Enhanced Nitrogen Fixation in a Rhizobium etli ntrC Mutant That Overproduces the Bradyrhizobium japonicum Symbiotic Terminal Oxidasecbb ₃

ABSTRACT

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 ntrCmutant (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 theR. etli fixNOQP operon, which codes for terminal oxidasecbb ₃, were not affected by the ntrCmutation. However, the production levels of the two c-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when theBradyrhizobium 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.