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

Laura Van Niftrik,Mary Van Helden,Huub J M Op Den Camp,Mike S M Jetten,John A Fuerst,Richard I Webb,Marc Strous,Elly G Van Donselaar,Silke Kirchen,Harry R Harhangi

doi:10.1111/j.1365-2958.2010.07242.x

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.

Highlights

Anammox bacteria perform anaerobic ammonium oxidation to dinitrogen gas and are applied for removal of ammonium from wastewater
Anaerobic ammonium-oxidizing bacteria are divided into three compartments by bilayer membranes: paryphoplasm, riboplasm and anammoxosome
We investigated the location of membrane-bound ATPases in the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’

Summary

Introduction

Anammox bacteria perform anaerobic ammonium oxidation (anammox) to dinitrogen gas and are applied for removal of ammonium from wastewater. They are important in nature where they contribute significantly to oceanic nitrogen loss (Devol, 2003; Kuypers et al, 2003; 2005; Arrigo, 2005; Brandes et al, 2007). Chemical analysis, genome sequencing and resistance to beta-lactam and other cell wall-targeting antibiotics have revealed that planctomycetes lack the otherwise universal bacterial cell wall polymer peptidoglycan (König et al, 1984; Liesack et al, 1986; Stackebrandt et al, 1986; Fuerst, 1995) and an outer membrane typical of Gram-negative bacteria. The outermost membrane in planctomycetes has been defined as the cytoplasmic membrane based on the detection of RNA directly on its inner side by immunogold labelling and the position of the membrane and membrane surfaces relative to the cell wall visualized in thin sections of cryofixed and freeze-substituted cells and in

Results

Discussion

Conclusion