RHIZOBIUM-LEGUMINOSARUM NODULATION GENE (NOD) EXPRESSION IS LOWERED BY AN ALLELE-SPECIFIC MUTATION IN THE DICARBOXYLATE TRANSPORT GENE DCTB

To identify host genes that might influence nod (nodulation) gene expr ession in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carry ing nodD) was introduced into a chemically mutagenized population of a R. leguminosarum strain lacking a symbiotic plasmid. The transconjuga nts were screened for expression of alkaline phosphatase (PhoA) on pla tes containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified. When the nodC-phoA plasmid was cure d from the mutant and the symbiotic plasmid pRL1JI introduced, the mut ant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal. When the nodC-phoA allele was introduced on pRL1JI a law le vel of nod gene induction was found. The reduced nodC expression appea red to be caused by a decrease in expression of the regulatory gene no dD, since expression of a nodD-IacZ fusion was also lower in the mutan t than in the control. These mutant phenotypes and the low nitrogen fi xation were complemented with a plasmid (pIJ1848) from a R. leguminosa rum cosmid library. DNA hybridization confirmed that pIJ1848 was not f rom the symbiotic plasmid and showed that a DNA insertion was present in the mutant. The complementing region of pIJ1848 was defined by tran sposon mutagenesis; DNA sequencing revealed that it carried the dicarb oxylic acid transport (dct) genes. However, the mutant grew well with succinate as sole C-source. Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mut ation caused the reduction in nod gene expression. The effect was alle le-specific since other mutations in dctB did not influence nod gene e xpression. Surprisingly, the mutant had a constitutive high level of s uccinate transport, indicating that the mutation caused unregulated ex pression of dctA the structural gene for dicarboxylic acid transport, This in some way appears to have lowered the expression of nodD, indic ating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB.