Updated project metadata. The physiology of the planctomycetal anammox bacteria makes them particularly special because they share features with all three domains of life. Anammox bacteria have been reported recently to produce surface-layer proteins, which represent the outermost layer and provide structure, shape and protection under extreme conditions. Furthermore, we report on the unique cell surface-layer glycosylation of the anammox bacterium Ca. Kuenenia stuttgartiensis as revealed by a newly established glycoproteomics approach. This approach enables untargeted exploration of prokaryotic protein glycosylation from (high-resolution) shotgun proteomics data directly.