Dataset Information

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.

SUBMITTER: van Niftrik L

PROVIDER: S-EPMC2936114 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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

van Niftrik Laura L van Helden Mary M Kirchen Silke S van Donselaar Elly G EG Harhangi Harry R HR Webb Richard I RI Fuerst John A JA Op den Camp Huub J M HJ Jetten Mike S M MS Strous Marc M

Molecular microbiology 20100609 3

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 Kuenen ...[more]

PMID: 20545867

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Project description:Bacteria capable of anaerobic oxidation of ammonium (anammox) form a deep branching clade within the Planctomycetes. Although the core metabolic pathway of anammox bacteria is largely resolved, many questions still remain. Data mining of the (meta) genomes of anammox bacteria is a powerful method to address these questions or identify targets for further study. The availability of high quality reference data greatly aids such analysis. Currently, only a single "near complete" (?98%) reference genome of an anammox bacterium is available; that of model organism "Candidatus Kuenenia stuttgartiensis." Here we present a comparative genomic analysis of two "Ca. K. stuttgartiensis" anammox bacteria that were independently enriched. The two anammox bacteria used are "Ca. K. stuttgartiensis" RU1, which was originally sequenced for the reference genome in 2002 and "Ca. K. stuttgartiensis" CH1, independently enriched from a Chinese wastewater treatment plant. The two different "Ca. Kuenenia" bacteria have a very high sequence identity (>99% at nucleotide level) over the entire genome, but 31 genomic regions (average size 11?kb) were absent from strain CH1 and 220?kb of sequence was unique to the CH1 assembly. The high sequence homology between these two bacteria indicates that mobile genetic elements are the main source of variation between these geographically widely separated strains. Comparative analysis of the RU1 and CH1 assemblies led to the identification of 49 genes absent from the reference genome. These include a leucyl-tRNA-synthase, the absence of which led to the estimation of the 98% completeness of the reference genome. Finally, a set of 244 genes was present in the reference genome, but absent in the RU1 and CH1 assemblies. These could represent either identical gene duplicates or assembly errors in the published genome. We are confident that this analysis has further improved the most complete available high quality reference genome of an anammox bacterium and will aid further studies on this globally important group of organisms.

Project description:BACKGROUND: Anaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute approximately 50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria. RESULTS: Two training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive. CONCLUSIONS: A physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K. stuttgartiensis. This result provides strong in silico support for the existing experimental evidence for the existence of an organelle in this bacterium, and is an important step forward in unravelling a geochemically relevant case of cytoplasmic differentiation in bacteria. The predicted dual location of the Sec-translocation system and the apparent absence of a specific N- or C-terminal signal in the organellar proteins suggests that additional chaperones may be necessary that act on an as-yet unknown property of the targeted proteins.

Dataset Information

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

Publications

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

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