Exopolysaccharide Synthesis, Translocation, and Potential Interplay with Protein Glycosylation in Sulfolobus acidocaldarius (SweetSaci)

• Applicant: Professorin Dr. Bettina Siebers
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 393406057

Exopolysaccharides (ePS) play crucial roles in microbial stress responses and biofilm formation. Despite their significance, the synthesis pathways and functions of archaeal ePS are still largely unexplored. In SweetSaci, we aim to elucidate ePS synthesis, translocation, and the potential interplay between ePS and protein glycosylation pathways in the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci). In our previous project (ArchaeaEPS), we developed an efficient biofilm cultivation technique and analytical methods for analyzing EPS components (extracellular polymeric substances) as well as ePS composition and size. Analysis of gene deletion mutants showed that the gene cluster saci_1904-1927, which encodes various glycosyltransferases and membrane proteins, including a putative homolog of bacterial Wzx flippases, significantly influences biofilm formation and ePS synthesis. However, phenotypes similar to those reported for mutants in archaeal N-glycosylation, such as reduced motility, were also observed. Bioinformatic analyses suggest similarities of proteins encoded by this gene cluster to components of the bacterial Wzx/Wzy-dependent ePS synthesis pathway (Wzx flippase) as well as to GT-C fold proteins from bacterial three-component systems and eukaryotic N-glycosylation (oligosaccharyltransferase (OST) complex), indicating a potential connection between ePS and protein glycosylation pathways. SweetSaci aims to elucidate the mechanisms and potential interplay of these crucial biological processes. As an important foundation, the primary structure of Saci ePS will be deciphered with regard to the monosaccharide sequence, the absolute and anomeric configuration, glycosidic bonds, and non-carbohydrate side chains. Furthermore, we will examine changes in EPS and ePS composition and dynamics during the biofilm cycle and in response to stress conditions such as temperature stress or nutrient deficiency. This will help elucidate Saci’s adaptation strategies to changing environmental conditions and gain insights into the physiological functions of ePS. We will also explore the interplay between ePS and N-glycan synthesis by phenotypically characterizing deletion mutants of dolichol phosphate glycosyltransferases (DolP-GTs) and membrane proteins. These data will be complemented by the enzymatic characterization of DolP-GTs. Finally, we will investigate protein interactions and potential membrane protein complex formation, providing valuable insights into their involvement and cooperation in glycan synthesis processes. Altogether, SweetSaci will contribute to the understanding of archaeal biofilms and provide important insights into the interplay of glycosylation pathways in the third domain of life.

• DFG Programme: Research Grants