Analysis of C4-Dicarboxylic Acid Transport Genes in Rhizobium Meliloti

Abstract

Rhizobium meliloti mutants defective in C₄-dicarboxylic acid transport (Dct⁻) were previously isolated by Tn5 mutagenesis, and divided into two groups based on complementation of Dct- with cosmid clones. In this work further characterization was carried out on the two loci. Group I mutants were found to be defective in dicarboxylate transport (Dct⁻), nitrate utilization, and symbiotic nitrogen fixation. Subcloning and complementation work confined the Group I mutations to a 3.5 kbp BamHI-EcoRI fragment containing the ntrA gene. Group V mutants were defective in dicarboxylate transport and demonstrated varying levels of nitrogen fixation. Complementation and site-directed Tn5 mutagenesis revealed three transcriptional units, corresponding to dctA, dctB, and dctD, localized within a 6 kbp HindIII fragment. The use of dctA::TnphoA fusions determined the expression of dctA to be ntrA, dctB, and dctD dependent. Dct+ revertants of dctB and dctD mutants were selected which carried second-site mutations responsible for restoring the Dct⁺ phenotype.