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Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae.

ABSTRACT: Glucose dehydrogenase (GDH) of Gram-negative bacteria is a membrane bound enzyme catalyzing the oxidation of glucose to gluconic acid and is involved in the solubilization of insoluble mineral phosphate complexes. A 2.4 kb glucose dehydrogenase gene (gcd) of Enterobacter asburiae sharing extensive homology to the gcd of other enterobacteriaceae members was cloned in a PCR-based directional genome walking approach and the expression confirmed in Escherichia coli YU423 on both MacConkey glucose agar and hydroxyapatite (HAP) containing media. Mineral phosphate solubilization by the cloned E. asburiae gcd was confirmed by the release of significant amount of phosphate in HAP containing liquid medium. gcd was over expressed in E. coli AT15 (gcd::cm) and the purified recombinant protein had a high affinity to glucose, and oxidized galactose and maltose with lower affinities.The enzyme was highly sensitive to heat and EDTA, and belonged to Type I, similar to GDH of E. coli.

SUBMITTER: Tripura C

PROVIDER: S-EPMC3450103 | biostudies-other | 2007 Jun

REPOSITORIES: biostudies-other

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Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae.

Tripura C C Sudhakar Reddy P P Reddy M K MK Sashidhar B B Podile A R AR

Indian journal of microbiology 20070601 2

Glucose dehydrogenase (GDH) of Gram-negative bacteria is a membrane bound enzyme catalyzing the oxidation of glucose to gluconic acid and is involved in the solubilization of insoluble mineral phosphate complexes. A 2.4 kb glucose dehydrogenase gene (gcd) of Enterobacter asburiae sharing extensive homology to the gcd of other enterobacteriaceae members was cloned in a PCR-based directional genome walking approach and the expression confirmed in Escherichia coli YU423 on both MacConkey glucose ag ...[more]

PMID: 23100654

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Project description:An in vitro study was conducted to assess the impact of organochlorine pesticides (OCPs) on cellular growth, morphology, cell viability, biofilm-formation activity, and growth-regulating substances of a soil bacterium. Phosphate-solubilizing EAM 35 isolated from rhizosphere soil was molecularly identified as Enterobacter cloacae (accession number MT672578.1). Strain EAM 35 tolerated varying levels of OCPs, viz., benzene hexachloride (BHC), chlorpyrifos (CP), dieldrin (DE), and endosulfan (ES). The toxicity of OCPs to strain EAM 35 was displayed in a concentration-dependent manner. Among the OCPs, ES at a concentration of 200 ?M showed a higher toxicity, where it maximally reduced the bacterial synthesis of indole-3-acetic acid (IAA), salicylic acid (SA), and 2,3-dihydroxy-benzoic acid (DHBA) by 73% (p ? 0.001), 85% (p ? 0.005), and 83% (p ? 0.001), respectively, over the control. While comparing the toxicity of OCPs to P-solubilizing activity of E. cloacae after 10 days of growth, the toxicity pattern followed the order ES (mean value = 82.6 ?g mL-1) > CP (mean value = 93.2 ?g mL-1) > DE (mean value = 113.6 ?g mL-1) > BHC (mean value = 127 ?g mL-1). Furthermore, OCP-induced surface morphological distortion in E. cloacae EAM 35 was observed as gaps, pits on both cellular facets, and fragmented and disorganized cell structure under a scanning electron microscope (SEM). The membrane-compromised cells increased as the concentrations of OC pesticides increased from 25 to 200 ?M. Additionally, microbial counts (log10 CFU/mL) were also affected after pesticide exposure and decreased with increasing concentrations. While assessing the impact of OCPs on inhibition (%) of log10 CFU/mL, 150, 175, and 200 ?M concentrations of ES completely reduced the growth of E. cloacae. Similarly, while comparing the toxicity of higher concentrations of OCPs to bacterial growth, sensitivity followed the order ES > DE > CP > BHC. In addition, the biofilm-formation ability of strain EAM 35 was inhibited in a pesticide-dose-dependent manner, and it was statistically (p ? 0.05, p ? 0.005, and p ? 0.001) significant. Conclusively, the present study clearly suggests that before applying pesticides to soil, their recommended dose should carefully be monitored.

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.

Dataset Information

Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae.

Publications

Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae.

Similar Datasets

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