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Doc number: 28 Abstract Background: YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity. Results: In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme's reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher. Conclusion : Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.

Background Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense . Results Deletion of Mgfnr not only resulted in decreased N 2 production due to reduced N 2 O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr. Conclusions We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O 2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization.

The structure of the respiratory nitrate reductase (NapAB) from Rhodobacter sphaeroides, the periplasmic heterodimeric enzyme responsible for the first step in the denitrification process, has been determined at a resolution of 3.2 A. The di-heme electron transfer small subunit NapB binds to the large subunit with heme II in close proximity to the [4Fe-4S] cluster of NapA. A total of 57 residues at the N- and C-terminal extremities of NapB adopt an extended conformation, embracing the NapA subunit and largely contributing to the total area of 5,900 A(2) buried in the complex. Complex formation was studied further by measuring the variation of the redox potentials of all the cofactors upon binding. The marked effects observed are interpreted in light of the three-dimensional structure and depict a plasticity that contributes to an efficient electron transfer in the complex from the heme I of NapB to the molybdenum catalytic site of NapA.

Copper is typically coordinated by histidine, cysteine, or methionine in proteins, and these residues are particularly sensitive to oxidation. However, it remains unclear whether copper-coordinating residues are more prone to oxidation than non-coordinating ones, and how their susceptibility changes between the apo and copper-bound states. The copper chaperone PcuC, important for cytochrome c oxidase assembly in bacteria, contains a canonical binding site composed of two histidines and two methionines (H51xnM63x22H86xM88), as well as a disordered C-terminal extension enriched in methionine and histidine. To quantify methionine oxidation sensitivity in both apo- and Cu-bound PcuC, we used a methionine-specific oxaziridine probe combined with mass spectrometry and compared labeling patterns to those generated by 18O-labeled hydrogen peroxide. We show that methionine residues display distinct oxidation sensitivities in the apoprotein, and that the oxaziridine reacts similarly to H218O2. Importantly, this probe enables quantification of methionine oxidation independently of hydroxyl radicals generated by copper-driven Fenton chemistry, which lacks residue specificity. In the copper-bound form, Cu binding strongly alters methionine reactivity, with a marked increase in oxidation of the coordinating Met63 and Met88. Structural analysis revealed that two copper ions occupy the canonical site, while the C-terminal extension does not contribute to coordination. Comparison of structural features and oxidation values showed that methionine sensitivity correlates with solvent exposure in the folded domain, but with local positive charge in the disordered region. These findings demonstrate that copper coordination modulates methionine oxidation, and that oxaziridine-based probes provide powerful tools for mapping oxidation sensitivity in (metallo)proteins.

Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.

Abstract Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.