Project description:Anaerobic ammonium-oxidising (anammox) bacteria, members of the ‘Candidatus Brocadiaceae’ family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-bound compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic- and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbour multiple copies of ammonium-, nitrite- and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite- and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells under ammonium-limitation showed that three of the seven ammonium transporter genes and one of the six nitrite transporter genes were significantly upregulated, while another ammonium and nitrite transporter gene were downregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.
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: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.