Journal of Proteome Research bioﬁlm formation increased in response to unfavorable environ- mental conditions, including high metal concentrations, pH, and temperature changes.7 Recently, bioﬁlm characterization was carried out in the mesoacidophilic archaeon Ferroplasma acid- armanus. This euryarchaeon is predominantly found in bioﬁlm- associated structures in the environment, and two distinct bioﬁlm morphologies were described: a multilayer ﬁlm that formed on both an inert glass as well as pyrite that acts as energy source and 5 mm-long ﬁlaments that formed on the sintered glass spargers in a gas lift bioreactor. Proteomic studies on these bioﬁlms showed that 6 out of the 10 up-regulated proteins were involved in the adaptation to anaerobic growth indicating anaerobic zones in the multilayered Ferroplasma bioﬁlms.8 We have chosen the crenarchaeal model organism Sulfolobus spp. to initiate comprehensive studies on archaeal bioﬁlms. Sulfolobus species are hyperthermoacidophiles growing optimally at 70 85 C and pH 2 3 that are found worldwide in geother- mically active environments such as solfataric ﬁelds. Our previous work has provided evidence that cell surface structures such as ﬂagella and pili are essential for the initial attachment of Sulfolobus solfataricus to abiotic surfaces from shaking cultures.9 Furthermore, by means of a microtiter plate assay adapted to high temperatures, we established that bioﬁlm formation occurs more broadly in S. acidocaldarius, S. solfataricus and S. tokodaii. Additionally, it was determined that S. acidocaldarius most readily engaged in bioﬁlm formation in comparison to the other investigated Sulfolobus strains. Confocal laser scanning micro- scopy showed that the three strains form very diﬀerent commu- nity morphologies, ranging from simple carpet structures in S. solfataricus to high density tower-like forming structures in S. acidocaldarius. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, ga- lactose, mannose and N-acetylglucosamine once bioﬁlm forma- tion was initiated. Whereas ﬂagella mutants showed no pheno- type in three day old static bioﬁlms of S. solfataricus, a UV induced pili deletion mutant showed bioﬁlm impairment.10 Bacterial bioﬁlms have previously been examined using tran- scriptomic, proteomic and in vivo expression approaches for Escherichia coli and Pseudomonas spp.11 13 To date, it has been demonstrated that the transition from a planktonic lifestyle to a sedentary bioﬁlm lifestyle requires the coordinated regulation of genes involved in the development of bioﬁlms. These functional genomics analyses have revealed that hundred genes, most of which are uncharacterized, are diﬀerentially expressed during sessile lifestyle.12,14,15 However, after comparison of the diﬀer- entially expressed gene sets identiﬁed in several recent DNA microarray studies, a common expression pattern for bioﬁlms has yet to emerge, highlighting the particularity of bioﬁlm physiology among the diﬀerent studied models.11,14,16 Proteomics has also supplied a broader perspective on gene expression and has been used successfully to study bioﬁlms.17 20 Recently, a combined approach including proteomic and Fourier transform infrared (FT-IR) spectroscopy analysis immensely assisted the investiga- tion of the distinctiveness of bioﬁlm formation in Bordetella pertussis21 and E. coli.20 These studies have both demonstrated that bioﬁlm formation is a rather complex but distinct process and that deep insights into the bioﬁlm physiology can be pro- vided by the combined use of whole cell metabolic ﬁngerprinting by FT-IR, multivariate statistical analysis, and proteomics.20,21 Here, we have carried out a comparative study of three Sulfolobus strains (S. acidocaldarius, S. solfataricus and S. tokodaii) to gain insights into the physiological adjustments that may take ARTICLE place when these archaeons are grown as bioﬁlms. We used a combination of spectroscopic, proteomic and transcriptomic approaches to describe physiological and regulatory features involved in the bioﬁlm lifestyle for each strain. Furthermore, we present the data as a comparative analysis, to highlight common features in bioﬁlm formation among the three Sulfolobus strains under study. ’ EXPERIMENTAL PROCEDURES Strains and Growth Conditions The shaking precultures of Sulfolobus strains Sulfolobus solfa- taricus P2 (DSM1617), Sulfolobus acidocaldarius (DSM639) and Sulfolobus tokodaii (DSM16993) were grown for two days aerobi- cally at 76 C. The media described by Brock et al. (1972) were adjusted with sulphuric acid to a pH of 3 and supplemented with 0.1% w/v tryptone. Biofilm Growth and Cell Harvesting Bioﬁlms of the Sulfolobus strains were grown in large Petri dishes (150/20 mm gamma-sterile with Ventilation Cams, Sarstedt, N€umbrecht) for two days in Brock media as a standing culture. Four biological replicates were performed for each of the three strains. For all three strains, as was determined by Koerdt et al. (2010), diﬀerent OD600 inoculations were used: for S. solfataricus an OD of 0.03, for S. acidocaldarius an OD of 0.01, and for S. tokodaii an OD of 0.06. The Petri dishes were put in a specially designed metal box (25 cm L 20 cm W 20 cm D) with ∼500 mL of water in the bottom to minimize evapora- tion of the media, as described by Koerdt et al. (2010). After 48 h the planktonic and the bioﬁlm cells were harvested. The supernatant of the Petri dishes containing the planktonic cells was carefully removed. The bioﬁlm was washed with 50 mL of Brock media. Then, 15 mL Brock media was added and the bioﬁlm was harvested with a cell scraper (Cell Scraper, 28 cm length, Greiner bio-one, Frickenhausen). The bioﬁlm and plank- tonic cells were spun down for 20 min at 4 C and 2000 g. The liquid supernatant was removed and the pellets were frozen in liquid nitrogen and stored at 80 C. Fourier Transformation Infrared Spectroscopy Fourier transformation infrared spectroscopy (FTIR) spec- troscopy was conducted using a diamond Attenuated Total Reﬂectance (ATR) apparatus (Pike Technologies, Madison, WI) attached to a Shimadzu IRPrestige-21 Fourier Transforma- tion Infrared Spectrophotometer (Shimadzu, U.K). A blank spectrum, using the ATR without any biological samples, was run as a background and the baseline shift of the spectra was corrected using the IR solution software provided with the Shimadzu instrument. For each biological sample (bioﬁlm or planktonic), a small amount of the cell biomass was mounted on the ATR, covering the entire diamond surface, and allowed to dry at room temperature before analysis. At least 64 scans, with resolution of 4 cm 1 using the Happ-Genzel apodization func- tion, were collected for all samples. As biological macromolecules show characteristic peak absor- bance between 800 and 1800 cm 1,22 further spectral processing, including atmospheric correction was focused in this region. Spectral processing was carried out using IR solution software and each signiﬁcant peak was interpreted using the software “Knowitall” (http://www.knowitall.com/academic/welcome.asp) and corresponding absorption band assignments for functional groups of macromolecules previously reported for bacteria. 4106 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119