Cyclic organization of the carbohydrate metabolism in <i>Sinorhizobium meliloti</i>

Portais, Jean‐Charles; Tavernier, Patricia; Gosselin, Isabelle; Barbotin, Jean‐Noël

doi:10.1046/j.1432-1327.1999.00778.x

Cited by 24 publications

(36 citation statements)

References 27 publications

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“…Reaction 5 principally enables a cyclic flux through the ED pathway that could not be resolved by the present 13 C-labeling experiments; nevertheless, a weak flux was obtained through reaction 5 in P. putida (see Table S1 in the supplemental material). The network models for A. tumefaciens and S. meliloti were basically the same as that for P. fluorescens (1,12,21,25,36). Since we did not detect gluconate or 2-ketogluconate accumulation, however, glucose uptake was assumed to be direct in both organisms.…”

Section: Methodsmentioning

confidence: 93%

“…As a fast-growing rhizobium with a generation time of less than 6 h on complex medium, S. meliloti was reported to be able to convert glucose to gluconate, which then enters metabolism (36,43). Since Ͻ1 mM gluconate accumulated and the labeling data did not allow the resolution of the different uptake routes, both were lumped for net-flux analysis.…”

Section: Vol 187 2005 Experimental Identification Of Metabolic Netwmentioning

confidence: 99%

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Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

2005

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The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13 C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, ␥-proteobacteria, and ␣-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively. Based on13 C tracer experiments, metabolic-flux analysis emerged as a key methodology to identify the network topology of active reactions and to quantify the in vivo distribution of molecular fluxes throughout metabolism (38, 47). In contrast to global protein, mRNA, or metabolite concentration analyses that assess network composition, flux methods directly assess the operation of metabolic networks by quantifying in vivo reaction velocities. The general principle is based on mass spectrometry (MS) or nuclear magnetic resonance detection of 13 C patterns in products of metabolism. Often, protein-bound amino acids that preserve the carbon backbone of eight metabolic key intermediates are used. The detected 13 C isotope patterns then reflect the activity of intracellular pathways and reactions, whose fluxes can be quantified from the isotope data by using mathematical models with various levels of complexity. In the simplest approach, algebraic equations are used to determine strictly local ratios of converging fluxes analytically by so-called metabolic-flux ratio (METAFoR) analysis (3,16,41,44). Absolute intracellular fluxes in millimoles per gram of biomass per hour may be estimated indirectly by combining such 13 C data with quantitative physiological ...

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Section: Methodsmentioning

confidence: 93%

Section: Vol 187 2005 Experimental Identification Of Metabolic Netwmentioning

confidence: 99%

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

2005

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“…Although somewhat obvious, this cell compartmentation of C polymers does not seem to be the only explanation for the difference in C channeling in response to the C/N ratio. P(3HB) is more reduced than mannitol, CPS, or EPS, and polysaccharide synthesis by overflow metabolism might involve a recycling of carbon through dehydrogenating pathways (42), thus contributing much less to the disposal of excess reducing power than P(3HB) synthesis does. This could indicate that N-limited cultures accumulated less reducing power, probably as a consequence of their lower biomass.…”

Section: Discussionmentioning

confidence: 99%

Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

et al. 2001

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In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day ؊1 in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH 4 ) 2 SO 4 ] limited to only 20 M. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH 4 ) 2 SO 4 . The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.

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“…2 and Table 2). Carbon cycling has been reported for a number of other bacterial species, including Sinorhizobium meliloti (26)(27)(28). RL0753 and RL1315 encode putative glucose-6-phosphate-1-dehydrogenases ( Fig.…”

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confidence: 98%

Role of O ₂ in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

et al. 2017

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Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O 2 was used to understand how O 2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547[phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O 2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omegaamino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O 2 , conditions similar to those experienced by N 2 -fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O 2 and cell death.IMPORTANCE Rhizobium leguminosarum, a soil bacterium that forms N 2 -fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O 2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O 2 tensions during growth in soil and N 2 fixation in symbiosis with legumes.KEYWORDS INSeq, nodules, Rhizobium leguminosarum, legumes, nitrogen fixation, Pisum sativum R hizobia are alphaproteobacteria able to form symbioses with legumes, on which they induce development of root nodules. Nodules provide an environment with both a low O 2 concentration and the correct nutrients to enable differentiation of rhizobia into nitrogen (N 2 )-fixing bacteroids (1). Bacteroids fix atmospheric N 2 into ammonium (NH 4 ϩ ), which is secreted to the plant host, and are, in turn, provided with

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Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti

Cited by 24 publications

References 27 publications

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

Role of O ₂ in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

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Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti

Cited by 24 publications

References 27 publications

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

Role of O 2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

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Role of O ₂ in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate