma-Aldrich, Munich, Germany) up to the (-)-p-Bromotetramisole Oxalate logarithmic phase (OD578 = 0.5 – 0.6). Both cultures were mixed in a donor:recipient ratio of 1:1; 2:1; 5:1 or 10:1 according to the optical density (OD578) of the cultures. Cells were sedimented by centrifugation for 2 min at 8,000 × g at 20°C, resuspended in the residual liquid and used to inoculate hMB agar, LB+hs agar and hLB+hs agar respectively, all supplemented with 50 mg/ml ALA, in form of a spot. The plates were incubated at 30°C for 24 h and 48 h. Subsequently, cells were scraped from the plate and resuspended in 1 ml MB by vigorous shaking. Disruption of cell aggregates was confirmed via microscopic inspection of the resulting single cells. A dilution series in 1.7% (w/v) sea salt solution was prepared and plated on hMB with the appropriate antibiotic concentration to determine the number of transconjugants per ml. Since the plates did not contain ALA the auxotrophic donor E. coli strain was not able to grow. In parallel, transconjugants were also plated on hMB without antibiotics to determine the number of viable cells per ml.