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:The dataset provides the whole proteome of the anammox bacterium "Candidatus Kuenenia Stuttgartiensis" strain CSTR1 growing planctonically in semi-CSTR reactor. The bacteria were growing at high growth rate (0.33 d-1) (reactor HRT 3d).
Project description:Little is known about the cell physiology of anammox bacteria growing at extremely low growth rates. Here, "Candidatus Brocadia sinica" and "Candidatus Scalindua sp." were grown in continuous anaerobic membrane bioreactors (MBRs) with complete biomass retention to determine maintenance energy (i.e., power) requirements at near-zero growth rates. After prolonged retentostat cultivations, the specific growth rates (μ) of "Ca. B. sinica" and "Ca. Scalindua sp." decreased to 0.000023 h-1 (doubling time of 1255 days) and 0.000157 h-1 (184 days), respectively. Under these near-zero growth conditions, substrate was continuously utilized to meet maintenance energy demands (me) of 6.7 ± 0.7 and 4.3 ± 0.7 kJ mole of biomass-C-1 h-1 for "Ca. B. sinica" and "Ca. Scalindua sp.", which accorded with the theoretically predicted values of all anaerobic microorganisms (9.7 and 4.4 kJ mole of biomass-C-1 h-1at 37 °C and 28 °C, respectively). These me values correspond to 13.4 × 10-15 and 8.6 × 10-15 watts cell-1 for "Ca. B. sinica" and "Ca. Scalindua sp.", which were five orders of magnitude higher than the basal power limit for natural settings (1.9 × 10-19 watts cells-1). Furthermore, the minimum substrate concentrations required for growth (Smin) were calculated to be 3.69 ± 0.21 and 0.09 ± 0.05 μM NO2- for "Ca. B. sinica" and "Ca. Scalindua sp.", respectively. These results match the evidence that "Ca. Scalindua sp." with lower maintenance power requirement and Smin are better adapted to energy-limited natural environments than "Ca. B. sinica", suggesting the importance of these parameters on ecological niche differentiation in natural environments.
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 ammon ium oxidizing (anammox) bacteria oxidize ammonium and reduce nitrite, producing N2, and could play a major role in energy-optimized wastewater treatment. However, sensitivity to various environmental conditions and slow growth currently hinder their wide application. Here, we attempted to determine online the effect of environmental stresses on anammox bacteria by using an overnight batch activity test with whole cells, in which anammox activity was calculated by quantifying N2 production via headspace-pressure monitoring. A planktonic mixed culture dominated by "Candidatus Kuenenia stuttgartiensis" strain CSTR1 was cultivated in a 30-L semi-continuous stirring tank reactor. In overnight resting-cell anammox activity tests, oxygen caused strong inhibition of anammox activity, which was reversed by sodium sulfite (30 µM). The tested antibiotics sulfamethoxazole, kanamycin, and ciprofloxacin elicited their effect on a dose-dependent manner; however, strain CSTR1 was highly resistant to sulfamethoxazole. Anammox activity was improved by activated carbon and Fe2O3. Protein expression analysis from resting cells after anammox activity stimulation revealed that NapC/NirT family cytochrome c (KsCSTR_12840), hydrazine synthase, hydrazine dehydrogenase, hydroxylamine oxidase, and nitrate:nitrite oxidoreductase were upregulated, while a putative hydroxylamine oxidoreductase HAO (KsCSTR_49490) was downregulated. These findings contribute to the growing knowledge on anammox bacteria physiology, eventually leading to the control of anammox bacteria growth and activity in real-world application. KEY POINTS: • Sulfite additions can reverse oxygen inhibition of the anammox process • Anammox activity was improved by activated carbon and ferric oxide • Sulfamethoxazole marginally affected anammox activity.
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.