The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa

Abstract

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5. Rhizobium meliloti induces nitrogen (N2)-fixing nodules on the roots of alfalfa (Medicago sativa). This symbiotic relationship begins on the root surface, proceeds with the invasion of root hairs, and culminates in the development of an effective N2-fixing nodule. R. meliloti nodulation (nod) genes are located on a large symbiotic plasmid termed pSym (6, 37) and have been cloned and characterized. The common nodulation genes (nodABC) are structurally and functionally conserved among several Rhizobium and Bradyrhizobium species (23,27). Other nodulation genes, such as nodFEGHPQ, are believed to confer specificity for nodulation (12, 21). It has been proposed that the common genes nodABC and the specific genes nodHPQ produce alfalfaspecific signals that trigger a host response (16,26,38). The nodD gene codes for a protein that activates the expression of the other nod genes in the presence of inducing compounds exuded by alfalfa seeds or roots (20,30 In addition, uncharacterized auxotrophic mutants with altered symbiotic properties have been described (14). In this study we have examined the symbiotic properties of two Ilv-mutants of R. meliloti and found that in only one strain the same mutation (ilvC) causes the mutant to be symbiotically defective. We have demonstrated that the mutation in the llv-Nod-mutant maps in the gene coding for the enzyme acetohydroxy acid isomeroreductase (isomero-* Corresponding author. reductase) (E.C. 1.1.1.89) (ilvC). We have determined the primary sequence of the R. meliloti ilvC gene and have shown that the Escherichia coli ilvC gene complements both defective phenotypes in the Ilv-Nod-mutant of R. meliloti. MATERIALS AND METHODSBacterial strains, plasmids, and media. The strains and plasmids used in this study are listed in Table 1. E. coli strains were maintained on LB agar. R. meliloti was grown on either TY medium or minimal medium (28).Amino acids and precursors were purchased from Sigma Chemical Co. unless stated otherwise and were used as netessary in the following concentrations (milligrams per liter): isoleucine, 50; valine, 50; a-ketobutyrate, 125; a-ketoisovalerate, 90; calcium D-pantothenate, 8.4; racemic acetolactate

Biosynthesis of branched-chain amino acids is essential for effective symbioses between betarhizobia and Mimosa pudica

“…Both mutant strains were mutated in genes involved in BCAA biosynthesis. These data are consistent with the results obtained from previous studies on alpharhizobial mutants with altered BCAA biosynthesis, which were also defective in nodulating their host legumes (Aguilar & Grasso, 1991;de las Nieves Peltzer et al, 2008;Ferraioli et al, 2002;Hassani et al, 2002;Kerppola & Kahn, 1988;Ló pez et al, 2001;Pobigaylo et al, 2008;Sanjuán-Pinilla et al, 2002;Truchet et al, 1980 Prell, unpublished data). In B. phymatum STM815 only one ilvE gene was identified and no paralogue could be found.…”

“…Several mutants of members of the genus Rhizobium (and related alpharhizobia genera) that are affected in their biosynthesis of isoleucine, valine and leucine have repeatedly been demonstrated to be defective in either nodulating their host plants or fixing nitrogen when nodules are formed (Aguilar & Grasso, 1991;de las Nieves Peltzer et al, 2008;Ferraioli et al, 2002;Hassani et al, 2002;Kerppola & Kahn, 1988;Ló pez et al, 2001;Pobigaylo et al, 2008;Sanjuán-Pinilla et al, 2002;Truchet et al, 1980). This is often generally the case for auxotrophic mutants, because the compounds that cannot be synthesized by the rhizobium might also not be provided by the plant in the rhizosphere or during the nodulation process.…”

Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici , Including Acid Tolerance

“…Plasmid DNA was prepared by the alkaline method as described by Kragelund et al (28). Total genomic R. tropici DNA isolation, restriction enzyme digestions, ligations, and Southern blotting experiments were carried out according to procedures previously described (2,43).…”

Structure and expression of a cyanobacterial ilvC gene encoding acetohydroxyacid isomeroreductase

“…The highest similarity (63.1% identity in a 1,018-bp overlap) was to the R. meiloti ilvC gene (1) (Fig. 3).…”

“…In a third reaction, S. typhimurium AHAIR is able to catalyze the reduction of a-ketopantoate to pantoate, an intermediate in the biosynthesis of coenzyme A (17). Finally, the ilvC gene, which encodes AHAIR, is required for nodulation in Rhizobium meliloti, for unknown reasons not involving isoleucine and valine biosynthesis (1).…”