A new addition to the cell plan of anammox bacteria: "Candidatus Kuenenia stuttgartiensis" has a protein surface layer as the outermost layer of the cell

Abstract

Anammox bacteria perform anaerobic ammonium oxidation (anammox) and have a unique compartmentalized cell consisting of three membrane-bound compartments (from inside outwards): the anammoxosome, riboplasm, and paryphoplasm. The cell envelope of anammox bacteria has been proposed to deviate from typical bacterial cell envelopes by lacking both peptidoglycan and a typical outer membrane. However, the composition of the anammox cell envelope is presently unknown. Here, we investigated the outermost layer of the anammox cell and identified a proteinaceous surface layer (S-layer) (a crystalline array of protein subunits) as the outermost component of the cell envelope of the anammox bacterium "Candidatus Kuenenia stuttgartiensis." This is the first description of an S-layer in the phylum of the Planctomycetes and a new addition to the cell plan of anammox bacteria. This S-layer showed hexagonal symmetry with a unit cell consisting of six protein subunits. The enrichment of the S-layer from the cell led to a 160-kDa candidate protein, Kustd1514, which has no homology to any known protein. This protein is present in a glycosylated form. Antibodies were generated against the glycoprotein and used for immunogold localization. The antiserum localized Kustd1514 to the S-layer and thus verified that this protein forms the "Ca. Kuenenia stuttgartiensis" S-layer.

FIG 1

Cell plan of the anammox cell showing the three different compartments and their surrounding membranes. The riboplasm compartment has been defined the pirellulosome in Planctomycetes.

FIG 2

“Ca. Kuenenia stuttgartiensis” cells display S-layers as observed by TEM after multiple types of sample preparation. (A) “Ca. Kuenenia stuttgartiensis” cells are covered by a hexagonal S-layer, as observed after freeze-etching. (B) The FFT power spectrum of a part of the S-layer seen in panel A reflects the regular pattern of the S-layer. The p6 symmetry and center-to-center spacing of 20 nm for the S-layer of “Ca. Kuenenia stuttgartiensis” are reflected by the FFT power spectrum. (C) A freeze fracture through the S-layer gives an inside view into the “Ca. Kuenenia stuttgartiensis” cell underneath. (D) The S-layer (indicated by the arrow) forms a zigzag layer on top of the cytoplasmic membrane in “Ca. Kuenenia stuttgartiensis” cells that were cryofixed; freeze-substituted in acetone containing 2% osmium tetroxide, 0.2% uranyl acetate, and 1% water; Epon embedded; and thin sectioned. Scale bars, 200 nm.

FIG 3

The S-layer of “Ca. Kuenenia stuttgartiensis” visualized by image processing. (A) Correlation averaging shows that the S-layer of “Ca. Kuenenia stuttgartiensis” has hexagonal symmetry with a unit cell consisting of six protein densities surrounding a central pore. Scale bar, 20 nm. (B) Relief reconstruction gives an impression of the surface of the S-layer in three dimensions. White represents high and black represents low areas. Scale bar, 10 nm.

FIG 4

S-layer patches are present after S-layer enrichment of “Ca. Kuenenia stuttgartiensis.” (A) S-layer patch with typical hexagonal symmetry observed by freeze-drying. (B) A three-dimensional model of the isolated S-layer shows the same relief as observed for the S-layers present on “Ca. Kuenenia stuttgartiensis” cells. (Inset) Correlation averaging used to determine the three-dimensional representation. Scale bar, 70 nm.

FIG 5

Analysis of crude extract and enriched S-layers of “Ca. Kuenenia stuttgartiensis” by SDS-PAGE. (A) SDS-PAGE gel stained with Coomassie G shows all proteins present in the “Ca. Kuenenia stuttgartiensis” crude extract. (B) SDS-PAGE gel stained with Coomassie G shows the proteins present after S-layer enrichment. (C) SDS-PAGE gel stained with periodic acid-Schiff's reagent shows glycoproteins present after S-layer enrichment. Arrows indicate protein bands in which Kustd1514 was detected via LC-MS/MS analysis.

FIG 6

Immunoblot analysis of the antiserum directed against the “Ca. Kuenenia stuttgartiensis” S-layer glycoprotein Kustd1514 tested against a “Ca. Kuenenia stuttgartiensis” cell extract. Lane 1, marker; lane 2, incubation with preimmune serum; lane 3, incubation with anti-Kustd1514. The arrow indicates the expected target size (about 250 kDa).

FIG 7

(A to C) Immunogold localization of the antiserum directed at Kustd1514 localizes the protein to the S-layer surrounding the cells in rehydrated “Ca. Kuenenia stuttgartiensis” cryosections. In panel A, for clarification, white circles indicate gold labels that are localized inside the cytoplasmic membrane (so not directly on the S-layer). Considering the length of the antibody-protein A-gold complex (25 nm), these gold labels most likely also correspond to S-layer labeling (except for the one in the anammoxosome). (D) Negative control incubated with preimmune serum instead of antiserum. Scale bars, 500 nm.