Insertion of transposon Tn5tac1 in the Sinorhizobium meliloti malate dehydrogenase (mdh) gene results in conditional polar effects on downstream TCA cycle genes

• Published in: Molecular plant-microbe interactions Vol. 17; no. 12; pp. 1318 - 1327
• Main Authors: Dymov, S.I, Meek, D.J.J, Steven, B, Driscol, B.T
• Format: Journal Article
• Language: English
• Published: St Paul, MN APS Press 01.12.2004; The American Phytopathological Society
• Subjects: Agronomy. Soil science and plant productions; alfalfa; Amino Acid Sequence; amino acid sequences; Bacterial plant pathogens; Biological and medical sciences; Citric Acid Cycle - genetics; DNA Transposable Elements - genetics; Economic plant physiology; enzyme activity; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; genes; insertion mutants; insertional mutagenesis; malate dehydrogenase; Malate Dehydrogenase - chemistry; Malate Dehydrogenase - genetics; Malate Dehydrogenase - metabolism; mdh gene; Medicago - microbiology; Medicago sativa; Molecular Sequence Data; mutants; Mutation; nodulation; nucleotide sequences; Phenotype; Phytopathology. Animal pests. Plant and forest protection; Rhizobium leguminosarum; Sequence Alignment; Sequence Homology, Amino Acid; Sinorhizobium meliloti; Sinorhizobium meliloti - enzymology; Sinorhizobium meliloti - genetics; Sinorhizobium meliloti - growth & development; Symbiosis; Symbiosis (nodules, symbiotic nitrogen fixation, mycorrhiza...); symbiotic nitrogen fixation; transposons; tricarboxylic acid cycle; Plant; Gene; Microbiology; Enzyme; Transposon; Bacteria; Malate dehydrogenase; Transposable element; Oxidoreductases; Rhizobiaceae; Sinorhizobium meliloti
• ISSN: 0894-0282; 1943-7706
• DOI: 10.1094/MPMI.2004.17.12.1318

To isolate Sinorhizobium meliloti mutants deficient in malate dehydrogenase (MDH) activity, random transposon Tn5tac1 insertion mutants were screened for conditional lethal phenotypes on complex medium. Tn5tac1 has an outward-oriented isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter (Ptac). The insertion in strain Rm30049 was mapped to the mdh gene, which was found to lie directly upstream of the genes encoding succinyl-CoA synthetase (sucCD) and 2-oxoglutarate dehydrogenase (sucAB and lpdA). Rm30049 required IPTG for wild-type growth in complex media, and had a complex growth phenotype in minimal media with different carbon sources. The mdh:: Tn5tacl insertion eliminated MDH activity under all growth conditions, and activities of succinyl-CoA synthetase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase were affected by the addition of IPTG. Reverse-transcriptase polymerase chain reaction (RT-PCR) studies confirmed that expression from Ptac was induced by IPTG and leaky in its absence. Alfalfa plants inoculated with Rm30049 were chlorotic and stunted, with small white root nodules, and had shoot dry weight and percent-N content values similar to those of uninoculated plants. Cosmid clone pDS15 restored MDH activity to Rm30049, complemented both the mutant growth and symbiotic phenotypes, and was found to carry six complete (sdhB, mdh, sucCDAB) and two partial (IpdA, sdhA) tricarboxylic acid cycle genes.