Malic enzyme cofactor and domain requirements for symbiotic N2 fixation by Sinorhizobium meliloti

The NAD(+)-dependent malic enzyme (DME) and the NADP(+)-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N(2) (Fix(-)) in alfalfa root nodules, whereas tme mutants are unimpaired in their N(2)-fixing ability (Fix(+)). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N(2) fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N(2) fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD(+)-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N(2) fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H(+) to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N(2)-fixing bacteroids.