Journal of Proteome Research most radical spectral change in comparisons of bioﬁlm-associated versus planktonic samples. In line with this premise, XPS analysis also showed that S. acidocaldarius bioﬁlm-associated cells experi- enced an increase of polysacharide-containing molecules on their cell surfaces (Table 1). Interestingly, a putative glycosyl tranferase-encoding gene were regulated in S. acidocaldarius (Table 3). Glycosyltransferases (GTs) play an important role modifying both lipid and protein components of biological membranes by the covalent addition of polysaccharides. In addition, the speciﬁc function of GTs in biosynthesis of high- molecular-weight sugar-rich heteropolymeric EPS molecules has been described in bacterial systems.44 Moreover, GT encoding genes have been found to be overexpressed in bacterial bioﬁlms, and their disruption alters the ability to synthesize the EPS matrix.45 The increased expression of EPS production related genes observed in S. acidocaldarius bioﬁlm cells might be correlated to its particular cell surface chemical composition. Future analyses will focus on determining their involvement in the EPS biosynthetic pathway, which is expected to involve further enzymatic activities. Commonly Biofilm-Regulated Genes among the Three Strains The description of bioﬁlm-regulated genes and proteins common to all three examined Sulfolobus species yielded some interesting ﬁndings. All three displayed increased levels of a putative transcriptional regulator belonging to the Lrs14-like proteins (Saci_1223, SSO1101 and ST0837) (Table 2). These putative proteins are homologous to the Lrs14 protein (SSO1108) of S. solfataricus, the protein levels of which were also increased during the bioﬁlm lifestyle (Table 3). It has been described that S. solfataricus Lrs14 (SSO1108) is autoregulated in a negative manner and accumulates in the midexponential and late growth phases.46 Archaeal Lrs14-like regulators are thought to be related to the Lrp-AsnC bacterial transcriptional regulator family (leucine-responsive regulatory protein).46 The Lrp E. coli regulon includes genes involved in amino acid biosynthesis (ilvIH, serA), in the biosynthesis of cell surface structures such as pili (pap, fan, ﬁm), and in the assimilation of ammonia (glnA, gltBD).47 However, Lrp-like regulators have not been yet directly identiﬁed as being involved in bioﬁlms. In bioﬁlm-associated bacteria, several global gene regulators are known to control a wide range of adaptive physiological and regulatory circuits within sessile community and to be up-regulated as a response to environmental conditions, that is, nutrient limitation, oxygen availability and osmotic stress.15 Thus, the expression of these homologous transcriptional regulators might constitute a master regulatory factor involved in Sulfolobus bioﬁlm development. The functional role of Lrs14-like proteins in Sulfolobus bioﬁlms is being currently investigated. One gene, 3-oxoacyl-(acyl-carrier-protein) reductase (FabG) (Table 3), was found to be down-regulated in bioﬁlm-associated cells (and up-regulated in planktonic cells) in all three Sulfolobus strains. FabG enzymatic activity is involved in the production of the Quorum Sensing (QS) autoinducer (AI) 3-oxo-dodecanoyl- HSL (3-oxo-C12-HSL) in P. aeuruginosa.36 In bacteria, QS phe- nomena is known to be closely interrelated to bioﬁlm formation. QS provides the means to coordinate the activities of cells so that they function as a multicellular community. Interestingly, FabG levels were also found to be heightened in P. aeuruginosa planktonic cells in comparison to their bioﬁlm counterparts.17 Although, it seems that Sulfolobus genomes do not encode ARTICLE LasI-homologous proteins, an utterly diﬀerent mechanism em- ployed by an unknown activity might be involved in tandem with FabG to produce putative archaeal AI molecules. In this regard, studies in bioﬁlms of the archaeon F. acidarmanus Fer1 showed no evidence for quorum sensing or signaling molecules.8 How- ever, the production of AI molecules by Sulfolobus cells still has to be proven. Furthermore, in the future, it will be of interest to determine the potential occurrence of cell signaling and com- munication within Sulfolobus bioﬁlm-associated communities. ’ ASSOCIATED CONTENT bS Supporting Information DNA microarrays signiﬁcant data of each Sulfolobus strains is listed in the exel ﬁle “Transcriptomic signiﬁcant data.xlsx”. Proteomic data analysis of each Sulfolobus strains is listed in the exel ﬁles: “Proteomic suplementary-1.xlsx” for S. solfataricus P2, “Proteomic suplementary-2.xlsx” for S. acidocaldarius and “Proteomic suplementary-3.xlsx” for S. tokodaii. Sheet 1 shows details of peptides lists and Sheet 2 shows details of quantiﬁed proteins lists. Table S1 shows binding energies, assignments and composition (%) of XPS spectral bands of Sulfolobus acidocaldarius, solfataricus and tokodaii grown planktonically and as bioﬁlm. Table S2, iTRAQ labeling of samples. Table S3 shows all signiﬁcant result obtained from iTRAQ proteomic analysis. Table S4, deter- mination of gene expression by qRT-PCR. Table S5 lists qPCR oligonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *S.-V. Albers, e-mail: albers@mpi-marburg.mpg.de. Tel.: +496421178426. Fax: +496421178429. Author Contributions ‡These authors contributed equally to this manuscript ’ ACKNOWLEDGMENT A.K., A.O. and S.-V.A. were supported by intramural funds of the Max Planck Society. S.-V.A. received additional support by a VIDI grant from the Dutch Science Organization (NWO). EPSRC (S&I Grant EP/E036252/1), BBSRC (under the Sys- MO Programme SulfoSys; BBF0034201) ’ REFERENCES (1) Lopez, D.; Vlamakis, H.; Kolter, R. Bioﬁlms. Cold Spring Harb. Perspect. Biol. 2010, 2 (7), a000398. (2) Kruger, M.; Blumenberg, M.; Kasten, S.; Wieland, A.; Kanel, L.; Klock, J. H.; Michaelis, W.; Seifert, R. A novel, multi-layered methano- trophic microbial mat system growing on the sediment of the Black Sea. Environ. Microbiol. 2008, 10 (8), 1934–47. (3) Zhang, C. L.; Ye, Q.; Huang, Z.; Li, W.; Chen, J.; Song, Z.; Zhao, W.; Bagwell, C.; Inskeep, W. P.; Ross, C.; Gao, L.; Wiegel, J.; Romanek, C. S.; Shock, E. L.; Hedlund, B. P. Global occurrence of archaeal amoA genes in terrestrial hot springs. Appl. Environ. Microbiol. 2008, 74 (20), 6417–26. (4) Rinker, K. D.; Kelly, R. M. Growth Physiology of the Hyperther- mophilic Archaeon Thermococcus litoralis: Development of a Sulfur-Free Deﬁned Medium, Characterization of an Exopolysaccharide, and Evi- dence of Bioﬁlm Formation. Appl. Environ. Microbiol. 1996, 62 (12), 4478–85. 4117 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119