Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.     

Enhancement of alkaline phytase production in Pichia pastoris: influence of gene dosage, sequence optimization and expression temperature

Supplementation of animal feed with phytases has proven to be an effective strategy to alleviate phosphorous contamination of soil and water bodies. The inability of non-ruminant animals to digest phytates in corn and soybeans contributes to environmental contamination. Alkaline phytase from lily pollen (LlALP) exhibits unique catalytic and thermal stability properties that could be useful as a feed supplement. rLlALP2 was successfully expressed in Pichia pastoris; however, enzyme yields were modest (8-10 mg/L). In this paper, we describe our efforts to enhance rLlALP2 yield by investigating the influence of the following potential limiting factors: transgene copy number, codon bias, sequence optimization, and temperature during expression. Data presented indicate that increasing rLlAlp2 copy number was detrimental to heterologous expression, clones with one copy of wt-rLlAlp2 produced the highest activity, clones with two, four and seven or more copies produced 70%, 25% and 10% respectively, of enzyme activity implying that gene dosage is not limiting rLlALP2 yield. Use of a sequence-optimized rLlAlp2 increased the yield of the active enzyme by 25-50% in one/two copy clones, suggesting that translational efficiency is not a major bottleneck for rLlALP2 expression. Reducing the temperature during heterologous expression led to increases of 1.2-20-fold suggesting that protein folding and post-translational processes may be the dominant factors limiting rLlALP2 expression. Early knowledge of the transgene copy number allowed us to develop a more rational strategy for yield enhancement. Cumulatively, sequence optimization and temperature reduction led to the doubling of rLlALP2 enzyme activity in P. pastoris.     

SPR and ITC determination of the kinetics and the thermodynamics of bivalent versus monovalent sugar ligand–lectin interactions

A kinetic study of the interaction of bivalent and monovalent sugar ligands with a lectin was undertaken with the aid of surface plasmon resonance (SPR) method. The study involved a series of bivalent α-d-mannopyranoside containing sugar ligands, with systematic variation in the distance between the sugar ligands. The detailed kinetic studies showed that bivalent ligands underwent a faster association (k _on) and a slower dissociation (k _off) of the ligand–lectin complexes, in comparison to the monovalent ligand–lectin complexes. The kinetic constants were complemented further by assessing the thermodynamic parameters with the aid of isothermal titration calorimetry (ITC). The initiation of cross-linking of ligand–lectin interactions emerge from the early stages of the complexation. The dynamic light scattering (DLS) and the transmission electron microscopy (TEM) techniques allowed judging the sizes and morphologies of the complex in the solution and solid states, respectively.     

Establishment of adventitious root co-culture of Ginseng and Echinacea for the production of secondary metabolites

We have successfully established the co-culture of ginseng (Panax ginseng C.A. Meyer) and echinacea [Echiancea purpurea (L.) Moench.] adventitious roots for the production secondary metabolites. Adventitious roots of ginseng and echinacea were cultured in different proportions (5 g L⁻¹; 4:1, 3:2 and 2:1 ginseng and echinacea, respectively) in 5-L capacity airlift bioreactors containing 4 L Murashige and Skoog medium supplemented with 25 μM indole-3-butyric acid and 50 g sucrose L⁻¹ and maintained at 25°C in the dark for 40 days. Results showed the negative effect of echinacea adventitious roots on the growth of ginseng roots, however, by limiting the inoculum density of echinacea, it was possible to establish the co-cultures. To enhance the accumulation of secondary metabolites, co-cultures were treated with 200 μM methyl jasmonate after 30 days of culture initiation. Methyl jasmonate elicitation promoted the accumulation of ginsenosides in the co-cultures. It was possible to produce ginsenosides and caffeic acid derivatives in higher amounts by establishing co-cultures with higher inoculum proportion of ginseng to echinacea (4:1 and 3:2) followed by elicitation treatment. This work demonstrates the effectiveness of interspecies adventitious root co-cultures for the production of plant secondary metabolites.     

Micropropagation of Salix pseudolasiogyne from nodal explants

The effect of phytohormones on the breaking of dormancy of axillary buds in Salix pseudolasiogyne and their subsequent proliferation from nodal explants were examined. Nodal explants obtained from a 20–year-old S. pseudolasiogyne tree were cultured either on woody plant basal medium (WPM) or WPM supplemented with benzyladenine (BA, 2.2/4.4 μM), zeatin (1.1/2.2 μM), gibberillic acid (GA₃, 2.9 and 14.5 μM), and GA₃ + BA (2.9 + 4.4 μM). Although axillary shoots developed in all the media, a higher percentage bud break occurred on BA supplemented media. To corroborate the results, endogenous levels of cytokinins [Cks, N ⁶-isopentenyladenine (iP), zeatin riboside (t-ZR), dihydrozeatinriboside (DHZR)] and abscisic acid (ABA) were determined. On BA supplemented media, the levels of zeatin type (Z-type) of Cks were higher than those of isopentenyladenine type of Ck in the explants, while the ABA level was low. Axillary shoots did not grow well and became necrotic upon subculture to fresh basal WPM. In order to improve shoot growth, they were subcultured twice at a 4-week interval on to WPM supplemented with BA (2.2/4.4 μM), GA₃ (1.4 μM), or GA₃ + BA (1.4 + 4.4/2.9 + 4.4 μM). Maximal shoot growth (93%) was achieved on WPM supplemented with 2.2 μM BA. Comparative analyses of endogenous Cks revealed that higher Cks (Z-type Cks) were present in actively growing shoots. Rooting was readily achieved when the shoots were subcultured to WPM without phytohormones. The rooted plants were acclimatized well upon transplantation.     

Silicon nanopillar substrates for enhancing signal intensity in DNA microarrays

The use of ordered, high-aspect ratio nanopillar arrays on the surface of silicon-based chips to enhance signal intensity in DNA microarrays is reported. These nanopillars consisting either of a single silicon dioxide substrate or a dual silicon/silicon dioxide substrate are fabricated using deep-UV lithography followed by reactive ion etching. These pillar type arrays provide a three-dimensional high surface-density platform that increases the immobilization capacity of captured probes, enhances target accessibility and reduces background noise interference in DNA microarrays, leading to improved signal-to-noise ratios, sensitivity and specificity. Consequently, it was found that the use of such nanopillars enhanced the hybridization signals by up to seven times as compared to silicon dioxide thin film substrates. In addition, hybridization of synthetic targets to capture probes that contained a single-base variation showed that the perfect matched duplex signals on dual-substrate nanopillars can be up to 23 times higher than the mismatched duplex signals, allowing the targets to be unambiguously identified. These results suggest that the nanopillars, particularly the dual-substrate pillars, are able to enhance the hybridization signals and discrimination power in nucleic acids-based detection, providing an alternative platform for improving the performance of DNA microarrays.     

Effects of salt stress on basic processes of photosynthesis

Salt stress causes decrease in plant growth and productivity by disrupting physiological processes, especially photosynthesis. The accumulation of intracellular sodium ions at salt stress changes the ratio of K : Na, which seems to affect the bioenergetic processes of photosynthesis. Both multiple inhibitory effects of salt stress on photosynthesis and possible salt stress tolerance mechanisms in cyanobacteria and plants are reviewed.This revised version was published online in March 2005 with corrections to the page numbers.