Journal of Proteome Research controlled via Nanodrop (NanoDrop ND-1000 Peqlab, Erlangen) and analytical gel electrophoresis. To remove all DNA, the samples were digested with DNase (TURBO DNA-free, Ambion Applied Biosystems, Darmstadt) according to the manual. Additionally RNase Inhibitor (RNasin Plus RNase Inhibitor, Promega, Mannheim) was added to inhibit the digestion of RNA. The purity of the RNA was tested via PCR with primers for a very small product before and after DNA digestion. Microarrays Experiments and Statistical Analysis Four biological replicates per strain were measured both for bioﬁlm and nonbioﬁlm grown cells. Geniom Biochips containing 4 arrays were used for the analysis. Each array on the chip had 15 000 spots with 50mer probes. For each gene, ﬁve to six diﬀerent probes were computed. The probe computation relies on freely available information of the DoE Joint Genome Institute. For background correction single “T” nucleotide probes were used. For further veriﬁcations, additional hybridiza- tion controls were added to the array template. Blank, labeling control and hybridization control probes are not included in the data. Febit (company, Heidelberg) used the MessageAmpII-Bac- teria Prokaryotic RNA Kit from Ambion for the labeling of RNA for mRNA expression analysis. The kit provides a transcription of RNA in cDNA, following a transcription in cRNA while enrich- ment of all nucleic acid molecules is included. For each array, 1 μg of total RNA was labeled according to the manufacturers instructions. After labeling, samples were dried in a vacuum concentrator and fragmented with a fragmentation buﬀer (see Febit protocol 20). Finally, Febit's proprietary standard Hybri- dization Buﬀer (20 μL per array) was added. Hybridization was done automatically overnight (16 h) at 45 C using the Geniom RT analyzer. After hybridization, the Geniom Biochip was washed automatically. For maximum sensitivity, Febit used biotin and its detection with strptavidin-phycoerythrin (SAPE), in combination with Febit's Consecutive Signal Enhancement (CSE) procedure. For a more detailed description please read Febit protocol 010. The feature recognition (using Cy3 ﬁlter set) and signal calculation were done automatically within millise- conds. Accurate detection of mRNA proﬁles correlates well with the qPCR data. There was no photo bleaching, thus enabling repeated measurements and multiple detection of each Biochip. The basis of the analysis was Febit's background corrected data sets. In these data sets, all negative values were replaced by 0. To reduce inﬂuences of sample binding problems, only the three spots with the highest intensities were used per gene in the following calculations. For each array the sum of all intensities was calculated. Subsequently all intensities of each array were multiplied with a factor to level the total sum to the highest. Afterward, the three intensities of each gene were reduced to the median, followed by quantile normalization. The following calculations were done in Microsoft Excel. The medians of the four biological replicates for bioﬁlm and planktonic cells were calculated and their logarithmic fold change calculated to the base 2. The signiﬁcance was computed by a statistical heteroscedastic t test with a two-tailed distribution. Regulated genes were chosen by a speciﬁc threshold value for each strain. For S. acidocaldarius, the standard threshold value of 0.05 was selected. For the two other strains, the values were adapted by the average of the calculated signiﬁcances (S. solfataricus 0.0631, S. tokodaii 0.0747). The resulting signiﬁcantly regulated genes were split in two groups (up-, down-regulated). ARTICLE To ﬁnd homologues for both groups, databases containing the amino acid sequences were created. Afterward, for each gene, a BLAST search in the speciﬁc database was performed with a cutoﬀ value of e 10. Quantitative RT-PCR (qRT-PCR) The cDNA Synthesis was performed with the iScript cDNA Synthesis Kit (Bio-Rad, Munich) according to the manual. qPCR was performed using SYBR Green qPCR Master Mix (Fermentas, St. Leon-Rot). Two-step cycling qPCR was carried out in 25 μL ﬁnal volume reaction according to the provider indications. A 20 times diluted cDNA of four biological replicates per strain were assayed both for bioﬁlm and nonbioﬁlm grown cells. Reactions were set up in a 7300/7500 Real Time PCR Systems Cycler (Applied Biosystems, Darmstadt Germany). Primers were designed to amplify a speciﬁc product of a length range of 90 120 bp (oligonnucleotide sequences are listed in Table S4 in the Supporting Information section). All primers were used at the ﬁnal concentration of 0.3 μM. The cycling program used for each primer pair was as follows: 10 min at 95 C, 40 cycles of 15 s at 95 C and 60 s at 60 C (annealing and extension in one step). Saci_0269, SSO0007 and ST2326 genes were used as standards for the relative quantiﬁcation. The Ct values were calculated automatically using software core applica- tion version 1.2.3 (Applied Biosystems). ’ RESULTS Spectroscopic Analysis of Biofilm versus Planktonic Cells Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Photoelectron Spectroscopy (XPS) Analysis. FT-IR spectroscopy has been successfully used as a rapid nondestructive technique to characterize the molecular composition of many different microbial systems,26 29 including environmental isolates30 and biofilms.20,30,31 ATR-FTIR can detect both surface and cyto- plasmic constituents of a biological sample. Furthermore, Jiang et al.29 established that variations in the ATR-IR spectrum essen- tially arose due to modifications on the cell surface. Therefore, it is proposed that any variations observed in the ATR-IR spectra conducted in this study, can be related to surface specific changes in functional groups. FT-IR spectra between 800 and 1800 cm 1 were recorded from S. acidocaldarius, S. solfataricus and S. tokodaii cell samples grown either in the bioﬁlm or planktonic mode, respectively (Figure 1). Principal component analysis (PCA) of the FT-IR spectra was applied to interpret the variations among the three strains in both lifestyles (bioﬁlm v/s planktonic). PCA analysis showed that cells associated with bioﬁlms clustered separately from their respective planktonic counterparts for each investi- gated strain (see Figure S1 in Supporting Information). These ﬁndings suggest that the FT-IR data provides spectroscopic evidence to support the premise that Sulofolobus spp. bioﬁlm population has characteristics that distinguish it from the plank- tonic cells population. To further investigate the potential chemical functional groups within the FT-IR spectrum that may have contributed to the observed diﬀerences in the PCA analysis, chemical functional groups were assigned to the FT-IR spectra (Figure 1) according to deﬁnitions from Eboigbodin and Biggs,26 Naumann22 and Bosch et al.32 Comparative analysis of normalized spectral data (Figure 1) mainly revealed a signiﬁcant increase in the intensity of absorption bands assigned to carbohydrate functional groups 4108 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119