NADP+ -dependent malic enzyme of Rhizobium meliloti.
The bacterium Rhizobium meliloti, which forms N2-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 N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-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 N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa. Moreover, the apparent NADP+ -dependent malic enzyme activity detected in wild-type (N2-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.
Driscoll
B T
BT
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Finan
T M
TM
D002848:Chromatography, DEAE-Cellulose; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009154:Mutation; D009586:Nitrogen Fixation; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013559:Symbiosis
D011993:Recombinant Fusion Proteins; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)