title: Copper starvation-inducible protein for cytochrome oxidase biogenesis in Bradyrhizobium japonicum.
abstract: Microarray analysis of Bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuABCDE, which are co-transcribed and co-regulated as an operon. The predicted gene products are periplasmic proteins (PcuA, PcuC, and PcuD), a TonB-dependent outer membrane receptor (PcuB), and a cytoplasmic membrane-integral protein (PcuE). Homologs of PcuC and PcuE had been discovered in other bacteria, namely PCu(A)C and YcnJ, where they play a role in cytochrome oxidase biogenesis and copper transport, respectively. Deletion of the pcuABCDE operon led to a pleiotropic phenotype, including defects in the aa(3)-type cytochrome oxidase, symbiotic nitrogen fixation, and anoxic nitrate respiration. Complementation analyses revealed that, under our assay conditions, the tested functions depended only on the pcuC gene and not on pcuA, pcuB, pcuD, or pcuE. The B. japonicum genome harbors a second pcuC-like gene (blr7088), which, however, did not functionally replace the mutated pcuC. The PcuC protein was overexpressed in Escherichia coli, purified to homogeneity, and shown to bind Cu(I) with high affinity in a 1:1 stoichiometry. The replacement of His(79), Met(90), His(113), and Met(115) by alanine perturbed copper binding. This corroborates the previously purported role of this protein as a periplasmic copper chaperone for the formation of the Cu(A) center on the aa(3)-type cytochrome oxidase. In addition, we provide evidence that PcuC and the copper chaperone ScoI are important for the symbiotically essential, Cu(A)-free cbb(3)-type cytochrome oxidase specifically in endosymbiotic bacteroids of soybean root nodules, which could explain the symbiosis-defective phenotype of the pcuC and scoI mutants.
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule Zürich (ETH Zürich), Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Fabio
authors.initials: F
authors.lastname: Serventi
authors.forename: Zeb Andrew
authors.initials: ZA
authors.lastname: Youard
authors.forename: Valérie
authors.initials: V
authors.lastname: Murset
authors.forename: Simona
authors.initials: S
authors.lastname: Huwiler
authors.forename: Doris
authors.initials: D
authors.lastname: Bühler
authors.forename: Miriam
authors.initials: M
authors.lastname: Richter
authors.forename: Ronny
authors.initials: R
authors.lastname: Luchsinger
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Fischer
authors.forename: Robert
authors.initials: R
authors.lastname: Brogioli
authors.forename: Martina
authors.initials: M
authors.lastname: Niederer
authors.forename: Hauke
authors.initials: H
authors.lastname: Hennecke
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D020369:Bradyrhizobium / Q000201:enzymology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis* / Q000737:chemistry; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D024101:Mitochondrial Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000737:chemistry; D009586:Nitrogen Fixation; D020411:Oligonucleotide Array Sequence Analysis; D019897:Periplasm / Q000378:metabolism; D010641:Phenotype; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D003300:Copper; D003576:Electron Transport Complex IV; D009584:Nitrogen