Functional characterization of the Sinorhizobium meliloti acetate metabolism genes aceA, SMc00767, and glcB

Abstract

The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium 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 in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, 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 and glcB 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 regulates aceA 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, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other alpha-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.

FIG. 1.

Functional role of aceA, SMc00767, and glcB in acetate utilization by S. meliloti. Bacteria were grown in M9 minimal medium supplemented with 5 mM potassium acetate. The OD₅₉₅ was measured every 24 h over a 96-h period. The bacterial strains used were wild-type strain Rm5000, aceA mutant RH190, SMc00767 mutant RH421, glcB mutant RH218, an aceA mutant complemented with the wild-type aceA gene (RH198), an SMc00767 mutant complemented with the SMc00786 gene (RH435), an SMc00767 mutant complemented with the wild-type SMc00767 gene and the intact aceA promoter region (RH442, glcB-SMc00767 double mutant RH419, a glcB-SMc00767 double mutant complemented with glcB (RH429), a glcB-SMc00767 double mutant complemented with SMc00767 (RH436), a glcB-SMc00767 double mutant complemented with the aceA promoter region and SMc00767 (RH443), aceA-glcB double mutant RH222, an aceA-glcB double mutant complemented with aceA-glcB (RH312), an aceA-glcB double mutant complemented with aceA-SMc00767 (RH327), an aceA mutant complemented with aceA-SMc00767 (RH326), and an aceA mutant complemented with aceA-glcB (RH462). Growth kinetics were determined at least four times, and the graphs show the averages of all experiments.

FIG. 2.

β-Glucuronidase activities of RH465 (wild-type strain Rm5000 containing the aceA::gusA fusion) and RH467 (SMc00767 mutant RH421 harboring the aceA::gusA fusion) grown in M9 medium containing acetate. The cultures were collected at OD₅₉₅s of 0.35, 0.7, and 1. The values are the means of three independent experiments performed in duplicate. PNP, p-nitrophenyl; GUS, β-glucuronidase.

FIG. 3.

Genome context of the SMc00767, aceA, SMc00769, potF, potG, potH, and potI genes in different members of the α-proteobacteria. In all cases, SMc00767 is clustered in this group of genes, suggesting that they are involved in a common process. Genes and intergenic distances are drawn in proportion. The figure was generated using GeCont (7).