Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone.
ABSTRACT: Escherichia coli is capable of synthesizing the apo-glucose dehydrogenase enzyme (GDH) but not the cofactor pyrroloquinoline quinone (PQQ), which is essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, E. coli does not produce gluconic acid. Evidence is presented to show that the expression of an Erwinia herbicola gene in E. coli HB101(pMCG898) resulted in the production of gluconic acid, which, in turn, implied PQQ biosynthesis. Transposon mutagenesis showed that the essential gene or locus was within a 1.8-kb region of a 4.5-kb insert of the plasmid pMCG898. This 1.8-kb region contained only one apparent open reading frame. In this paper, we present the nucleotide sequence of this open reading frame, a 1,134-bp DNA fragment coding for a protein with an M(r) of 42,160. The deduced sequence of this protein had a high degree of homology with that of gene III (M(r), 43,600) of a PQQ synthase gene complex from Acinetobacter calcoaceticus previously identified by Goosen et al. (J. Bacteriol. 171:447-455, 1989). In minicell analysis, pMCG898 encoded a protein with an M(r) of 41,000. These data indicate that E. coli HB101(pMCG898) produced the GDH-PQQ holoenzyme, which, in turn, catalyzed the oxidation of glucose to gluconic acid in the periplasmic space. As a result of the gluconic acid production, E. coli HB101(pMCG898) showed an enhanced mineral phosphate-solubilizing phenotype due to acid dissolution of the hydroxyapatite substrate.
Project description:We have recently shown that the ability of some gram-negative bacteria to dissolve poorly soluble calcium phosphates (Mps+ phenotype) is the result of periplasmic oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase (GDH), a component of the direct oxidation pathway. Escherichia coli K-12 derivatives synthesize apo-GDH but not the cofactor pyrroloquinoline-quinone (PQQ) essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, these strains do not produce gluconic acid and are Mps-. Evidence is presented to show that expression of a single 396-base Pseudomonas cepacia open reading frame (designated gabY) in E. coli JM109 (a K-12 derivative) was sufficient to induce the Mps+ phenotype and production of gluconic acid. We present the nucleotide sequence of this open reading frame which coded for a protein (GabY) with a deduced M(r) of 14,235. Coupled transcription-translation of a plasmid (pSLY4 or pGAB1) carrying gabY resulted in production of a protein with an M(r) of 14,750. Disruption of the open reading frame of gabY via site-directed mutagenesis changed the phenotype to Mps- and eliminated gluconic acid production. The deduced amino acid sequence of gabY has no apparent homology with those of previously cloned direct oxidation pathway genes but does share regions highly homologous with the histidine permease system membrane-bound protein HisQ as well as other proteins in this family. In the presence of 1 microM exogenous PQQ, both JM109(pSLY4) and JM109(pGAB1) produced 10 times as much gluconic acid as was seen with either the plasmid or exogenous PQQ alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Project description:Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator.Plant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.
Project description:A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.
Project description: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.
Project description:Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg(2+). Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis-Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for D-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (k(cat)), which were determined to be 2.2x10(3) and 6.8+/-0.8x10(3) s(-1) (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (k(f,PQQ)) and with Mg(2+) (k(f,Mg)) were determined to be 3.8+/-0.4x10(4) M(-1).s(-1) and 4. 1+/-0.9 M(-1).s(-1) respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg(2+) were determined as the dissociation constants K(d,PQQ(Mg)) and K(d,Mg) to be 1.0+/-0.1 nM and 0.14+/-0.01 mM respectively. The dissociation constants for Ca(2+) were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg(2+) in solution. EDTA was effective to remove Mg(2+) from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.
Project description:Rahnella aquatilis HX2, a biocontrol agent for grapevine crown gall caused by Agrobacterium vitis, produces an antibacterial substance that inhibits the growth of A. vitis in vitro. In this study, we show that MH15 and MH16, two Tn5-induced mutants of HX2, have lost their abilities to inhibit A. vitis and have reduced biocontrol activities; they grow in logarithmic phase at a rate similar to that of the wild type and have single Tn5 insertions. They are also impaired in producing pyrroloquinoline quinone (PQQ) or glucose dehydrogenase (GDH). Complementation of MH15 and MH16 with cosmid clones of CP465 and CP104 from an HX2 DNA library restored the antibiosis, biocontrol, and PQQ or GDH production phenotypes. A 6.7-kb BamHI fragment from CP465 that fully restored the MH15-affected phenotypes was cloned and sequenced. Sequence analysis of the mutated DNA region resulted in the identification of seven open reading frames (ORFs), six of which share significant homology with PQQ-synthesizing genes in other bacteria, designated pqqA through pqqF. Meanwhile, A 5.5-kb PstI fragment from CP104 fully complemented the MH16 mutant and contained a single ORF highly similar to that of genes coding for GDHs. An in-frame gdh deletion mutant has the same phenotypes as the Tn5 mutant of MH16. Complementation of both deletion and Tn5 gdh mutants restored the affected phenotypes to wild-type levels. Our results suggest that an antibacterial substance plays a role in biocontrol of A. vitis by HX2.
Project description: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.
Project description:Homeostatic interactions between the host and its resident microbiota is important for normal physiological functions and if altered, it could lead to dysbiosis, a change in the structure and function of the microbiota, and as a result to various pathophysiologies. Altered structure in bacterial community is associated with various pathophysiologies, but we are just beginning to understand how these structural changes translate into functional changes. Environmental factors including pathogenic infections can lead to altered interactions between the host and its resident microbiota. We used microarray analysis and a C. elegans model system to gain insights on the mechanisms of functional changes in host-commensal bacteria interaction in the presence or absence of G. duodenalis and identified expression pattern in commensal bacteria that are characteristic of homeostatic and dysbiotic interactions. E. coli HB101 exposed to C. elegans in the presence or absence of G. duodenalis conditioned S-basal complete media for 24 hours were used for RNA extraction and hybridization on Affymetrix microarrays. We collected expression data for E. coli HB101, E. coli HB101 exposed to C. elegans, E. coli HB101 exposed to Giardia conditioned media, and E. coli HB101 exposed to both C. elegans and Giardia conditioned media.
Project description:Aneurinibacillus aneurinilyticus strain CKMV1 was isolated from rhizosphere of Valeriana jatamansi and possessed multiple plant growth promoting traits like production of phosphate solubilization (260mg/L), nitrogen fixation (202.91nmolethylenemL-1h-1), indole-3-acetic acid (IAA) (8.1μg/mL), siderophores (61.60%), HCN (hydrogen cyanide) production and antifungal activity. We investigated the ability of isolate CKMV1 to solubilize insoluble P via mechanism of organic acid production. High-performance liquid chromatography (HPLC) study showed that isolate CKMV1 produced mainly gluconic (1.34%) and oxalic acids. However, genetic evidences for nitrogen fixation and phosphate solubilization by organic acid production have been reported first time for A. aneurinilyticus strain CKMV1. A unique combination of glucose dehydrogenase (gdh) gene and pyrroloquinoline quinone synthase (pqq) gene, a cofactor of gdh involved in phosphate solubilization has been elucidated. Nitrogenase (nif H) gene for nitrogen fixation was reported from A. aneurinilyticus. It was notable that isolate CKMV1 exhibited highest antifungal against Sclerotium rolfsii (93.58%) followed by Fusarium oxysporum (64.3%), Dematophora necatrix (52.71%), Rhizoctonia solani (91.58%), Alternaria sp. (71.08%) and Phytophthora sp. (71.37%). Remarkable increase was observed in seed germination (27.07%), shoot length (42.33%), root length (52.6%), shoot dry weight (62.01%) and root dry weight (45.7%) along with NPK (0.74, 0.36, 1.82%) content of tomato under net house condition. Isolate CKMV1 possessed traits related to plant growth promotion, therefore, could be a potential candidate for the development of biofertiliser or biocontrol agent and this is the first study to include the Aneurinibacillus as PGPR.
Project description:AIMS:To assess protective efficacy of genetically modified Escherichia coli Nissle 1917 (EcN) on metabolic effects induced by chronic consumption of dietary fructose. MATERIALS AND METHODS:EcN was genetically modified with fructose dehydrogenase (fdh) gene for conversion of fructose to 5-keto-D-fructose and mannitol-2-dehydrogenase (mtlK) gene for conversion to mannitol, a prebiotic. Charles foster rats weighing 150-200 g were fed with 20% fructose in drinking water for two months. Probiotic treatment of EcN (pqq), EcN (pqq-glf-mtlK), EcN (pqq-fdh) was given once per week 109 cells for two months. Furthermore, blood and liver parameters for oxidative stress, dyslipidemia and hyperglycemia were estimated. Fecal samples were collected to determine the production of short chain fatty acids and pyrroloquinoline quinone (PQQ) production. RESULTS:EcN (pqq-glf-mtlK), EcN (pqq-fdh) transformants were confirmed by restriction digestion and functionality was checked by PQQ estimation and HPLC analysis. There was significant increase in body weight, serum glucose, liver injury markers, lipid profile in serum and liver, and decrease in antioxidant enzyme activity in high-fructose-fed rats. However the rats treated with EcN (pqq-glf-mtlK) and EcN (pqq-fdh) showed significant reduction in lipid peroxidation along with increase in serum and hepatic antioxidant enzyme activities. Restoration of liver injury marker enzymes was also seen. Increase in short chain fatty acids (SCFA) demonstrated the prebiotic effects of mannitol and gluconic acid. CONCLUSIONS:Our study demonstrated the effectiveness of probiotic EcN producing PQQ and fructose metabolizing enzymes against the fructose induced hepatic steatosis suggesting that its potential for use in treating fructose induced metabolic syndrome.