Enhanced Nitrogen Fixation in a Rhizobium etli ntrC Mutant That Overproduces the Bradyrhizobium japonicum Symbiotic Terminal Oxidase cbb(3)

The ntrC gene codes for a transcriptional activator protein that modulates gene expression in response to nitrogen. The cytochrome production pattern of a Rhizobium etli ntrC mutant (CFN2012) was studied. CO difference spectral analysis of membranes showed that CFN2012 produced a terminal oxidase similar to the symbiotic terminal oxidase of bacteroids in free-living cells under aerobic conditions, with a characteristic trough at 553 nm. CFN2012 produced two c-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only a 32-kDa c-type cytochrome. The expression levels of the R. etli fixNOQP operon, which codes for terminal oxidase cbb(3), were not affected by the ntrC mutation. However, the production levels of the two c-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the Bradyrhizobium japonicum fixNOQP operon was expressed in CFN2012 from the nptII promoter (pMSfix(c)), suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of cbb(3) and that CFN2012/pMSfix(c) overproduced this terminal oxidase. CFN2012/pMSfix(c) showed a significant increase in its symbiotic performance as judged by the determination of nitrogenase activities of plants inoculated with this strain, suggesting that the overproduction of cbb(3) terminal oxidase correlates with an enhancement in symbiotic nitrogen fixation.