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Transgenic expression of glucose dehydrogenase in Azotobacter vinelandii enhances mineral phosphate solubilization and growth of sorghum seedlings.

ABSTRACT: The enzyme quinoprotein glucose dehydrogenase (GDH) catalyses the oxidation of glucose to gluconic acid by direct oxidation in the periplasmic space of several Gram-negative bacteria. Acidification of the external environment with the release of gluconic acid contributes to the solubilization of the inorganic phosphate by biofertilizer strains of the phosphate-solubilizing bacteria. Glucose dehydrogenase (gcd) gene from Escherichia coli, and Azotobacter-specific glutamine synthetase (glnA) and phosphate transport system (pts) promoters were isolated using sequence-specific primers in a PCR-based approach. Escherichia coli gcd, cloned under the control of glnA and pts promoters, was mobilized into Azotobacter vinelandii AvOP and expressed. Sorghum seeds were bacterized with the transgenic azotobacters and raised in earthen pots in green house. The transgenic azotobacters, expressing E. coli gcd, showed improved biofertilizer potential in terms of mineral phosphate solubilization and plant growth-promoting activity with a small reduction in nitrogen fixation ability.

SUBMITTER: Sashidhar B

PROVIDER: S-EPMC3815912 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Transgenic expression of glucose dehydrogenase in Azotobacter vinelandii enhances mineral phosphate solubilization and growth of sorghum seedlings.

Sashidhar Burla B Podile Appa Rao AR

Microbial biotechnology 20090521 4

The enzyme quinoprotein glucose dehydrogenase (GDH) catalyses the oxidation of glucose to gluconic acid by direct oxidation in the periplasmic space of several Gram-negative bacteria. Acidification of the external environment with the release of gluconic acid contributes to the solubilization of the inorganic phosphate by biofertilizer strains of the phosphate-solubilizing bacteria. Glucose dehydrogenase (gcd) gene from Escherichia coli, and Azotobacter-specific glutamine synthetase (glnA) and p ...[more]

PMID: 21255283

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Project description:The plant growth-promoting Acinetobacter sp. SK2 isolated from Vigna radiata rhizosphere was characterized for mineral phosphate solubilization (MPS). To understand the contribution of the membrane glucose dehydrogenase (mGDH) and soluble glucose dehydrogenase (sGDH) in glucose oxidation and MPS, insertional inactivation of the corresponding genes was carried out. The disruption of mGDH encoding gene gdhA resulted in complete loss of mGDH activity, which confirmed its role in periplasmic glucose oxidation and gluconate-mediated MPS phenotype. The inactivation of sGDH encoding gene gdhB resulted in loss of sGDH activity, which did not alter the MPS or mGDH activity. Thus, it was also concluded that the sGDH was dispensable in gluconate-mediated MPS. Supplementation of succinate in glucose-containing medium suppressed the activity of mGDH (and sGDH) and therefore repressed the MPS phenotype. The catabolite repression control protein (Crc) of Pseudomonas was implicated in Acinetobacter sp. for a similar function in the presence of preferred and non-preferred carbon sources. To understand the regulatory linkage between Crc and genes for glucose oxidation, crc mutants were generated. The inactivation of crc resulted in increased activity of the mGDH in glucose + succinate-grown cells, indicating derepression. An increase in phosphate solubilization up to 44% in glucose + succinate-grown crc - compared with glucose-grown cells was recorded, which was significantly repressed in the wild-type strain under similar conditions. It is therefore proposed that in Acinetobacter sp. SK2, Crc is involved in the succinate-provoked repression of the MPS phenotype. The gene expression data indicated that Hfq may also have a regulating role in preferential utilization of carbon source by perhaps modulating Crc-Hfq functionality. V. radiata plants inoculated with the wild type improved both root and shoot length by 1.3 to 1.4-fold. However, crc - increased the root and shoot length by 1.6-fold, compared with the uninoculated controls. In mimicking the soil condition (in the presence of multiple carbon sources, e.g., succinate along with glucose), the crc - strain of Acinetobacter sp. SK2 performed better in supporting the growth of V. radiata in pot experiments.

Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Dataset Information

Transgenic expression of glucose dehydrogenase in Azotobacter vinelandii enhances mineral phosphate solubilization and growth of sorghum seedlings.

Publications

Transgenic expression of glucose dehydrogenase in Azotobacter vinelandii enhances mineral phosphate solubilization and growth of sorghum seedlings.

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