P is number of quantiﬁed proteins). As a result, values of 1.04 10 4, 1.08 10 4 and 9.24 10 5 were calculated for S. solfataricus P2, S. acidocaldarius, S. tokodaii, respectively. However, we also considered proteins with p-values e0.05 regulated proteins for a conﬁrmatory test. Lists of signiﬁcantly regulated proteins are summarized in sheet 3 in proteomics Supporting Information, Excel ﬁles 1, 2, and 3 for S. solfataricus, S. acidocaldarius, S. tokodaii, respectively. In order to get a wider view for understanding the behavior of cells in bioﬁlm versus planktonic conditions, proteins with p-values less than R = 0.05 (without correction) (known as lists of potentially regulated proteins) were also used for further discussion. The lists of these potentially regulated proteins are shown in the sheet 4 in pro- teomics Supporting Information, Excel ﬁles 1, 2 and 3. In terms of identifying proteins that were diﬀerentially chan- ged during the bioﬁlm mode of life versus the planktonic coun- terparts, a protein comparison was performed. S. acidocaldarius had 30 bioﬁlm-regulated proteins (19 up- and 11 down-reg- ulated), S. solfataricus displayed 36 protein changes (17 up- and 19 down-regulated) and for S. tokodaii 67 proteins changed their relative abundances in the bioﬁlm lifestyle (41 up- and 26 down- regulated). All the statistically signiﬁcant changes are tabulated in Table S3 in the Supporting Information section. The most noteworthy ﬁndings are listed in Tables 2 and 3 and discussed in the next section. Additionally, a BLASTp analysis was carried out in order to identify common bioﬁlm-regulated proteins between the three Sulfolobus species (bioﬁlm core response). Amino acid sequences as queries of both signiﬁcantly up- regulated proteins and down-regulated proteins were used in this analysis, respectively. This analysis yielded three diﬀerent proteins which were commonly up-regulated, while four proteins were found to be down-regulated in all three Sulfolobus species (Figure 2, Table 2). Furthermore, the BLASTp analysis also yielded homologous proteins that were commonly regulated in at least two strains (Figure 2, Table 3). Identification of Differentially Expressed Proteins in Biofilm- Grown Sulfolobus Strains Identiﬁed proteins were categorized in functional groups using the assigned COG numbers. By means of this analysis, we were able to ﬁnd that Sulfolobus spp. bioﬁlm mode of growth altered not only the expression of proteins involved predominantly in cellular functions like energy production, energy conver- sion, adaptation to environmental changes and stress, and substrate transport/binding activities but also the expression of proteins implicated in cellular processes and regulatory events (Table 3). ARTICLE S. to 0.85 1.48 0.82 1.02 1.0 0.26 0.79 0.88 0.58 0.91 0.74 0.22 0.24 0.67 0.14 0.4 0.43 0.16 0.63 0.43 0.37 0.55 1.03 0.47 1.72 0.64 0.21 transcriptional regulator Lrs14-like protein DNA-binding protein Chaperone Small heat shock protein hsp20 ABC transporter ATP-binding protein -alpha subunit (thsA) -beta subunit (thsB) -gamma subunit V-type ATPase 3-oxoacyl-(acyl carrier protein) reductase (fabG-1) Table 2. Common Bioﬁlm-Regulated Proteins and Genes within S. acidocaldarius, S. solfataricus and S. tokodaii Strains Up-regulated in bioﬁlm proteomic analysis annotation ORF number fold change (log2) Saci_1223 SSO1101 ST0837 Saci_0064 SSO10610 STS077 Saci_1665 SSO2427 ST0555 Saci_2305 SSO0053 ST0535 Saci_1401 SSO0862 ST1253 Saci_0666 SSO0282 ST0321 Saci_1203 SSO3000 ST0820 Saci_1548 SSO0563 ST1436 Saci_1792 SSO0975 ST1299 Journal of Proteome Research Table 1. Quantiﬁcation of the Elemental Surface Composition (C, O and N) of S. acidocaldarius (S.aci), S. solfataricus (S.so) and S. tokodaii (S.to) Grown Planktonically and as Bioﬁlma planktonic cells S. so S. aci S. to S. aci bioﬁlm cells S. so C 62.23 ( 1.36 63.73 ( 1.10 52.97 ( 1.42 57.42 ( 0.74 59.53 ( 0.56 56.22 ( 2.41 O 29.90 ( 0.17 28.38 ( 1.49 40.81 ( 2.81 34.50 ( 1.04 31.36 ( 0.40 35.49 ( 2.43 N 7.88 ( 1.20 7.90 ( 0.40 6.23 ( 1.39 8.09 ( 0.30 9.12 ( 0.16 8.29 ( 0.03 N/C 0.13 ( 0.02 0.120 ( 0.004Δ 0.12 ( 0.02 0.140 ( 0.003 0.150 ( 0.004Δ 0.15 ( 0.01 O/C 0.48 ( 0.0R1 0.45 ( 0.03β 0.77 ( 0.07γ 0.60 ( 0.03R 0.53 ( 0.01β 0.63 ( 0.07γ a Numbers with similar Greek symbols are statistically signiﬁcant (90% conﬁdence interval; Students’ t test, p < 0.1). Outliers were detected and removed in the data by calculating the interquartile range and also by using Grubbs’ test at 99% conﬁdence interval. RNA microarray analysis Down-regulated in bioﬁlm proteomic analysis annotation ORF number fold change Thermosome RNA microarray analysis Proteomic data showed that adjustments in energetic meta- bolism are made during growth in a bioﬁlm. Putative cytochrome oxidase subunits were identiﬁed as up-regulated in S. solfataricus 4110 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119