Biochemical and Genetical Analysis of Rhizobium meliloti Mutants Defective in C4-dicarboxylate Transport

Engelke, Thomas; Jagadish, Mn; Pühler, Alfred

doi:10.1099/00221287-133-11-3019

Cited by 43 publications

(58 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The origin and nature of this factor remain unknown. A similar situation has been reported for the dctABD genes, which encode the structural and regulatory genes of the dicarboxylic acid transport system in R. meliloti (Engelke et al, 1987;Ronson et al, 1987;Birkenhead et al, 1990;Watson, 1990). In free-living R. meliloti, the sensor-effector couple dctBD regulates transcription of the dctA gene, which encodes a dicarboxylate permease, in response to the presence of dicarboxylic acids in the medium.…”

Section: Discussionsupporting

confidence: 72%

HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum

Soom

Wilde

Vanderleyden

1997

Molecular Microbiology

View full text Add to dashboard Cite

SummaryA chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion. The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.

show abstract

Section: Discussionsupporting

confidence: 72%

HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum

Soom

Wilde

Vanderleyden

1997

Molecular Microbiology

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene

McDermott

Kahn

1992

J Bacteriol

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 99%

“…Although sucrose is the major carbon source transported from the shoot to the nodule (44), most data suggest that it is not an important source of reductant for bacteroids (58) and that dicarboxylic acids are more likely to be their main carbon and energy sources (8,13,56). Succinate and malate are found in high concentrations in nodules (20,49,57) and bacteroids (15,29,45,51) and are actively transported across the symbiosome membrane (29,62). Bacterial mutants that lack dicarboxylate transport protein DctA are Fix- (5,17,48), demonstrating that the acquisition of dicarboxylic acids is important to the bacteria during symbiosis.…”

mentioning

confidence: 99%

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

Alfano

Kahn

1993

J Bacteriol

View full text Add to dashboard Cite

Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA. Genes for the latter two enzymes, WttA and batA, were previously isolated from R. meliloti. aatB is distinct from but hybridizes to aaL4, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da. The amino acid sequence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs. AatB expressed in E. coli has a Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed in aatB and hztA and transferred to the genome ofR. melioti 104A14. Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation.Bacteria of the genus Rhizobium fix nitrogen in a symbiotic association with leguminous plants. Carbon compounds produced by the plant are fed to the bacteria and metabolized to supply the energy and reductant needed for nitrogen fixation. Identifying which carbon compounds are transferred to the bacteria and how these compounds are metabolized is important in understanding the physiology of the symbiosis. Although sucrose is the major carbon source transported from the shoot to the nodule (44), most data suggest that it is not an important source of reductant for bacteroids (58) and that dicarboxylic acids are more likely to be their main carbon and energy sources (8,13,56). Succinate and malate are found in high concentrations in nodules (20,49,57) and bacteroids (15,29,45,51)

show abstract

Biochemical and Genetical Analysis of Rhizobium meliloti Mutants Defective in C4-dicarboxylate Transport

Cited by 43 publications

References 26 publications

HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum

HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum

Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene

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

Contact Info

Product

Resources

About