A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti

Povolo, Silvana; Casella, Sergio

doi:10.1007/s002030000171

Cited by 42 publications

(71 citation statements)

References 49 publications

(37 reference statements)

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“…The potential polypeptide translated from this gene showed high identity (up to 56%) with proteins translated from regions containing genes for PHB biosynthesis in a number of proteobacteria (3,11,12,36). For rhizobia, this gene has been designated aniA and implicated in a complex regulation of carbon flow in these bacteria (12,36). Orf3 also showed lower identities with PhaR proteins in P. denitrificans and R. eutropha, which have been implicated elsewhere in negative regulation of GAPs in these organisms (28,29,46).…”

Section: Resultsmentioning

confidence: 93%

“…orf3 was found upstream of phaA (23). The potential polypeptide translated from this gene showed high identity (up to 56%) with proteins translated from regions containing genes for PHB biosynthesis in a number of proteobacteria (3,11,12,36). For rhizobia, this gene has been designated aniA and implicated in a complex regulation of carbon flow in these bacteria (12,36).…”

Section: Resultsmentioning

confidence: 99%

“…PhaR has been implicated previously in playing a role in negative regulation of GAPs (phasins) in some bacteria (i.e., P. denitrificans and R. eutropha), while for others (rhizobia) a PhaR homolog, AniA, was shown to play a role in global regulation of carbon flow, and mutation in this gene caused highly pleiotropic effects (11,12,36). To test the role of PhaR in M. extorquens AM1, we generated a null mutation in phaR and investigated the mutant phenotype with respect to growth characteristics on various substrates, PHB accumulation during growth on various substrates, and expression of genes involved in PHB biosynthesis.…”

Section: Resultsmentioning

confidence: 99%

“…The evidence showing that PhaR is not a transcriptional regulator of phasins suggested that it might play a role more similar to that of AniA. A number of results in the literature point toward AniA, a homolog of PhaR playing a role in directing carbon flow in rhizobial species (12,36). However, the key metabolite in this regulation or the targets of such a regulation still remain elusive…”

Section: Resultsmentioning

confidence: 99%

“…In Sinorhizobium meliloti, a homolog of PhaR was called AniA, because of its expression during anaerobic growth. An even more complex function was suggested for this protein, in partitioning of carbon flow in cells, affecting not only PHB production but also production of extracellular polymeric substances and nitrogen fixation (36). AniA was also investigated in another rhizobial species, Rhizobium etli, and suggested to play a role not only in PHB production but also as a global regulator of protein expression and of carbon and energy fluxes, based on the pleiotropic effect of mutation in AniA on metabolism of this bacterium (11,12).…”

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confidence: 99%

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Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Korotkova¹,

Chistoserdova²,

Lidstrom

2002

J Bacteriol

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Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-␤-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon-and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C . In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR. We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C 2 compounds, while they show wild-type growth characteristics on C 1 or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C 1 compounds and has defects in PHB accumulation but grows normally on C 3 and C 4 compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C 2 compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C 1 , C 2 , and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Korotkova¹,

Chistoserdova²,

Lidstrom

2002

J Bacteriol

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Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using ¹³C‐label tracing and mass spectrometry

Dien

Strovas

Lidstrom

2003

Biotech & Bioengineering

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The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm. The combination of labeled methanol with unlabeled CO(2), which enters central metabolism in two different reactions, provided the discriminatory power necessary to allow quantification of the unknown fluxes within a reasonably small confidence interval. In wild-type M. extorquens AM1, no measurable flux was detected through pyruvate dehydrogenase or malic enzyme, and very little flux through alpha-ketoglutarate dehydrogenase (1.4% of total carbon). In contrast, the alpha-ketoglutarate dehydrogenase flux was 25.5% of total carbon in the regulatory mutant strain phaR, while the pyruvate dehydrogenase and malic enzyme fluxes remained insignificant. The success of this technique with growth on C(1) compounds suggests that it can be applied to help characterize the effects of other regulatory mutations, and serve as a diagnostic tool in the metabolic engineering of methylotrophic bacteria.

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Effect of poly‐3‐hydroxybutyrate synthase mutation on the metabolism of Ensifer (formerly Sinorhizobium) meliloti

Povolo¹,

Casella²

2009

J. Basic Microbiol.

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In order to investigate the effect of poly-3-hydroxybutyrate synthase mutation (phbC) on the synthesis of exopolysaccharides (EPS) and glycogen, on the symbiotic properties and on the survival under specific conditions of Ensifer meliloti (formerly Sinorhizobium), a new stable phbC mutant of Ensifer meliloti 41 was isolated and characterized.Under poly-3-hydroxybutyrate accumulation conditions, the phbC -minus mutant (strain 41003) accumulates more glycogen and less exopolysaccharides as compared to the wild-type strain, and grows poorly in pyruvate as carbon source. The inactivation of aniA, encoding for a global carbon flux regulator, restores in E. meliloti 41003 the ability to grow on pyruvate, indicating a new role for this gene. Survival studies of E. meliloti 41 and 41003 under carbon free medium in both liquid and soil microcosms showed prolonged survival of E. meliloti 41 under these adverse conditions as compared to the mutant strain unable to accumulate the polyester. On the other hand, the accumulation of P(3HB) gave no significant advantage in survival under oxygen-limiting conditions. In both strains, E. meliloti 41 and 41003, nodule-inducing ability on alfalfa plants and acetylene reduction activity did not significantly differ from each other, although the mutant strain was less competitive in terms of root colonization.

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A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti

Cited by 42 publications

References 49 publications

Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using ¹³C‐label tracing and mass spectrometry

Effect of poly‐3‐hydroxybutyrate synthase mutation on the metabolism of Ensifer (formerly Sinorhizobium) meliloti

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A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti

Cited by 42 publications

References 49 publications

Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C‐label tracing and mass spectrometry

Effect of poly‐3‐hydroxybutyrate synthase mutation on the metabolism of Ensifer (formerly Sinorhizobium) meliloti

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Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using ¹³C‐label tracing and mass spectrometry