Copper Metallochaperones are Required for the Assembly of Bacteroid Cytochrome c Oxidase Which is Functioning for Nitrogen Fixation in Soybean Nodules

Abstract

Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium for Glycine max, has complex respiratory electron transport chains. Bll4880 contained a copper-binding motif for metallochaperone, H(M)X(10)MX(21)HXM. A mutant strain, Bj4880, induced nodules with lower acetylene reduction activity. A double mutant, Bj4880-1131, which had inserted mutations both in blr1131, a gene of the Sco1-like protein, and in bll4880, induced nodules of significant Fix(-) phenotype and low cytochrome c oxidase (Cco) activity in the bacteroid. Our data suggest that bll4880 protein is involved in copper ion delivery to Cco through blr1131 protein, and the expression of both proteins was induced under microaerobic conditions.

Copper Starvation-inducible Protein for Cytochrome Oxidase Biogenesis in Bradyrhizobium japonicum

“…Intuitively, one would like to propose that the soluble, periplasmic PcuC protein disseminates copper to the membrane-bound ScoI protein and perhaps to other membranebound recipients (e.g. CoxG/Cox11, FixI/CcoI, and CcoA), as has been suggested in part also by others (15,21,23,24). However, more biochemical work, such as establishing a copper transfer assay with purified components in vitro, will be needed as proof.…”

“…Subsequent work with B. japonicum ScoI has adopted this view, supported by the fact that a scoI knock-out mutant was clearly defective in cytochrome aa 3 but not in cytochrome cbb 3 when the latter was tested in cells that had been grown microoxically or anoxically (10). The symbiosis defect caused by the B. japonicum scoI mutant remained enigmatic (10,21). Other reports have shown, however, that SenC and PrrC play a role in the formation of active cytochrome cbb 3 (22)(23)(24).…”

“…Intriguingly, the authors of this study noticed a role of PCu A C also in the formation of the Cu B center of cytochrome cbb 3 . Homologous genes for PCu A C-like proteins had been recognized in the B. japonicum genome (10,21), but detailed studies on their biochemical function had not been done.…”

Two MicroRNAs Linked to Nodule Infection and Nitrogen-Fixing Ability in the Legume Lotus japonicus

“…Cu 2+ availability seems crucial to the functionality of this complex, as deletion of the fix-GHIS genes in the soybean symbiont B. japonicum, encoding for the copper-transporting ATPase FixI, results in defective nitrogen fixation (Preisig et al, 1996a). The same effect is found with B. japonicum carrying mutations in the bll4880 gene, encoding a metallochaperone proposed to transfer Cu 2+ to cbb 3 , further emphasizing the need for Cu 2+ trafficking from the plant to the nodule and bacteroids (Arunothayanan et al, 2010). Moreover, a nodule-specific expression pattern has been reported for a putative plant copper transporter in M. truncatula (Fedorova et al, 2002).…”

“…At the biochemical level, nitrogen fixation requires low concentrations of free oxygen, which could otherwise inactivate the oxygensensitive nitrogenase enzymatic complex. On the other hand, a high respiratory rate is needed in nodules to meet the high demands in ATP, which is satisfied through strategies that include the action of the oxygen carrier leghemoglobin and the use of a specialized respiratory electron transport chain terminated by a Cu 2+ -containing bb3-type cytochrome oxidase (cbb 3 ) with high affinity for oxygen (Preisig et al, 1996b;Delgado et al, 1998;Ott et al, 2005;Arunothayanan et al, 2010). Cu 2+ availability seems crucial to the functionality of this complex, as deletion of the fix-GHIS genes in the soybean symbiont B. japonicum, encoding for the copper-transporting ATPase FixI, results in defective nitrogen fixation (Preisig et al, 1996a).…”