Project description:Nitric oxide (NO) has several important functions in biology and atmospheric chemistry as a toxin, signaling molecule, ozone depleting agent and the precursor of the greenhouse gas nitrous oxide (N2O). Even though NO is a potent oxidant, and was available on earth earlier than oxygen, its direct use by microorganisms for growth was not demonstrated before. Using physiological experiments, metatranscriptomics and metaproteomics, here we show that anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis grow by coupling ammonium oxidation to NO reduction, and produce only N2. Such a metabolism could have existed on early earth, and has implications in controlling N2O and NO emissions both from natural and manmade ecosystems, where anammox bacteria contribute significantly to N2 release to the atmosphere.
Project description:Anaerobic ammonium oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome predictions. Here, we experimentally resolved the central carbon metabolism using metabolomics (LC-MS and GC-MS), metabolic flux analysis and proteomics (shot-gun proteomics).
Project description:This SuperSeries is composed of the following subset Series: GSE28549: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Phenol vs. Benzoate) GSE30798: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Acetate) GSE30799: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Phenol) GSE30801: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Benzoate) Refer to individual Series
Project description:Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance but research into the mechanisms has been stymied by a lack of a genetically tractable pure culture which unequivocally does not use molecular oxygen to activate benzene. Geobacter metallireducens grew in a medium in which benzene was the sole electron donor and Fe(III) was the sole electron acceptor with a stoichiometry of benzene loss and Fe(III) reduction consistent with benzene oxidation to carbon dioxide coupled with Fe(III) reduction. Phenol labeled with 18O was produced when the medium was labeled with H218O, as expected for a true anaerobic conversion of benzene to phenol. Gene expression patterns indicated that benzene was metabolized through a phenol intermediate rather than benzoate or toluene. Deletion of ppcB, which encodes a subunit of the phenylphosphate carboxylase, an enzyme required for phenol metabolism, inhibited metabolism of benzene. Deleting genes specific for benzoate or toluene metabolism did not. Comparison of gene expression patterns in cells grown on benzene versus cells grown on phenol revealed genes specifically expressed in benzene-grown cells. Deletion of one of these, Gmet_3376, inhibited anaerobic benzene oxidation, but not the metabolism of phenol, benzoate, or toluene. The availability of a genetically tractable pure culture that can anaerobically convert benzene to phenol with oxygen derived from water should significantly accelerate elucidation of the mechanisms by which benzene can be activated in the absence of molecular oxygen. Total RNA from three separate cultures of G. metallireducens grown with 250 µM benzene three separate cultures of G. metallireducens grown with 500 µM phenol three separate cultures of G. metallireducens grown with 1 mM benzoate three separate cultures of G. metallireducens grown with 500 µM toluene three separate cultures of G. metallireducens grown with 10 mM acetate were used to study [1] Anaerobic oxidation of benzene by G. metallireducens (Benzene vs. acetate, Benzene vs. benzoate, Benzene vs. phenol, Benzene vs. toluene) [2] Anaerobic oxidation of benzoate by G. metallireducens (Benzoate vs. acetate) [3] Anaerobic oxidation of phenol by G. metallireducens (Phenol vs. acetate) [4] Anaerobic oxidation of toluene by G. metallireducens (Toluene vs. acetate) Each chip measures the expression level of 3,627 genes from G. metallireducens DSM 7210 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
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.