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 ␣-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.

High-Resolution Transcriptomic Analyses of Sinorhizobium sp. NGR234 Bacteroids in Determinate Nodules of Vigna unguiculata and Indeterminate Nodules of Leucaena leucocephala

“…Moreover, the coincident expressions of glyoxylate cycle genes and a subset of nif genes in the chemoautotrophic culture of B. japonicum [84] imply potential coordination in transcriptions of these genes. However, S. meliloti mutants of two principle genes, aceA (encoding isocitrate lyase) and glcB (encoding malate synthase), in the glyoxylate cycle were not impaired in nodulation and nitrogen fixation on alfalfa [85]. NGR_c03920- NGR_c03950 expressed higher in V. unguiculata bacteroids (RPKM = 254–401) than in L. leucocephala bacteroids (RPKM = 55–105) and the free-living condition (RPKM = 24–40).…”

Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis

“…To evaluate the role of ICL, null mutants were constructed in S. meliloti and Rhizobium tropici and their symbiotic performance was evaluated on M. sativa and Phaseolus vulgaris, respectively. The results demonstrated that in both symbiotic models ICL is not involved in nodulation or nitrogen fixation (Ramírez-Trujillo et al, 2007).…”

Microarray analysis of striatal embryonic stem cells induced to differentiate by ensheathing cell conditioned media

“…GenArise carries out a number of transformations: background correction, lowest normalization, intensity filter, replicate analysis, and selection of differentially expressed genes. The goal of GenArise is to identify good evidence that the genes are differentially expressed, and it has been successfully used in recent studies (Méndez‐Ortiz et al,2006; Luna‐Moreno et al,2007; Ramirez‐Trujillo et al,2007). The software identifies differentially expressed genes by calculating an intensity‐dependent z‐score.…”