Bacterial adhesion assays A549 and Detroit 562 cells (ATCC, Manassas, VA) were maintained in F-12 media supplemented with 10% fetal bovine serum and in Minimal Essential Medium supplemented with 10% fetal bovine serum and 0.2% lactoalbumin hydrolase respectively. All cell lines were maintained at 37uC in 5% CO2. Adhesion assays were performed as previously described [30]. A549 cells and Detroit 562 cells were grown to 95% confluence in COSTAR 24- well polystyrene plates (,106 cells/well). Cells were washed with sterile phosphate buffered saline (pH 7.4) and exposed to F12 media without serum containing 107 CFU/ml of either biofilm or planktonic derived bacteria diluted from the frozen stocks. Cells were incubated for 1 h at 37uC in 5% CO2. Following incubation, non-adherent bacteria were removed by gently washing the cells three times with PBS and the number of adherent bacteria was determined by lysis of the cell monolayer with 0.1% Triton X-100 in PBS and plating the lysates on blood agar plates. Each experiment contained 3 biological replicates per condition and was repeated $3 times. Adhesion is expressed as a percentage compared to the planktonic counterpart. Isolation of pneumococcal RNA S. pneumoniae grown under biofilm conditions for 4, 12, 24, and 48 h were collected, immediately suspended in RNAprotect (Qiagen) and stored at 220uC. Isolation of bacterial RNA was performed using an RNeasy Mini kit (Qiagen) following the manufacturer instructions with exception to bacteria lysis. Briefly, bacterial cells were suspended in 350 ml buffer RLT and transferred to 2 ml cryogenic safe-lock tubes containing approx- imately 25 mg of 0.1 mm zirconia/silicon acid washed beads (BioSpec). Cells were lysed by mechanical disruption using a Bead- Beater (BioSpec) for 5 min at maximum speed. Following lysis, beads were removed by passage through a QiaShredder column (Qiagen) and the supernatant was transferred into a 2 ml tube containing an equal volume of ethanol (70%). The lysate was transferred directly onto an RNeasy Mini spin column and bacterial RNA was purified as per the kit’s directions. Control RNA samples were generated from the seed cultures used to inoculate the biofilm lines (OD620 = 0.5) at 37uC in 5% CO2. RNA quality and quantity was determined by (i) measurement of absorbance at 260/280 nm, (ii) visualization of RNA samples using a 1% formaldehyde gel, and (iii) analyzing RNA profiles generated on the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) using Prokaryote Total RNA Nano chips. Microarray analysis of pneumococcal gene transcription and analysis of hybridization data The S. pneumoniae microarrays used in this study consisted of 3482 70-mer oligonucleotide probes from the genome of 3 pneumococcal strains (TIGR4, R6 and G54) as well as 10 amplicons and 500 oligonucleotides (70-mers) from Arabidopsis thaliana which served as negative controls. Probes were printed 36 on aminosilane-coated slides (SCHOTT Nexterion). The microarrays (version 8) were kindly provided by the Pathogen Functional Genomics Resou- rce Center (http://pfgrc.jcvi.org/index.php/microarray/array_ description/streptococcus_pneumoniae/version8.html) and experi- ments were performed as previously described [31]. Aliquots of 2 mg of the total RNAs were reverse transcribed into single-stranded cDNA using 200 U Superscript II reverse transcriptase (Invitrogen), 6 mg random hexamers (Invitrogen), 16 first strand buffer (Invitrogen), 10 mM dithiothreitol (DTT), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP and 0.2 mM of aminoallyl-modified nucleotide (Invitrogen). The Altered Virulence of Biofilm Pneumococci mixture was incubated overnight at 42uC and the reaction stopped by addition of 10 ml 0.5 M EDTA and 10 ml 1 M NaOH. Amine- modified cDNA was purified using QIAquick PCR purification kit (QIAGEN) followed by chemical labeling with Cy3- or Cy5-NHS- ester fluorescent dyes (GE Healthcare, Piscataway, NJ) in a final step. Slides were prehybridized in a 50 ml solution of 56 SSC, 0.1% SDS and 1% BSA for at least 1 h at 42uC, washed 106 in water and once in isopropanol, then dried by brief centrifugation. Labeled probes were re-suspended in hybridization buffer (50% formamide, 56 SSC, 0.1% SDS, 1 mL 0.1 M DTT, 0.6 mg/mL salmon sperm DNA) and hybridized to the microarray slides in a 42uC water bath for 16–20 h. Slides were washed twice in a low stringency buffer (26 SSC, 0.1% SDS) at 55uC for 5 min, twice in a medium stringency buffer (0.16 SSC, 0.1% SDS) at room temperature for 5 min, twice in a high stringency buffer (0.16 SSC) at room temperature for 5 min, and finally in water for 2 min, and then dried by brief centrifugation. Synthesized cDNA from each RNA sample from 3 (4 and 12 h time points) or 2 (24 and 48 h time points) independent preparations was hybridized on separate microarray slides (biological replicates), and independently synthesized cDNA from each of these RNA samples was hybridized in a repeat dye-swap experiment (technical replicates, except for the 48 h time point where RNA quantities were limiting) to test technical reproduc- ibility. Hybridized slides were scanned using a GenePix 4000B dual- color laser scanner (Axon Instruments, CA, USA). Signal intensities, generated using TIGR Spotfinder program version 2.2.3, http://www.tm4.org/spotfinder.html) [32], were imported into TIGR MIDAS software (v2.19) for filtering and normaliza- tion. Spots with Cy3 or Cy5 fluorescence intensities ,10,000 were discarded. Intensities were normalized using iterative log-mean centering. Data from replicate experiments (only where n$7, except for the 48 h time point where n$4) were averaged using in- house developed Perl scripts. Data points that did not meet these requirements are labelled ‘‘NA’’ in Tables S1, S2 and S3. Oligonucleotides with for all conditions tested were omitted from the tables. The Gene Expression Omnibus (GEO, http:// www.ncbi.nlm.nih.gov/geo/) series accession number for the microarray data of this study is GSE26976. The significance of ratios of query (e.g. 4 h time point) over the reference (culture prior to inoculation of the biofilm reactor) was assessed using a one-class Student t-test with means of log2 of ratios tested against 0, p-values based on t-distribution with an overall threshold of 0.01 and Bonferroni correction. Confirmation of microarray expression levels with qRT-PCR Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed in a two-step reaction consisting of reverse transcription and real-time PCR. Reverse transcription was carried out using the QuantiTect Reverse Transcription Kit (Qiagen) in accordance with the manufacturer’s instructions. Briefly, 1 mg of total RNA was incubated in gDNA Wipeout Buffer (76) and RNase-free water and incubated at 42uC for 2 min to remove contaminating genomic DNA. The cDNA was synthesized from the RNA using Quantiscript reverse transcrip- tase, Quantiscript RT buffer and a primer mix consisting of long random primers and oligo-dT. The reaction was incubated at 42uC for 15 min and then at 95uC for 3 min to inactivate Quantiscript reverse transcriptase. Quantitative real-time PCR was performed as previously described [31]. Dilutions of the cDNA (0.2 ml of stock cDNA per 20 ml reaction) were used as PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28738