Analysis of C₄‐dicarboxylate transport genes in Rhizobium meliloti

Abstract

A 5.1 kbp DNA fragment was isolated which complemented C4-dicarboxylate transport mutants (dct) of Rhizobium meliloti. Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD. TnphoA-generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription. Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression. The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants. This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD. Three independent second-site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus. Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible. These results are consistent with the direct requirement of the ntrA gene product for dctA expression. Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen-fixing activity. Nodules induced by dctA mutants failed to fix nitrogen. These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

“…Although reduced in amount compared with effective nodules, the synthesis of AS mRNA in both plant genecontrolled and bacterially controlled ineffective nodules (Figures 5 and 6) raises questions regarding the signal(s) involved in nodule-enhanced AS expression. Developmentally, all of the ineffective nodules evaluated by RNA blot analysis in Figures 5 and 6 attain a relatively similar stage, which equates to a day 7 or 8 effective nodule (Hirsch et al, 1983;Virts et al, 1988;Egli et al, 1989;Yarosh et al, 1989;Driscoll and Finan, 1993). Bacteria are released from infection threads, infected and uninfected cells can be found in the nodule interior, and small amounts of Lb can be detected.…”

NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic Regulation and Cellular Expression1

“…Nodules formed by S. meliloti F642, which is deficient in dicarboxylic acid transport (Yarosh et al, 1989), are generally much smaller than wild-type nodules. These nodules contain S. meliloti; however, the S. meliloti does not differentiate into fully developed bacteroids and therefore cannot fix atmospheric dinitrogen.…”

“…For analysis of NADH-GOGAT expression in bacterial conditioned ineffective nodules, cv Saranac seeds were surfacesterilized (70% ethanol for 10 min) and planted in sterilized sand inoculated with ineffective S. meliloti F642, dctA (dicarboxylic acid uptake deficient; Yarosh et al, 1989) or 7154, exoH (acid exopolysaccharide succinylation deficient; Leigh et al, 1987). These strains were generous gifts of Turlough Finan (McMaster University, Hamilton, Ontario, Canada) and Ann Hirsch (University of California, Los Angeles), respectively.…”

Molecular Characterization of NADH-Dependent Glutamate Synthase from Alfalfa Nodules

“…In plant-controlled ineffective nodules, early stages of nodule organogenesis proceed normally and slight nitrogenase activity is detectable (Vance and Johnson, 1983;Egli et al, 1989). The Rhizobium mutants employed for this study have different genetic lesions and are arrested at various developmental stages of the symbiotic process, from infection thread release to bacteroid development within the nodule (Hirsch et al, 1983;Leigh et al, 1987;Virts et al, 1988;Yarosh et al, 1989;Driscoll and Finan, 1993), and all are unable to fix nitrogen. This suggests that early events in nodule development, including bacterial release from infection threads, are not directly involved in induction of NADH-GOGAT gene expression.…”

“…For analysis of GOGAT expression in bacterially conditioned ineffective nodules, Saranac seeds were planted in sterilized sand that was inoculated with the ineffective strains. The R. meliloti strains and their relevant genotypes are as follows: T202, oxr (oxygen regulation deficient; Virts et al, 1988); 1491, nifH(nitrogenase subunit mutant; Hirsch et al, 1983); F642, dctA (dicarboxylic acid uptake deficient; Yarosh et al, 1989); 7154, exoH (acid exopolysaccharide succinylation deficient; Leigh et al, 1987); and G456, dme (malic enzyme deficient; Driscoll and Finan, 1993). These strains were generous gifts of Don Helinski (University of California, San Diego), Ann Hirsch (University of California, Los Angeles), and Brian Driscoll and Turlough Finan (McMaster University, Hamilton, Ontario, Canada).…”