RESUMEN
Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25-116 kDa and pI values of 5-8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.
RESUMEN
Like bacteria, archaea predominately exist as biofilms in nature. However, the environmental cues and the molecular mechanisms driving archaeal biofilm development are not characterized. Here we provide data suggesting that the transcriptional regulators belonging to the Lrs14-like protein family constitute a key regulatory factor during Sulfolobus biofilm development. Among the six lrs14-like genes encoded by Sulfolobus acidocaldarius, the deletion of three led to markedly altered biofilm phenotypes. Although Δsaci1223 and Δsaci1242 deletion mutants were impaired in biofilm formation, the Δsaci0446 deletion strain exhibited a highly increased extracellular polymeric substance (EPS) production, leading to a robust biofilm structure. Moreover, although the expression of the adhesive pili (aap) genes was upregulated, the genes of the motility structure, the archaellum (fla), were downregulated rendering the Δsaci0446 strain non-motile. Gel shift assays confirmed that Saci0446 bound to the promoter regions of fla and aap thus controlling the expression of both cell surface structures. In addition, genetic epistasis analysis using Δsaci0446 as background strain identified a gene cluster involved in the EPS biosynthetic pathway of S. acidocaldarius. These results provide insights into both the molecular mechanisms that govern biofilm formation in Crenarchaea and the functionality of the Lrs14-like proteins, an archaea-specific class of transcriptional regulators.
