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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 . 

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 . 

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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 pepti...

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... oxidation, nammox bacteria thereby are converting able to perform ammonium anaerobic and nitrite ammonium to dini- trogen gas (1, 2). Anammox bacteria are applied in wastewater treatment to remove ammonium from wastewater (3) and play an important role in the biological nitrogen cycle (4, 5). They com- prise five genera (which all have a “ Candidatus ” status, since they are described from phylotypes that are not isolated in pure culture) that belong to the phylum Planctomycetes in the order Bro- cadiales (6). The species “ Candidatus Kuenenia stuttgartiensis” is the most extensively studied anammox bacterium, and its genome (7), proteome, and metabolism (8) were described previously. Functional gene analysis remains difficult since no genetic system is available for anammox bacteria. The phylum Planctomycetes is known for encompassing strik- ingly complex cell plans involving multiple cellular compartments and extensive membrane invaginations (9). Currently, the cell organization of Planctomycetes is under debate (10–13). Even within this phylum, the cell biology of anammox bacteria is remarkable, since anammox cells are divided into no fewer than three compartments, separated by bilayer membranes ( Fig. 1). The inner compartment, the anammoxosome, is a so-called “prokaryotic organelle” (14, 15) in which the anammox reaction is assumed to take place. During the anammox reaction (7, 8, 16), a proton motive force (PMF) is established over the anammoxosome membrane. Membrane-bound ATPases could utilize this PMF for ATP production in the riboplasm. The riboplasm (which is topologi- cally equivalent to the “pirellulosome” compartment in non- anammox planctomycete species) is the compartment that surrounds the anammoxosome, and it contains ribosomes and the nucleoid, thereby resembling the classical bacterial cytoplasm. The function of the outermost, apparently ribosome-free compartment, the paryphoplasm, has not yet been elucidated. The composition of the cell envelope which encloses the paryphoplasm is unknown but has been proposed to deviate from both the typical Gram-positive and Gram-negative bacterial cell envelope types because it is proposed to lack both peptidoglycan (17) and a typical outer membrane. The proposed lack of peptidoglycan is based on (i) the close relationship of anammox bacteria to other planctomycetes where the cell wall composition has been chemically analyzed (18, 19) and (ii) the fact that not all genes necessary for the biosynthesis of peptidoglycan are present in the genome. Interestingly, most peptidoglycan biosynthesis genes are harbored by the genome, except for those encoding penicillin binding protein (PBP1a) and PBP1b (7, 20), which are required for the insertion of peptidoglycan precursors into polymeric peptidoglycan. Although in the transcriptome, a small number of reads is found for all peptidoglycan genes, none of their respective proteins, besides the protein D -alanine– D -alanine ligase (Ddl), could be detected in the “ Ca . Kuenenia stuttgartiensis” proteome (8). Peptidoglycan has been proposed to contribute to the integrity of the cell and, in some cases, to the maintenance of cell shape (21). This raises the question of whether the cell envelope of anammox bacteria contains other structures that help to maintain the integrity of the cell. It is therefore of high interest to investigate the cell envelope of anammox bacteria in more detail. Another interesting feature of the anammox cell plan is the outermost membrane, which surrounds the paryphoplasm. This membrane has been defined as a cytoplasmic membrane, which is also consistent with the immunogold localization of an ATPase to this membrane (22). However, several outer membrane proteins and key proteins in outer membrane biosynthesis have been detected in both the genomes and proteomes of two anammox species (7, 8, 13, 22), although none of these have yet been localized to any particular cell structure. At the moment, the identity of the cytoplasmic membrane of anammox bacteria remains under debate and needs further investigation. In Gram-negative and Gram-positive bacteria as well as Archaea , a proteinaceous surface layer (S-layer) can be present as the outermost component of the cell envelope. S-layers constitute a two-dimensional (2D) crystalline array of (usually) identical protein subunits covering the entire cell surface (23, 24). The regular pattern formed by the S-layer can exist in oblique (p1 and p2), square (p4), or hexagonal (p3 and p6) symmetry, which is dictated by the arrangement and number of protein subunits (indicated by the number behind the p) that form the single morphological unit. The (self-)assembly of the proteins into the regular pattern is thought to be driven by entropical forces (25). S-layer proteins have a broad molecular mass range, between 40 and 200 kDa, and isoelectric points of between 3 and 6 (25). These proteins typically consist of 40 to 60% hydrophobic amino acids (26), although S-layer proteins with a predominant amount of hydrophilic amino acids have also been described (27). Many S-layer proteins are glycosylated (i.e., glycoproteins) by either N - or O -glycosylation. In some rare cases, both glycosylation types can be found on the same protein (28). One clear distinction between the various S-layers on different prokaryotic cells is the anchor to the underlying cell envelope component. In Gram-positive bacteria, S-layers are linked to the underlying cell wall components, including peptidoglycan and so-called secondary cell wall polymers (SC- WPs) (29–31). In Gram-negative bacteria, S-layers are anchored in the outer membrane, while in Archaea , S-layers are always found to be anchored in the cytoplasmic membrane (32–34). In the present study, we investigated the cell envelope of the anammox bacterium “ Ca . Kuenenia stuttgartiensis.” We found a proteinaceous S-layer as the outermost component of the cell (envelope) using transmission electron microscopy on freeze-etched cells as well as in thin sections of cryofixed, freeze-substituted, and Epon-embedded cells. The S-layer was found to have a hexagonal (p6) symmetry, in which each S-layer motif is formed by six identical proteins. Enrichment of the S-layer led to the identification of a candidate S-layer glycoprotein, which was used to generate specific antibodies. Immunogold localization showed the antibody to bind to the outermost rim of the cell, where the S-layer is located, and thus verified that this protein forms the ...

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... The S-layer was clearly discernable as a distinct layer ∼ 11 nm above the outer membrane. Instead of adhering to the cell body as usually seen in other microorganisms (van Teeseling et al., 2014;Bharat et al., 2017;Gambelli et al., 2019;Gaisin et al., 2020;von Kügelgen et al., 2020;Oatley et al., 2020. For examples of reviews see Albers and Meyer (2011);Pavkov-Keller et al. (2011), Rodrigues-Oliveira et al. (2017, and Pum et al. (2021), the S-layer of M. lanthanidiphila formed several distinct patches. ...
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... Interestingly, although the Brocadia species was the first anammox species to be reported [62], the Kuenenia species is known to be the most extensively studied anammox species in terms of cell structure, physiology, biochemistry, and comparative genomics research [63]. In particular, the key proteins involved in the genome of Kuenenia stuttgartiensis (e.g., hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, and an S-layer protein with an encoded hypothesized anammox pathway) have been experimentally validated in recent studies [64][65][66]. The most interesting feature of the anammox species was the experimental verification of a key intermediate hydrazine produced from HNO 2 [67]; this suggests that the Kuenenia species might include novel metabolic pathways for nitrogen. ...
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