Journal of Proteome Research ARTICLE Figure 3. Whole genome expression proﬁling of S. acidocaldarius, S. solfataricus and S. tokodaii bioﬁlms compared to planktonic cells after 2 days of growth. Genes whose expression levels signiﬁcantly changed were categorized into functional groups in terms of their COG assigned numbers. The bars show the numbers of genes belonging to each group that were altered in expression (up- and down-regulated). encoding for homologous Lrs14 proteins were also altered during the bioﬁlm mode of growth, as revealed by qRT-PCR experiments (Table S4, Supporting Information). Transcriptional Response of Biofilm-Grown Sulfolobus Strains To broaden our analysis from proteomics to include tran- scriptomics to identify bioﬁlm-regulated genes, the transcrip- tional proﬁle of S. acidocaldarius, S. solfataricus and S. tokodaii bioﬁlm-associated cells was compared to that of their planktonic counterparts using microarray analysis. Results from the micro- arrays indicates that the expression of 437, 244, and 152 trans- cripts changed signiﬁcantly for S. acidocaldarius, S. solfataricus and S. tokodaii, respectively, during the establishment of a two day old bioﬁlm (Supporting Information, Excel ﬁle “Transcriptomic signiﬁ- cant data.xlsx”). S. acidocaldarius transcriptomic data showed that 335 genes (∼15% of the S. acidocaldarius genome) displayed a 1.5-fold (log2 0.5) or more change in expression. These included 103 genes that were up-regulated in the bioﬁlm and 206 down-regulated genes. For S. solfataricus, 103 genes had an altered expression level of 1.5-fold or more (∼3.4% of the S. solfataricus genome), the majority of which (60) were down-regulated, while 43 were up-regulated. S. tokodaii transcriptomic data showed that the change in expression levels was lower than those from S. acidolcaldarius and S. solfataricus. Up to 32 genes were diﬀerentially regulated 1.5-fold or more in S. tokodaii (∼1% of the genome), 15 of which that were up-regulated in bioﬁlm and 17 of which were down-regulated. It was determined that 51%, 59% and 66% of the diﬀerentially expressed genes corresponded to those annotated as having hypothetical or unknown function for S. acidocaldarius, S. solfataricus and S. tokodaii, respectively. All bioﬁlm-regulated genes were catalogued into functional groups according to their COG category. The analysis yielded genes predominantly involved in energy production, energy conversion, amino acid metabolism, lipid and carbohydrate metabolism, transport related functions, and cell sur- face appendages (Figure 3). The key ﬁndings are discussed below (Table 3) and the complete data set is presented in the Supporting Information, Excel ﬁle: “Transcriptomic signiﬁcant data.xlsx”. RNA microarray experiments displayed bioﬁlm-regulated genes encoding terminal oxidases in Sulfolobus spp. cells. More speciﬁcally, the data show up-regulation of the gene encoding for the polypeptide I of the cytochrome c oxidase complex ba3 in both S. acidocaldarius (Saci_0097) and S. tokodaii (ST2595) bioﬁlm-associated cells. In addition, a quinol oxidase-2 gene (soxE) was also up-regulated in S. solfataricus (SSO2972). In S. acidocaldarius, SoxE forms part of the bb3 terminal oxidase complex SoxM (SoxEFGHIM).35 In general, the ﬁndings from proteomic analyses revealed no noteworthy correlation at the transcriptomic level, and only the down-regulation of V-type ATPase subunit B encoding gene (Saci_1549) correlated on both in transcriptomic and proteomic analyses (Table 3). As performed for the proteomic data, a BLASTp analysis was performed for each transcript using the encoded amino acid 4115 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119