Journal of Proteome Research sequences for queries of both signiﬁcantly up-regulated genes and signiﬁcantly down-regulated genes. By doing so, we were able to cluster bioﬁlm-regulated genes common to all three Sulfolobus species (Figure 3, Table 2). From the up-regulated data set, only one gene was determined to be overexpressed in all three Sulfolobus species in bioﬁlm-associated cells (Figure 2, Table 2). This change corresponds to an ATP-binding transpor- ter (Saci_2305, SSO0053 and ST0535), most likely involved in cation detoxiﬁcation. Further ORFs annotated as inorganic substrates transporters were found to be up-regulated in at least two Sulfolobus strains (SSO1078 and ST1577). On the other hand, the single common bioﬁlm down-regu- lated gene, or alternatively, up-regulated in planktonic cells, corresponded to a 3-oxoacyl-(acyl-carrier-protein) reductase (Saci_1792, SSO0975 and ST1299) (Table 3). Interestingly, this enzyme is involved in the production of the quorum sensing autoinducer 3-oxo-C12-HSL in P. aeuruginosa.36 Some of the genes proposed to be required for ﬂagella biosynthesis and assembly were up-regulated in S. acidocaldarius bioﬁlm-associated cells, as revealed by RNA microarrays (Table 3). The UV-inducible type IV pili related gene upsF (SSO0119) was up-regulated. upsF encodes a putative trans- membrane protein proposed to form part of the membrane platform of the pili structure (Table 3). The expression of some genes catalogued into the cell wall/ membrane/envelope biogenesis functional group was also al- tered in Sulfolobus bioﬁlm cells (Table 3). S. acidocaldarius displayed the overexpression of ORFs Saci_0134, which encodes a hypothetical protein sharing signiﬁcant sequence similarity to an annotated NAD-dependent epimerase/dehydratase of Metallosphaera sedula (Msed_0434). This enzyme is potentially involved in extracellular polysaccharides (EPS) production.37 ’ DISCUSSION General Overview We previously described that S. acidocaldarius, S. solfataricus, and S. tokodaii form very diﬀerent bioﬁlm morphologies, ranging from simple carpet structures in S. solfataricus to high-density tower-like forming structures in S. acidocaldarius.10 In this study, we have further characterized the process of Sulfolobus bioﬁlm formation by integrating spectroscopic analysis, transcriptomics and proteomics in order to determine how each of the three species is adapted to growth in bioﬁlms. As we showed through FTIR analysis, spectral data sets from bioﬁlm cells within Sulfolobus bioﬁlms diﬀered substantially from their planktonic counterparts. In addition, we were able to show that a bioﬁlm-associated lifestyle displayed distinct expression transcriptomic and proteomic proﬁles in all Sulfolobus species. These results resonate with those from studies of bacteria, in which proteomics and FT-IR spectroscopy with multivariate statistical analysis were combined to show a distinct, species- speciﬁc diﬀerences between the physiology of bioﬁlm-associated and planktonic bacterial cells.20,21,38,39 In addition, the expression proﬁle as per microarray analysis showed that the bioﬁlm lifestyle aﬀects each strain diﬀerently. While ca. 15% of the S. acidocaldarius genes’ expression was altered by a factor of 1.5 or more, the change in genes expression patterns represented only ∼3.4 and ∼1% in S. solfataricus and S. tokodaii, respectively. The percentage diﬀerences in bioﬁlm- regulated genes between the Sulfolobus species are consistent with what has been reported in transcriptomes from bioﬁlm-grown ARTICLE bacteria. For example, in E. coli, 5.5% of ORFs were determined to have diﬀerent expression patterns in bioﬁlm-associated cells, while 14% of genes in bioﬁlm-associated B. subtilis cells had a dif- ferent expression pattern when compared to planktonic cells.40,41 Furthermore, the numbers of bioﬁlm-regulated genes in other bacterial systems are even smaller, as P. aeruginosa transcriptomic experiments showed that only 1% of genes are diﬀerentially expressed in bioﬁlms.42 The diﬀerence in gene expression levels between Sulfolobus bioﬁlm-associated and planktonic cells were found to be less than that of eubacteria. Thus, it is tempting to suggest that lifestyle transition from planktonic to bioﬁlm does not radically alter the regulated transcript abundance in Sulfolobus bioﬁlms. However, even slight changes in gene expression may potentially have a profound eﬀect on cellular physiology, as has been described by analyses of transcriptomes from bioﬁlm-associated bacteria.14 Discrepancies observed between transcriptome and proteome proﬁling underscores the putative role that post-transcriptional and post-translational regulation mechanisms might play in Sulfolobus bioﬁlm formation. In this regard, the reduced regula- tion at the transcriptional level observed in S. tokodaii in comparison to the remarkable proteomic changes obtained suggests that the physiological eﬀect might correspond to intense post-transcriptional regulation. Moreover, in the future we expect to gain further understanding of the role of regulatory noncoding RNAs in bioﬁlm-associated Sulfolobus cells. Energetic Adjustments during the Biofilm Mode of Life Genes and proteins involve in energetic metabolism were highly altered in Sulfolobus spp. bioﬁlm-associated population. These changes included mainly genes related to respiratory complexes, Tricarboxylic acid cycle (TCA) enzymes and V-AT- Pases subunits (Table 3, Figure 3). Genes encoding cytochrome o ubiquinol oxidase subunits have been also described as up- regulated in both Salmonella enterica serovar Tiphymurium and E. coli K-12 bioﬁlm-associated cells, suggesting that the environ- ment of these bioﬁlms was aerobic,13,15 which might also be the case for Sulfolobus bioﬁlms. On the other hand, previous studies have shown that terminal respiratory complexes work as proton pumps for maintaining the intracellular pH and generating proton motive force in certain Sulfolobales.43 Moreover, it has been proposed that the SoxM complex might serve as a pH sensor and it would assume its highest activity when the pH rises to values greater than 5 in the extracellular medium.35 As we previously observed, the pH in the extracellular medium during bioﬁlm development progressively raises above 5 in Sulfolobus spp. cells (Koerdt et al., unpublished results). Consequently, the overexpression of Sox complex-related genes might be a response to keep the ambient pH down. Furthermore, several genes playing a role in TCA cycle were also altered, being most of them down-regulated in bioﬁlm- associated cells, suggesting a decreased metabolic activity in this cell population in comparison to the planktonic counterparts. V-ATPases subunits were also down-regulated in S. acidocaldar- ius bioﬁlm-associated cells (Table 3). Conversely, the ATP synthase subunit E was found up-regulated in S. solfataricus bioﬁlm-associated cells (Table 3). Cell Surface Modifications As we have previously described, S. acidocaldarius more readily engages in community formation than other Sulfolobus species.10 This descriptive observation is conducive with and complemen- tary to our spectroscopic analysis. S. acidocaldarius showed the 4116 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119