We report structural and functional analyses of the Bradyrhizobium japonicum fixGHIS genes, which map immediately downstream of the fixNOQP operon for the symbiotically essential cbb 3-type heme-copper oxidase complex. Expression of fixGHIS, like that of fixNOQP, is strongly induced in cells grown microaerobically or anaerobically. A fixGHI deletion led to the same prominent phenotypes as those known from a fixNOQP deletion: defective symbiotic nitrogen fixation (Fix–) and decreased cytochrome oxidase activity in cells grown under oxygen deprivation. Only traces, if any, of cytochrome cbb 3 subunits were present in membranes isolated from the ΔfixGHI strain, as revealed by Western blot analysis with subunit-specific antibodies. This effect was not due to lack of fixNOQP transcription. The results suggested a critical involvement of the fixGHIS gene products in the assembly and/or stability of the cbb 3-type heme-copper oxidase. On the basis of sequence similarities between the FixI protein and a Cu-transporting P-type ATPase (CopA) of Enterococcus hirae, and between FixG and a membrane-bound oxidoreductase (RdxA) of Rhodobacter sphaeroides, we postulate that a membrane-bound FixGHIS complex might play a role in uptake and metabolism of copper required for the cbb 3-type heme-copper oxidase.
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