This Article Full Text Full Text (PDF) Supplemental material Other Versions of this Article: JB.00385-07v1 189/16/5875    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ramírez-Trujillo, J. A. Articles by Hernández-Lucas, I. Search for Related Content PubMed PubMed Citation Articles by Ramírez-Trujillo, J. A. Articles by Hernández-Lucas, I.

Functional Characterization of the 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,¹ Centro de Ciencias Genómicas, Universidad Nacional Autónoma de Mexico, Cuernavaca, Morelos, México,² Department of Biochemistry, University of Missouri, Columbia, Missouri 65211³

Received 15 March 2007/ Accepted 17 May 2007

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.

Published ahead of print on 25 May 2007.

Supplemental material for this article may be found at http://jb.asm.org/.