Biofilm formation and EPS analysis of the thermoacidophilic Archaeon Sulfolobus acidocaldarius

In their natural environment up to 99% of all microorganisms are assumed to live in microbial aggregates, so called biofilms. Bacterial as well as eukaryotic biofilms have been extensively studied for decades, while the formation of biofilms in the third domain of life, Archaea, only recently became of research interest. In the present study the biofilm formation and synthesis of extracellular polymeric substances (EPS) of the thermoacidophilic Archaeon Sulfolobus acidocaldarius was investigated. The cultivation of S. acidocaldarius in biofilms represented a major challenge, since the organism grows at temperatures of 75°C to 80°C and a pH of 2 - 3. Several cultivation methods frequently applied for bacterial biofilm formation, e.g. growth on glass slides or on solidified growth medium, were applied to establish a method, which yields sufficient biofilm mass required for subsequent analysis. Of the tested methods, growth as unsaturated biofilm on polycarbonate membrane filters or on gellan gum-solidified Brock medium plates, were successfully adapted to the conditions required for S. acidocaldarius biofilm growth. Microscopy studies of the biofilm formed on polycarbonate membrane filters revealed the occurrence of densely packed cells in a 110 µm thick biofilm, which were stainable with the N-acetylglucosamine specific lectin wheat germ agglutinin. Unsaturated biofilms grown on Brock medium plates were used to evaluate different EPS isolation methods. Five methods frequently applied for the isolation of EPS from bacterial biofilms and activated sludge, namely shaking, shaking in presence of a cation exchange resin (CER) as well as treatment with either NaOH, EDTA and crown ether were tested. The suitability of the five methods for the isolation of EPS from S. acidocaldarius biofilms was determined by comparing the concentration of isolated carbohydrates, proteins and eDNA. Moreover, interferences of the isolation methods with subsequent analytical methods and impact on cell integrity were determined. Applying these criteria, the CER method was determined the best suited EPS isolation procedure resulting in EPS carbohydrate, protein and eDNA concentrations of 4.7 ± 0.1 fg cell-1, 4.2 ± 1.1 fg cell-1 and 0.91 ± 0.48 fg cell-1, respectively. Visualization of the extracellular proteome of CER isolated EPS revealed approximately 1,000 protein spots mainly with a molecular mass of 25 kDa to 116 kDa and a pI of 5 to 8. Functional analysis of the EPS proteins using fluorogenic substrates as well as zymography demonstrated the presence of diverse groups of hydrolytic enzymes within the EPS of S. acidocaldarius, capable of degrading a variety of macromolecules. In-gel activity staining for proteases and esterases suggests expression of at least one extracellular protease, the so called thermopsin, and one extracellular esterase. Presence and the diversity of extracellular enzymes indicate the importance of EPS as an external digestive system for S. acidocaldarius biofilms. First analysis concerning the composition of the EPS polysaccharides within the biofilm matrix using acid hydrolyzation of the exopolysaccharides and thin layer chromatography revealed D-glucose as the main component. Additionally, a yet unidentified sugar was detected. The involvement of glycosyltransferases (GTs) of a gene cluster comprising 12 GTs, in the formation of the exopolysaccharides was proven by quantitative analysis of the carbohydrate concentration of GT deletion mutants. EPS composition of certain GT gene deletion mutants revealed significant changes of EPS protein and carbohydrate concentrations and ratios compared to the reference strain. In conclusion, this study revealed first insights into the general composition and function of EPS from the thermoacidophilic Archaeon S. acidocaldarius. For the first time, a method to isolate EPS from Archaea was successfully applied. This study identified polysaccharides as the main component of S. acidocaldarius EPS, followed by proteins and eDNA and revealed the individual importance of these polymers for the integrity of the biofilm. The EPS isolation method established in this study was used in a collaboration with the MPI Marburg revealing the involvement of an Lrs14 regulator in the S. acidocaldarius submersed biofilm formation (Orell et al. 2013b) as well as the involvement of an α-mannosidase in the EPS composition of S. solfataricus unsaturated biofilms (Koerdt et al. 2012). Furthermore, several GTs were identified to play crucial roles in the synthesis of exopolysaccharides. The expression and characterization of GTs with respect to substrate specificity will reveal their role in the formation of exopolysaccharides of S. acidocaldarius.


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