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A novel bacterial system involved in copper tolerance
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The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase
Publication . Carreira, Cíntia; Nunes, Rute F.; Mestre, Olga; Moura, Isabel; Pauleta, Sofia R.; LAQV@REQUIMTE; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Springer
Abstract: Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state. Graphic abstract: [Figure not available: see fulltext.].
Source and reduction of nitrous oxide
Publication . Pauleta, Sofia R.; Carepo, Marta S. P.; Moura, Isabel; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; LAQV@REQUIMTE; Elsevier
Nitrous oxide is a potent greenhouse gas with a global warming impact 300-fold higher than carbon dioxide. Due to its exponential increase in the atmosphere and its implications in climate change there is the need to develop strategies to mitigate its emissions and to reduce it to the inert dinitrogen gas. Only three enzymes have been reported to be able to reduce nitrous oxide, namely nitrogenase, one multicopper oxidase and nitrous oxide reductase, with the latter being the only one with a relevant physiological activity. In this enzyme, reduction of nitrous oxide occurs in a unique catalytic tetranuclear sulfide center, named “CuZ” center, a complex center required to overcome the high activation barrier of this reaction. Nitrous oxide reductase can be isolated with “CuZ” center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), that differ in their catalytic and spectroscopic properties. Recently, another step towards a better understanding of the catalytic and activation mechanism of this enzyme was taken by identifying and spectroscopically characterizing an intermediate species of its catalytic cycle, CuZ 0 . A different approach for N 2 O reduction can be attained using model compounds. The unique structural motif present in “CuZ” center, a Cu 4 (µ 4 -S), has been a challenge for inorganic synthesis but several synthetic clusters that mimic different forms of “CuZ” center have been reported. Model compounds for the oxidation states involved in N 2 O reduction are also available. The advances in this area will be discussed in light of the recent data, with structural and functional model compounds of N 2 OR active site.
Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes
Publication . Carreira, Cíntia; Mestre, Olga; Nunes, Rute F.; Moura, Isabel; Pauleta, Sofia R.; LAQV@REQUIMTE; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; PeerJ Inc.
Background. Denitrification is one of the main pathways of the N-cycle, during which nitrate is converted to dinitrogen gas, in four consecutive reactions that are each catalyzed by a different metalloenzyme. One of the intermediate metabolites is nitrous oxide, which has a global warming impact greater then carbon dioxide and which atmospheric concentration has been increasing in the last years. The four denitrification enzymes have been isolated and biochemically characterized from Marinobacter hydrocarbonoclasticus in our lab. Methods. Bioinformatic analysis of the M. hydrocarbonoclasticus genome to identify the genes involved in the denitrification pathway. The relative gene expression of the gene encoding the catalytic subunits of those enzymes was analyzed during the growth under microoxic conditions. The consumption of nitrate and nitrite, and the reduction of nitric oxide and nitrous oxide by whole-cells was monitored during anoxic and microoxic growth in the presence of 10 mM sodium nitrate at pH 7.5. Results. The bioinformatic analysis shows that genes encoding the enzymes and accessory factors required for each step of the denitrification pathway are clustered together. An unusual feature is the co-existence of genes encoding a q- and a c-type nitric oxide reductase, with only the latter being transcribed at similar levels as the ones encoding the catalytic subunits of the other denitrifying enzymes, when cells are grown in the presence of nitrate under microoxic conditions. Using either a batch- or a closed system, nitrate is completely consumed in the beginning of the growth, with transient formation of nitrite, and whole-cells can reduce nitric oxide and nitrous oxide from mid-exponential phase until being collected (time-point 50 h). Discussion. M. hydrocarbonoclasticus cells can reduce nitric and nitrous oxide in vivo, indicating that the four denitrification steps are active. Gene expression profile together with promoter regions analysis indicates the involvement of a cascade regulatory mechanism triggered by FNR-type in response to low oxygen tension, with nitric oxide and nitrate as secondary effectors, through DNR and NarXL, respectively. This global characterization of the denitrification pathway of a strict marine bacterium, contributes to the understanding of the N-cycle and nitrous oxide release in marine environments.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-PRO/098882/2008