5 min respectively and was ended by one step of 72°C for 5 min. The amplified fragment was cleaned

using the Qiagen PCR purification kit (Qiagen Benelux B.V.) and restricted with BamHI and EcoRI. This restricted epsC gene fragment was ligated into BamHI-EcoRI restricted pGEX-6p-3 plasmid to yield pGEX-PG0120. The 1.2 Kb EryF erythromycin resistance cassettes for use in P. gingivalis was amplified from plasmid pEP4351 using primers EryF ClaI F and EryF ClaI R. and after restriction with ClaI this fragment was ligated into the ClaI-restricted pGEX-PG0120 plasmid yielding pΔEpsC. The ScaI-linearized CP673451 nmr pΔEpsC plasmid was used for insertional inactivation of epsC in P. gingivalis strain W83. Complementation of the epsC mutant The 120 bp artificial constitutive CP25 promoter [37] was amplified from plasmid pDM15 [38] using primers CP25 ClaI F and CP25 AscI R. The intact epsC 1.2 Kb gene was amplified from genomic DNA of P. gingivalis strain W83 using primers epsC AscI F and epsC SpeI R. After ligation of these fragments into cloning vector pJET1.2 (Fermentas, GmbH, St. Leon-Rot, Germany) the constructed expression cassette was cut out with XhoI and HindIII and ligated into the GSK2126458 molecular weight SalI and HindIII digested pT-COW shuttle plasmid [39] to yield the complementation construct pT-PG0120. Transformation of P. gingivalis BHI+H/M was inoculated

with P. gingivalis W83 from a 6-day-old blood agar plate. This pre-culture was anaerobically incubated at 37°C for 2 days. 2 ml of the pre-culture was used to inoculate a 100 ml culture. The next day this culture was used to inoculate 2 × 100 ml of fresh

BHI+H/M to an OD690 of 0.2. After six hours of anaerobic incubation at 37°C the cells were harvested by centrifugation in mid-exponential phase. The pellet was washed two times in 20 ml EPB (10% glycerol, 1 mM MgCl2) and after that resuspended in 2 ml of EPB. Aliquots of 200 μl were stored at -80°C and used for electroporation. 200 ng of PstI digested pΔEpsC was added to 200 μl of W83 P. gingivalis cells. The mixture was transferred to a 2 mm electroporation cuvette and electroporated using an Electro Cell Manipulator Temsirolimus research buy 600 (BTX Instrument Division, Holliston, MA, USA; 25 μF, 2.5 kV, 186 Ω). 1 ml of BHI+H/M was added immediately after the pulse. The cells were left for recovery anaerobically at 37°C for 18 hours. The suspension was plated on BA+H/M plates with 5 μg/ml erythromycin for selection of the transformants. The authenticity of the insertional knockout epsC mutants was verified using primer combinations epsC BamHI F × PG0119 R and EryF ClaI F × epsC EcoRI R. Furthermore, using Real-Time PCR, the expression of the downstream gene hup-1 in both W83 and the epsC mutant was monitored using primers hup-1 F and hup-1 R to exclude polar effects. W83 and the epsC mutant were grown till early exponential phase. The cell Seliciclib concentration pellets were collected by centrifugation and resuspended in RLT buffer (Qiagen, Benelux B. V.