NADP(+)-dependent malic enzyme of Rhizobium meliloti

The bacterium Rhizobium meliloti, which forms N-2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP(+) dependent, while a second has maximal activity when NAD(+) is the coenzyme, The diphosphopyridine nucleotide (NAD(+))-dependent malic enzyme (DME) is required for symbiotic N-2 fixation, likely as part of a pathway for the conversion of C-4-dicarboxylic acids to acetyl coenzyme A in N-2-fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide [NADP(+)]-dependent malic enzyme) to a 3.7-kb region. We constructed strains carrying insertions within the tme gene region and showed that the NADP(+)-dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE-cellulose chromatography column, We found that NADP(+)-dependent malic enzyme activity was not required for N-2 fixation, as tme mutants induced N-2-fixing root nodules on alfalfa. Moreover, the apparent NADP(+)-dependent malic enzyme activity detected in wild-type (N-2-fixing) bacteroids was only 20% of the level detected in free-living cells, Much of that residual bacteroid activity appeared to be due to utilization of NADP(+) by DME. The functions of DME and the NADP(+)-dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants.