Functional Characterization of the Sinorhizobium meliloti Acetate Metabolism Genes aceA , SMc00767, and glcB

The genes encoding malate synthase (glcB ) and isocitrate lyase (aceA ) and a 240-bp open reading frame (SMc00767) located downstream ofaceA were isolated and functionally characterized inSinorhizobium meliloti . Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed.aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient inglcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed thataceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants.aceA -glcB ,aceA -SMc00767, andglcB -SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-typeaceA -glcB oraceA -SMc00767 loci were introduced into the double mutants. These data confirm the functional role ofaceA and SMc00767 and show thatglcB , in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains.aceA andglcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulatesaceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, includingaceA ; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism inS. meliloti . Genetic and functional analyses indicated thataceA and SMc00767 constitute a functional two-gene operon, which is conserved in other α-proteobacteria. Alfalfa plants infected with theaceA andglcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in theRhizobium -legume symbiosis.