4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Jin, H. N.; Dilworth, M. J.; Glenn, A. R.

doi:10.1007/bf00248427

Cited by 28 publications

(36 citation statements)

References 28 publications

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“…This inhibition appears to occur in the bacteroid due to a low 0, tension and definitely occurs in TCA-cycle mutants, It seems likely that different strains and species of Rhisobium and Bradyrhisobium will partition carbon and reducing equivalents between these two and possibly other overflow pathways to varying extents, including bypass pathways such as the GABA shunt, which catabolizes glutamate, and the enzyme aspartase, which hydrolyses aspartate to fumarate and ammonia. Both of these pathways have been postulated to occur in Rhizobiurn and at least some of the enzyme activities have been demonstrated (Poole et af., 1984;Jin et al, 1990;Kouchi et al, 1991;Fitzmaurice & O'Gara, 1993).…”

Section: Discussionmentioning

confidence: 99%

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

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Reading RG6 6AJ, UKMutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to s u d and suct), which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex ( s u d B ) . Mutation of either SUCC or SUCD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of s u d or suct), the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of R. leguminosamm was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (SUCD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and reductant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.

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Section: Discussionmentioning

confidence: 99%

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

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“…One of the possible routes of NH3 liberation from glutamate is a chain of glutamateoxaloacetate aminotransferase and aspartate-pyruvate aminotransferase with alanine ' dehydrogenase as suggested in Rhizobium sp. MNF2030 (Jin et al, 1990). Even though such a chain of transaminases with a terminal amino acid dehydrogenase may be also partly…”

Section: Discussionmentioning

confidence: 99%

“…GABA-AT in bacteroids was more active with pyruvate and oxaloacetate than with 2-oxoglutarate, unlike the situation in cowpea Rhizobium sp. MNF2030 (Jin et al, 1990) and R . meliloti bacteroids (Miller et al, 199 1).…”

Section: I4c Andmentioning

confidence: 99%

Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules

Kouchi

Fukai

Kihara

1991

Journal of General Microbiology

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Bacteroids were isolated anaerobically from root nodules of soybean (Glycine m a L. Merr.) and supplied with 4C-or sN-labelled glutamate or aspartate under microaerobic conditions where bacteroids retained a high activity of N2 fixation. Clutamate and aspartate significantly stimulated N2 fixation (C2H2 reduction) and respiration (C02 evolution) of isolated bacteroids. They were both utilized as substrates for bacteroid respiration and their nitrogen was rapidly released from the bacteroids as NH3. Amino-oxyacetate (AOA) strongly inhibited glutamate utilization, but aspartate utilization was only slightly affected by AOA. Substantial activity of aspartate ammonia-lyase (aspartase) was detected in the cell-free extract of bacteroids. These results suggest that the major pathway of glutamate utilization in bacteroids is transamination to form aspartate followed by direct deamination of aspartate by aspartase. It is also suggested that the 4-aminobutyrate pathway (GABA-shunt) is partly responsible for the catabolism of glutamate by soybean nodule bacteroids. The results are discussed in terms of the possible relationships between C,-dicarboxylates and glutamate in carbon and nitrogen metabolism in soybean nodule bacteroids.

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“…Isocitrate lyase (EC 4.1.3.1) activity was measured by the continuous assay of Dixon and Kornberg (9). Glutamate decarboxylase (EC 4.1.1.15) was assayed by the method of Jin and coworkers (18) in which the GABA formed was determined enzymatically (17). Protein content of the extracts was determined by the method of Bradford (4 Growth on alternate nitrogen substrates was conducted in the same manner except that the cells were washed with and inoculated into Tully's medium with the ammonium sulfate omitted; an alternate nitrogen source was then added to a final concentration of 15 mM.…”

Section: Methodsmentioning

confidence: 99%

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate

Green

Emerich

1997

J Bacteriol

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The sucA gene, encoding the E1 component of ␣-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of ␣-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, ␤-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of ␣-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.Bradyrhizobium japonicum is a gram-negative soil bacterium that forms nitrogen-fixing symbioses with soybean. During the development of the symbiosis, the bacteria induce and invade specialized organs, called nodules, on the root of the host plant (5,26,44). The nodules house the bacteria and provide an environment conducive to nitrogen fixation, including a reduced oxygen tension to protect nitrogenase and a steady supply of nutrients to the endosymbiont. Concomitantly with its invasion of the nodule, B. japonicum differentiates from a free-living to a symbiotic form (bacteroid), a process involving extensive adjustments to its nitrogen and carbon metabolism (25,40).Bacteroid respiration during nitrogen fixation depends entirely on reduced carbon supplied by the host plant. The main carbon compounds used by fully differentiated bacteroids are four-carbon dicarboxylic acids, the metabolism of which has been assumed to occur via the tricarboxylic acid (TCA) cycle (25,40). However, some studies indicate that the TCA cycle may not be operating to full capacity during bacteroid respiration. For example, the O 2 limitation of bacteroid respiration inside the soybean nodule can lead to increased NADH/NAD ratios and thus to significant inhibition of ␣-ketoglutarate dehydrogenase (34). Such studies imply that the respiration rate of B. japonicum bacteroids may in part be limited by carbon flux through certain steps of the TCA cycle.To maintain carbon flux under oxygen-limiting conditions, bacteroids coul...

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4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Cited by 28 publications

References 28 publications

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate

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