Transcriptome analysis of Paracoccus denitrificans during Cu-limitation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Agilent-026900 Paracoccus denitrificans
ORGANISM(S): Paracoccus Denitrificans Pd1222
SUBMITTER: 
Matthew John Sullivan
SECONDARY ACCESSION(S): PRJNA210735
REPOSITORIES: GEO
Dataset's files
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Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type grown to mid-exponential phase in minimal media with either 13 uM (Cu-H) or 0.5 uM (Cu-L) Cu regimes. The goal was to define the effects of Cu-limitation on denitrification genes Two growth conditions, three biological replicates of each condition. Each sample hybridised in a two-channel hybridization against Paracoccus denitrificans genomic DNA as the comparator/reference, which also acted as a control for spot quality. Cu-concentration 13 uM (Cu-H) versus 0.5 uM Cu (Cu-L) in anaerobic growth conditions.
2013-11-18 | E-GEOD-48577 | ArrayExpress
Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type grown to mid-exponential phase in minimal media with either 13 uM (Cu-H) or 0.5 uM (Cu-L) Cu regimes. The goal was to define the effects of Cu-limitation on denitrification genes Two growth conditions, three biological replicates of each condition. Each sample hybridised in a two-channel hybridization against Paracoccus denitrificans genomic DNA as the comparator/reference, which also acted as a control for spot quality. Cu-concentration 13 uM (Cu-H) versus 0.5 uM Cu (Cu-L) in anaerobic growth conditions.
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Project description:The final step of denitrification is the reduction of nitrous oxide (N<sub>2</sub> O) to N<sub>2</sub> , mediated by Cu-dependent nitrous oxide reductase (N<sub>2</sub> OR). Its metal centers, Cu<sub>A</sub> and Cu<sub>Z</sub> , are assembled through sequential provision of twelve Cu<sup>I</sup> ions by a metallochaperone that forms part of a nos gene cluster encoding the enzyme and its accessory factors. The chaperone is the nosL gene product, an 18 kDa lipoprotein predicted to reside in the outer membrane of Gram-negative bacteria. In order to better understand the assembly of N<sub>2</sub> OR, we have produced NosL from Shewanella denitrificans and determined the structure of the metal-loaded chaperone by X-ray crystallography. The protein assembled a heterodinuclear metal site consisting of Zn<sup>II</sup> and Cu<sup>I</sup> , as evidenced by anomalous X-ray scattering. While only Cu<sup>I</sup> is delivered to the enzyme, the stabilizing presence of Zn<sup>II</sup> is essential for the functionality and structural integrity of the chaperone.
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Project description:Dissimilatory nitrite reductase (NIR) is a key enzyme in denitrification, catalyzing the first step that leads to gaseous products (NO, N(2)O, and N(2)). We have determined the crystal structure of a Cu-containing NIR from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans, at 2.2-A resolution. The overall structure of this H. denitrificans NIR reveals a trigonal prism-shaped molecule in which a monomer consisting of 447 residues and three Cu atoms is organized into a unique hexamer (i.e., a tightly associated dimer of trimers). Each monomer is composed of an N-terminal region containing a Greek key beta-barrel folding domain, cupredoxin domain I, and a C-terminal region containing cupredoxin domains II and III. Both cupredoxin domains I and II bind one type 1 Cu and are combined with a long loop comprising 31 amino acid residues. The type 2 Cu is ligated at the interface between domain II of one monomer and domain III of an adjacent monomer. Between the two trimeric C-terminal regions are three interfaces formed by an interaction between the domains I, and the type 1 Cu in the domain is required for dimerization of the trimer. The type 1 Cu in domain II functions as an electron acceptor from an electron donor protein and then transfers an electron to the type 2 Cu, binding the substrate to reduce nitrite to NO. The discussion of the intermolecular electron transfer process from cytochrome c(550) to the H. denitrificans NIR is based on x-ray crystallographic and kinetic results.
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Project description:Nitrous oxide reductase (N2OR) is the terminal enzyme of the denitrification pathway of soil bacteria that reduces the greenhouse gas nitrous oxide (N2O) to dinitrogen. In addition to a binuclear CuA site that functions in electron transfer, the active site of N2OR features a unique tetranuclear copper cluster bridged by inorganic sulfide, termed CuZ. In copper-limited environments, N2OR fails to function, resulting in truncation of denitrification and rising levels of N2O released by cells to the atmosphere, presenting a major environmental challenge. Here we report studies of nosL from Paracoccus denitrificans, which is part of the nos gene cluster, and encodes a putative copper binding protein. A Paracoccus denitrificans ?nosL mutant strain had no denitrification phenotype under copper-sufficient conditions but failed to reduce N2O under copper-limited conditions. N2OR isolated from ?nosL cells was found to be deficient in copper and to exhibit attenuated activity. UV-visible absorbance spectroscopy revealed that bands due to the CuA center were unaffected, while those corresponding to the CuZ center were significantly reduced in intensity. In vitro studies of a soluble form of NosL without its predicted membrane anchor showed that it binds one Cu(i) ion per protein with attomolar affinity, but does not bind Cu(ii). Together, the data demonstrate that NosL is a copper-binding protein specifically required for assembly of the CuZ center of N2OR, and thus represents the first characterised assembly factor for the CuZ active site of this key environmental enzyme, which is globally responsible for the destruction of a potent greenhouse gas.
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Project description:Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.
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