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

Abstract

Tn5-induced mutants of <i>Rhizobium meliloti</i> 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 5.5-kb <i>Eco</i>RI fragment.- Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb <i>Eco</i>RI fragment restored the wild-type phenotype in the Ilv⁻ Nod⁻ mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for <i>Escherichia coli</i> DJvC and <i>Saccharomyces cerevisiae</i> Ilv5. Genes <i>ilvC</i> and <i>ilv5</i> code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the <i>ilvC</i> genes of <i>Rhizobium meliloti</i> and <i>E. coli</i> are functionally equivalent. We demonstrated that in <i>ilvC</i> mutant 1028 the common nodulation genes <i>nodABC</i> are not activated by the inducer luteolin. <i>E. coli</i> <i>ilvC</i> complemented both defective properties (Ilv⁻ and Nod⁻) found in mutant 1028. These findings demonstrate that <i>R. meliloti</i> requires an active isomeroreductase enzyme for successful nodulation of alfalfa.