Organelle Transcriptomes in Plants

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Interaction of a putative transcriptional regulatory protein and the thermo-inducible cts-52 mutant repressor in the Bacillus subtilis phage φ105 genome

A 144 amino acid residue cts-52 mutant repressor (mtcφ105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage φ105 is involved in the control mechanism of a thermo-inducible expression system. Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor. On the basis of the protein sequence alignment, a DNA-binding α helix-β turn-α helix (HTH) motif was identified in the N-terminal region (residues 18-37) of the repressor as well as in the polypeptide of ORF4 (residues 22-41). In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. To study their DNA binding properties, the wild-type repressor (wtcφ105) and the mutant repressor mtcφ105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays. Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR). Our data showed that a temperature shift does not influence the wtcφ105-operators' binding affinity, while the binding of mtcφ105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtcφ105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures. mRNA levels monitored by real-time RT-PCR indicated a suppression of mtcφ105 expression, but a stimulation of ORF4 transcription after thermo-induction. Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P M and P R within the immF region. © 2003 Elsevier Ltd. All rights reserved.postprin

Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the Φ105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production. © 2003 Elsevier Inc. All rights reserved.postprin

Properties of beta-propeller phytase expressed in transgenic tobacco

Phytases are enzymes that liberate inorganic phosphates from phytate. In a previous study, a beta-propeller phytase (168phyA) from Bacillus subtilis was introduced into transgenic tobacco, which resulted in certain phenotypic changes. In the study described herein, the recombinant phytase (t168phyA) was purified from transgenic tobacco to near homogeneity by a three-step purification scheme. The biochemical properties and kinetic parameters of t168phyA were compared with those of its counterpart from B. subtilis. t168phyA was glycosylated, and it showed a 4 kDa increase in molecular size in SDS-PAGE (44 kDa vs. 40 kDa). Although its thermostability remained unchanged, its temperature optimum shifted from 60°C to 45-50°C and its pH optimum shifted from pH 5.5 to 6.0. Kinetic data showed that the t168phyA had a lower Kcat, but a higher Km than the native enzyme. Despite these changes, t168phyA remained catalytically active and has a specific activity of 2.3 U/mg protein. These results verify the activity of recombinant Bacillus phytase that is expressed in plants. © 2005 Elsevier Inc. All rights reserved.postprin

Isolation of leptin-binding peptides from a random peptide phage library

Leptin plays a role in regulating the body weight in mice. Injection of recombinant mouse leptin expressed in Escherichia coli reduced the food intake and body weight in normal, ob/ob and diet-induced obesity mice. Hyperglycemia, hyperinsulinemia and hypothermia can also be corrected in ob/ob mice after leptin injection. Leptin is a 16-kDa secretory protein comprising 167 amino acids produced in adipose tissue and is secreted to blood stream. In this study, a recombinant mouse leptin was generated and purified from a baculovirus expression system. This protein was used to identify putative ligands using a phage library of random peptides. Three leptin-binding phage clones were found, which were characterized by DNA sequencing and ELISA methods. The amino acid sequences of the reactive peptides are: LAYCSDPVRCLVWWY, MFWlSAVSFVDHALV and LVLVLSAFLCCGVG. All three clones bound to recombinant human and mouse leptins. These peptides may be useful tools to study leptin-receptor interaction, food intake and body weight regulation.postprin

Pathway of phytate dephosphorylation by β-propeller phytases of different origins

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by the P-propeller phytase of Shewanella oneidensis was established, which was then compared with that of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 β-propeller phytases. The data demonstrate that all of these β-propeller phytases dephosphorylate myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P 5, Ins(2,4,5,6)P 4 to finally Ins(2,4,6)P 3. Thus, the β-propeller phytases prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does not support previous phytate degradation models proposed by J. Kerovuo, J. Rouvinen, and F. Hatzack (2000. Biochem. J. 352: 623-628) and R. Greiner, A. Farouk, M. Larsson Alminger, and N.G. Carlsson (2002. Can. J. Microbiol. 48: 986-994), but seems to fit with the structural model given by S. Shin, N.C. Ha, B.C. Oh, T.K. Oh, and B.H. Oh (2001. Structure, 9: 851-858). © 2007 NRC.published_or_final_versio

Hydrolysis of precipitated phytate by three distinct families of phytases

While genetically modified plants that secrete histidine acid phosphatases (HAPs), β-propeller phytases (BPPs) and purple acid phosphatases (PAPs) have been shown to assimilate soluble phytate, little is known about whether these plants have the ability to hydrolyze precipitated phytate. In this study, the ability of representative members of these three classes of phytases to hydrolyze metal-phytate salts and to hydrolyze phytate adsorbed to aluminum precipitates was compared. All three phytases were able to hydrolyze Ca 2+-, Mg 2+-, and Mn 2+-phytates, but were unable to hydrolyze Al 3+-, Fe 2+-, Fe 3+-, Cu 2+-, and Zn 2+-phytates. When these ions were present, the hydrolysis of Ca 2+-phytate was prevented. Citrate was more potent than malate and oxalate in solubilizing some of these phytate salts for enzyme hydrolysis. Phytate adsorbed to aluminum precipitates was resistant to all three enzymes, except when organic acids were added (citrate>oxalate>malate). While increasing concentrations of organic acids were inhibitory to enzyme activity (oxalate >citrate>malate), PAP was more resistant to citrate than HAP. As desorption of phytate from a solid surface by organic acids is essential for phytase activity, the genetic engineering of plants that enhances the secretion of both citrate and phytases from the root may be a feasible approach to improving soil phytate assimilation. © 2005 Elsevier Ltd. All rights reserved.postprin

RNA editing of cytochrome c maturation transcripts is responsive to the energy status of leaf cells in Arabidopsis thaliana

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De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing

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MESSI: metabolic engineering target selection and best strain identification tool

Metabolic engineering and synthetic biology are synergistically related fields for manipulating target pathways and designing microorganisms that can act as chemical factories. Saccharomyces cerevisiae’s ideal bioprocessing traits make yeast a very attractive chemical factory for production of fuels, pharmaceuticals, nutraceuticals as well as a wide range of chemicals. However, future attempts of engineering S. cerevisiae’s metabolism using synthetic biology need to move towards more integrative models that incorporate the high connectivity of metabolic pathways and regulatory processes and the interactions in genetic elements across those pathways and processes. To contribute in this direction, we have developed Metabolic Engineering target Selection and best Strain Identification tool (MESSI), a web server for predicting efficient chassis and regulatory components for yeast bio-based production. The server provides an integrative platform for users to analyse ready-to-use public high-throughput metabolomic data, which are transformed to metabolic pathway activities for identifying the most efficient S. cerevisiae strain for the production of a compound of interest. As input MESSI accepts metabolite KEGG IDs or pathway names. MESSI outputs a ranked list of S. cerevisiae strains based on aggregation algorithms. Furthermore, through a genome-wide association study of the metabolic pathway activities with the strains’ natural variation, MESSI prioritizes genes and small variants as potential regulatory points and promising metabolic engineering targets. Users can choose various parameters in the whole process such as (i) weight and expectation of each metabolic pathway activity in the final ranking of the strains, (ii) Weighted AddScore Fuse or Weighted Borda Fuse aggregation algorithm, (iii) type of variants to be included, (iv) variant sets in different biological levels. Database URL: http://sbb.hku.hk/MESSI