Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium 'Candidatus Kuenenia stuttgartiensis'

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium 'Candidatus Kuenenia stuttgartiensis'. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism.

Fig. 1

Electron micrograph (left) of an anammox bacterium and schematic overview (right) of the different scenarios regarding its cell plan. MB, membrane. Modified from van Niftrik et al. (2008b).

Fig. 2

Postulated model for catabolic anammox reactions coupled over the anammoxosome membrane in anammox bacteria resulting in a proton-motive-force and subsequent ATP synthesis. bc1, cytochrome bc1 complex; cyt, cytochrome; hao, hydrazine/hydroxylamine oxidoreductase; hh, hydrazine hydrolase; nir, nitrite reductase; Q, co-enzyme Q. Modified from Strous et al. (2006).

Fig. 3

Schematic model of (A) a prokaryotic F-ATPase and (B) a prokaryotic V-ATPase. Assembled from Grüber et al. (2001), Lolkema et al. (2003), Yokoyama and Imamura (2005) and Lau and Rubinstein (2010).

Fig. 4

Comparison of the four ATPase gene clusters identified in the K. stuttgartiensis genome to their closest homologue. A. Gene cluster 1 (kuste3787–3796): F-ATPase similar to E. coli F-ATPase with the exception of an unknown ORF between gene I and subunit a and two ORFs encoding subunit c. B. Gene cluster 2 (kuste4592–4600) and 3 (kustc0572–0579): F-ATPases similar to M. barkeri F-ATPase. Gene cluster 3 lacks the unknown ORF, urf2, between gene I and subunit a. C. Gene cluster 4 (kuste3864–3871): V-ATPase similar to B. burgdorferi prokaryotic V-ATPase with the exception of an unknown ORF between subunit B and subunit D. Genes marked with a star are annotated as (conserved) hypothetical proteins. These genes have a sequence identity of over 20% to a protein with an undefined function or to a protein with a defined function but without similar sequence length. ORF, open reading frame.

Fig. 5

Immunoblot analysis of the antiserum directed at the catalytic beta subunit of the F-ATPase-1 gene cluster found in the K. stuttgartiensis genome. Lane 1: Marker (PageRuler™ Prestained Protein Ladder Plus, Fermentas); lane 2: incubation with anti-F-ATPase-1; lane 3: incubation with the pre-immune serum; lane 4: incubation with only the secondary antibody. Arrow: expected target size (52 kDa).

Fig. 6

Immunogold labelling distribution (average gold particles per cell per location) in K. stuttgartiensis using the antiserum directed at F-ATPase-1 and its pre-immune serum. All three membranes of the anammox cell are significantly labelled compared with the incubation with the pre-immune serum (the negative control). OM, outermost membrane (membrane 1); MM, middle membrane (membrane 2); IM, innermost (anammoxosome) membrane (membrane 3); A, anammoxosome; R, riboplasm; ***, extremely statistically significant; *, statistically significant.

Fig. 7

A–D. Immunogold localization of the antiserum directed at the catalytic beta subunit of the F-ATPase-1 gene cluster localizes this ATPase to all anammox membranes; the outermost membrane (membrane 1), middle membrane (membrane 2) and innermost (anammoxosome) membrane (membrane 3) in K. stuttgartiensis rehydrated cryosections. See Fig. S3 for annotation of specific gold labels. E and F. Negative control: incubation with the pre-immune serum instead of the antiserum. Scale bars: 250 nm.