The S-Layer Protein of the Anammox Bacterium Kuenenia stuttgartiensis Is Heavily O-Glycosylated.

Muriel C F Van Teeseling,Friedrich Altmann,Rudolf Figl,Laura Van Niftrik,Paul Messner,Cornelia B Rath,Mike S M Jetten,Daniel Maresch,Christina Schäffer

doi:10.3389/fmicb.2016.01721

Abstract

Anaerobic ammonium oxidation (anammox) bacteria are a distinct group of Planctomycetes that are characterized by their unique ability to perform anammox with nitrite to dinitrogen gas in a specialized organelle. The cell of anammox bacteria comprises three membrane-bound compartments and is surrounded by a two-dimensional crystalline S-layer representing the direct interaction zone of anammox bacteria with the environment. Previous results from studies with the model anammox organism Kuenenia stuttgartiensis suggested that the protein monomers building the S-layer lattice are glycosylated. In the present study, we focussed on the characterization of the S-layer protein glycosylation in order to increase our knowledge on the cell surface characteristics of anammox bacteria. Mass spectrometry (MS) analysis showed an O-glycan attached to 13 sites distributed over the entire 1591-amino acid S-layer protein. This glycan is composed of six monosaccharide residues, of which five are N-acetylhexosamine (HexNAc) residues. Four of these HexNAc residues have been identified as GalNAc. The sixth monosaccharide in the glycan is a putative dimethylated deoxyhexose. Two of the HexNAc residues were also found to contain a methyl group, thereby leading to an extensive degree of methylation of the glycan. This study presents the first characterization of a glycoprotein in a planctomycete and shows that the S-layer protein Kustd1514 of K. stuttgartiensis is heavily glycosylated with an O-linked oligosaccharide which is additionally modified by methylation. S-layer glycosylation clearly contributes to the diversification of the K. stuttgartiensis cell surface and can be expected to influence the interaction of the bacterium with other cells or abiotic surfaces.

Highlights

From early on in the study of Planctomycetes, their cell biology has been a topic that has sparked much interest (Fuerst and Webb, 1991; Fuerst, 1995; Lindsay et al, 1997, 2001)
Recently peptidoglycan was revealed in several planctomycetes (Jeske et al, 2015; van Teeseling et al, 2015) and a proteinaceous S-layer was described in the anammox bacterium K. stuttgartiensis
As at the time of the discovery of the S-layer, the peptidoglycan layer in these bacteria had not yet been found, it was hypothesized that the S-layer provided integrity and strength to the anammox cells

Summary

Introduction

From early on in the study of Planctomycetes, their cell biology has been a topic that has sparked much interest (Fuerst and Webb, 1991; Fuerst, 1995; Lindsay et al, 1997, 2001). Anammox bacteria are present in marine environments, freshwater and soil (Schmid et al, 2007; Dale et al, 2009; Humbert et al, 2010; Zhu et al, 2013; Sonthiphand et al, 2014) where they contribute significantly to the loss of fixed nitrogen They are applied in sustainable nitrogen removal systems worldwide (Kartal et al, 2010). The anammox cell plan comprises a third membrane-enclosed compartment called anammoxosome (Lindsay et al, 2001) This prokaryotic organelle is the location of the anammox reaction that supports the energy for growth of these bacteria (Neumann et al, 2014). In this case the membrane curvature is hypothesized to provide an enlarged surface area for the many membraneassociated proteins involved in the anammox reaction (van Niftrik et al, 2004; van Teeseling et al, 2013)

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