9 h and reached steady State approximately 10 days after inoculation. The cell density of the culture remained constant, after it had reached steady State, at an OD650 nm BKM120 of 2.69 ± 0.21 and 2.80 ± 0.52 for the first and second biological replicates respectively. Robust biofilm was obtained on the FK228 supplier vertical surfaces of the fermentor vessel walls and at 40 days of culture the planktonic and biofilm cells from the fermentor vessel were harvested

for analysis. The glass microscope slides that were fixed to the fermentor vessel walls were used for physical characterization of the biofilm. CLSM revealed that the surface of the biofilm featured variable structures and the average percentage of viable cells within the biofilm was 91.2 ± 7.3% [15]. The biofilms were on average 240 ± 88 μm thick. Our continuous culture system allowed us to obtain a direct paired comparison I-BET151 price of transcriptomic profiles of both the planktonic and biofilm grown cells that were cultivated in the same fermentor vessel and therefore were subjected to identical gross environmental influences (such as media composition and temperature). Identification of genes differentially regulated during biofilm growth Microarray hybridizations were conducted using the paired planktonic cell and biofilm total RNA samples obtained from the two independent continuous cultures.

For each culture planktonic cell and biofilm pair, four technical replicates of array hybridizations were performed (2 array slides for each dye swap) yielding 16 measurements per gene as each gene was represented in quadruplicate on each slide. We designated all genes with an average expression ratio of 1.5-fold (up or down) differentially regulated, a threshold reported to be biologically significant [21, 22]. Moreover, we used the GeneSight 4.1 (Biodiscovery) confidence

analyzer to discriminate genes that had a 99% likelihood of being differentially regulated at above or below the 1.5 threshold. A total of 561 and 568 genes were identified to be differentially regulated (1.5 fold or more, P-value < 0.01) between the biofilm and planktonic Cediranib (AZD2171) cells of the first and second replicates respectively (data not shown). Of the identified genes, 377 belonged to a common data set (67% and 66% of the total genes identified for the first and second replicates respectively). Of the 377 genes in the common dataset 191 were up-regulated and 186 were down-regulated (see Additional files 1 and 2). This represents approximately 18% of the P. gingivalis genome. To validate the microarray data real time-PCR of selected genes PG0158, PG0270, PG0593, PG0914, PG1055, PG1431 and PG1432 was performed. Six of the genes were selected from the up-regulated group and one from the down-regulated group in biofilm cells. The expression of galE was detected to remain unchanged during biofilm and planktonic growth (data not shown) and was used for normalization.