Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold

Tobias Irmscher,Yvette Roske,Igor Gayk,Valentin Dunsing,Salvatore Chiantia,Udo Heinemann,Stefanie Barbirz

doi:10.1016/j.jbc.2021.100286

Abstract

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel β-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.

Highlights

This article contains supporting information. ‡ Authors contributed . * For correspondence: Udo Heinemann, the access of external substances such as antimicrobial drugs, functioning clearly beyond simple diffusion barriers [3]
WceF shows high structural similarity to bacteriophage tailspike proteins (TSPs) and is enzymatically active on stewartan, proposing a role in exopolysaccharide modification when P. stewartii is in the mucoid, biofilm producing state
WceF is a native trimer of parallel, right-handed β-helices

Summary

Results

We recombinantly expressed, purified, and crystallized WceF lacking the N-terminal 28 amino acids of the Tat-signal peptide that encodes for export via the Tat-pathway for folded proteins [22]. Among all hits were many bacteriophage TSP containing trimers of parallel, right-handed β-helices structurally similar to WceF (Table S3). The bacterial protein WceF is notably less stable in the presence of detergent compared with the P22TSP of bacteriophage origin This is in agreement with a calculated overall lower WceF trimer interface stabilization (Fig. S9). Constructs lacking more N-terminal residues, i.e., the neck domain or the α-helix cap preceding the β-helix were insoluble This is in contrast to P22TSP, where the N-terminal head domain was dispensable for trimer stabilization [33], we conclude that Wcef is a stable native trimer only in the presence of all its N-terminal domains. Due to the different glycosidic bond cleavage velocities found for both enzymes, we compared oligosaccharide products after different reaction times, i.e., 197 h for WceF and 6 min for uEa1HTSP, respectively.

Discussion

Conclusion

Experimental procedures