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J. Bacteriol. doi:10.1128/JB.00385-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico.; Centro de Ciencias Genómicas, Universidad Nacional Autónoma de Mexico, Cuernavaca, Morelos, México.; Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA

^* To whom correspondence should be addressed. Email: ismaelh{at}ibt.unam.mx.

	Abstract

The genes encoding malate synthase (glcB), 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 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 show that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the respective single mutants. Double aceA-glcB, aceA-SMc00767 and glcB-SMc00767 knockouts were also unable to grow on acetate, and recovered this ability when the wild type aceA-glcB or aceA-SMc00767 loci were introduced in each double mutant. 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 are able to complement the enzymatic activity lacking in the respective single mutants. The enzymatic activities also show that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirm the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant versus wild type Rm5000 show that SMc00767 repress the expression of a moderate number of ORFs 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.