Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene

Abstract

The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon TnS and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. melloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures.In the symbiosis between legumes and rhizobia, the host plant provides the bacteria with reduced carbon as an energy source. The bacteria use this energy to reduce atmospheric nitrogen to ammonia, which they release to the plant. The qualitative nature of this energy source has been the focus of much research. Sucrose is the major photosynthate transported from the shoot to the nodule (41), but biochemical evidence suggests that neither sucrose nor hexoses obtained from sucrose degradation are important sources of energy for the bacteroids (reviewed in references 7 and 34). In support of this conclusion, mutants of various species of rhizobia with defects in sugar metabolism have been found to be effective in symbiosis (7,34). By contrast, dicarboxylic acids appear to be important carbon sources in the establishment of an effective symbiosis. Succinate and malate are found at high concentrations in nodules (14,45,55), are actively transported across the peribacteroid membrane (20,56), are taken up by bacteroids (11,20,42,50), and are quickly oxidized to CO2 after uptake (47). Dicarboxylic acid transport (dct) mutants of Rhizobium meliloti (5) and R. leguminosarum biovars viciae (3, 12) and trifolii (44) We are interested in how the bacteroid TCA cycle is regulated and have selected ICD for our initial studies because it is a regulated enzyme at a branch point in the TCA cycle (15), because it is regulated by aerobiosis in other gram-negative bacteria (23, 24), and because it is a relatively simple enzyme with a single type of subunit that, in other bacteria, is encoded by a single gene (2). In this study, we report the isolation of the gene that encodes ICD in R meliloti and the symbiotic properties of mutants with defects in this gene. MATERIALS AND METHODSBacterial strains and plasmids. The strains of R. meliloti and Es...

Nutrient Sharing between Symbionts

“…Isocitrate dehydrogenase mutants of B. japonicum are only slightly delayed in nodule formation on soybean and fix nitrogen at similar rates to wild type (Shah and Emerich, 2006), while S. meliloti isocitrate dehydrogenase mutants are ineffective, although they form normal nodules full of bacteroids (McDermott and Kahn, 1992). This emphasizes the difference between soybean nodulating bacteria and those that nodulate temperate legumes such as alfalfa and pea.…”

Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti

“…Nondenaturing (native) polyacrylamide gels were used to separate aminotransferases. The nondenaturing gel system used has been described elsewhere (40). Native gels contained 10% acrylamide in the resolving phase and 5% acrylamide in the stacking phase.…”

“…The resulting plasmid, pJA15, was subcloned into the BamHI site of pMK413 (40) and conjugated into R. meliloti by use of the E. coli helper strain S17-1 (53). The loss of the plasmid and marker exchange were selected for by using the method described by McDermott and Kahn (40).…”

“…For selection for the rescue of DL39(Mucts) by the R. meliloti gene library, DL39(Mucts) was grown on minimal medium lacking aspartate, tyrosine and phenylalanine, or leucine; the minimal medium used was modified from that of Kuramitsu et al (38) and has been described elsewhere (61 (50). The R. meliloti gene library used in this study was constructed in pUC18 and has been described elsewhere (40 To provide a region of homology for recombination between this plasmid and plasmids from the pUC18-derived gene library, pUC18 and pMK409 were digested with BamHI and HindIII, ligated together, and transformed into C-2110 (31), a strain that does not permit the replication of pUC18. Tetr Penr transformants were screened for Suc'.…”

“…By using an in vitro assay, Udvardi et al (63) found that glutamate was not taken up in physiologically relevant concentrations by isolated soybean symbiosomes. However, an R. meliloti isocitrate dehydrogenase mutant that is a glutamate auxotroph (40) grew to normal concentrations in alfalfa nodules, suggesting that in alfalfa nodules glutamate is available to bacteroids.…”