A novel metabolic network leads to enhanced citrate biogenesis in Pseudomonas fluorescens exposed to aluminum toxicity

Mailloux, Ryan J.; Lemire, Joseph; Kalyuzhnyi, S.; Appanna, Vasu D.

doi:10.1007/s00792-008-0150-1

Cited by 37 publications

(22 citation statements)

References 38 publications

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“…Blue native polyacrylamide gel electrophoresis (BN-PAGE) and in-gel activity staining were performed as described previously (17). Membrane proteins were prepared in a native buffer (50 mM bis-Tris, 500 mM ε-aminocaproic acid [pH 7.0]) containing 1% maltoside at a final concentration of 4 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

et al. 2009

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␣-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH 4 ) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media.13 C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.All aerobic organisms have to contend with the dangers associated with reactive oxygen species (ROS), toxic moieties that are routinely generated as a consequence of ATP production via oxidative phosphorylation (34). The transfer of electrons from NADH and reduced flavin adenine dinucleotide to oxygen is mediated by the respiratory complexes, the major sites of intracellular ROS generation (1). These by-products of oxidative phosphorylation are very harmful and have to be nullified if organisms are to survive in an aerobic environment (24). If left unchecked, ROS can damage biological macromolecules, leading to the demise of the cell. Hence, it is not surprising that all aerobic organisms have devised intricate antioxidative defense strategies in an effort to proliferate in the presence of oxygen.Enzymes such as superoxide dismutase and catalase are uniquely bestowed with the task of eliminating superoxide and hydrogen peroxide, two important ROS (11, 12, 21). Glutathione (GSH), a tripeptide, also plays a pivotal role in the detoxification of ROS (22). However, to be effective, all these ROS disposal processes have to be regenerated with the aid of NADPH. This nicotinamide dinucleotide is the main power behind all antioxidative defense strategies, as it provides the reducing fuel necessary to recharge all effectors involved in combating ROS (32). Thus, various enzyme systems and metabolic networks that orchestrate the biogenesis of NADPH have to be activated if an organism is to...

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Section: Methodsmentioning

confidence: 99%

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

et al. 2009

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“…fluorescens ATCC 13525 exhibited enhanced citric acid biosynthesis to overcome aluminium toxicity when grown on malate as the carbon source, by employing a unique metabolic shift involving decreased activities of enzymes utilizing oxaloacetate and citrate (phosphoenolpyruvate carboxykinase, aconitase and ICDH) with a parallel increase in activities of enzymes involved in oxaloacetate and citrate biosynthesis: malic enzyme, malate dehydrogenase and CS (Mailloux et al, 2008). On the other hand, phosphate-solubilization requires~5-10 mM citric acid to be secreted by fluorescent pseudomonads utilizing sugars.…”

Section: Discussionmentioning

confidence: 99%

“…and Citrobacter koseri have been reported to secrete citric acid along with various other organic acids (Gyaneshwar et al, 1998). Apart from mineral phosphate solubilization, citric acid secretion by PSB could also be implicated in mediating aluminium tolerance, as in Pseudomonas fluorescens ATCC 13525 (Mailloux et al, 2008), and as a siderophore, as in Bradyrhizobium and Pseudomonas aeruginosa (Guerinot et al, 1990;Carson et al, 1992;Marshall et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene

Buch

Archana

2009

Microbiology

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Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant~2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2-and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3-and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525. Significantly low extracellular citrate levels as compared to the intracellular levels in Pf(pAB7) suggested a probable limitation of efficient citrate transport.

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“…Beberapa spesies dalam genus ini juga sitrat (Buch et al 2008) yang dikaitkan dengan toleransi bakteri ini terhadap cekaman Al (Mailloux et al 2008;Lemire et al 2010). Hal ini menjadi alasan para peneliti mengisolasi dan mengekspresikan gen sitrat sintase Pseudomonas ke dalam tanaman atau khamir (de la Fuente et al 1997;Baron et al 2008).…”

Section: Pendahuluanunclassified

Introduksi Gen Sitrat Sintase Pseudomonas aeruginosa ke Nicotiana tabacum dan Jatropha curcas untuk Meningkatkan Toleransi terhadap Cekaman Aluminium

Tistama

Widyastuti

Suharsono

2017

BAF

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Cekaman aluminium merupakan faktor penghalang utama untuk pengembangan komoditas tanaman unggulan di lahan-lahan masam. Sintesis dan sekresi asam organik oleh akar merupakan salah satu mekanisme mengurangi cekaman aluminium. Penelitian ini bertujuan mengintroduksikan gen sitrat sintase bakteri P. aeruginosa (PaCS) ke dalam tanaman tembakau (N. tabacum) dan Jatropha curcas. PaCS berhasil diintroduksi dengan batuan Agrobacterium tumefaciens baik ke dalam jaringan tembakau maupun J. curcas.. Toleransi tembakau transgenik ditandai dengan pertambahan panjang akar yang lebih tinggi dibanding tipe liarnya pada cekaman 0,3 mM AlCl3. Toleransi tembakau transgenik dapat mengurangi masuknya Al ke dalam jaringan akar. Sifat toleran gen PaCS diturunkan mengikuti Hukum Mendel. Persentase introduksi gen PaCS ke dalam J. curcas hanya 1% dari jumlah eksplan yang ditranformasi. Kata kunci : introduksi gen, sitrat sintase, cekaman aluminium, N. tabacum, J. curcas

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A novel metabolic network leads to enhanced citrate biogenesis in Pseudomonas fluorescens exposed to aluminum toxicity

Cited by 37 publications

References 38 publications

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene

Introduksi Gen Sitrat Sintase Pseudomonas aeruginosa ke Nicotiana tabacum dan Jatropha curcas untuk Meningkatkan Toleransi terhadap Cekaman Aluminium

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