Subproject A3 – Functional analysis of glycan-binding proteins and glycan transporters

Responsible: Matthias Höhne, Greifswald University

Putative proteins associated with glycan cycling were among the highest expressed gene products of bacteria during five consecutive years of algal blooms suggesting high relevance in marine carbon cycling. Many of these proteins belonged to polysaccharide utilization loci (PUL), genetic islands with putative SusD-like glycan binding proteins, TonB dependent receptors (TBDR=SusC like) and carbohydrate active enzymes (CAZymes) yet their function i.e. the polysaccharide substrates remain unknown. The subproject A3 aims to define the substrates and provide biochemical characterization of the polysaccharide specificity for those bacterial PULs that showed highest expression during spring algal blooms and in substrate defined in vitro experiments. Recent studies with human gut bacteria indicated SusD-like glycan binding proteins, which are located on the surface of bacteria and encoded by PULs coevolved with the specificity of its CAZymes. Hence, to identify the function we propose to identify the binding partners of SusD-like proteins as they are likely the substrates of the entire PUL. We have already cloned a diverse collection of putative glycan binding proteins belonging to the SusD-like family and expressed the recombinant proteins in Escherichia coli. After characterizing their binding properties towards algal polysaccharides we can now start to unravel the molecular determinants that affect substrate recognition. These is being investigated on single protein as well as on the superfamiliy level. Bioinformatic analysis of the sequence (structure) function relationships enable us to increase the accuracy of PUL function predictions. Furthermore, we investigate the TonB-dependent transport of selected PULs. Laminarin is one model substrate, because putative laminarinases belonged to the most abundant enzymes during algal blooms and we identified laminarinase specific PULs by directed proteome analysis of key glycan degraders from our isolate collection.