Proteomic study of the impact of Hik33 mutation in Synechocystis sp. PCC 6803 under normal and salt stress conditions.

Cyanobacteria are the only prokaryotes possessing plasma, thylakoid, and outer membranes. The plasma membrane of a cyanobacterial cell is essential for the biogenesis of cyanobacterial photosystems and serves as a barrier against environmental stress. We previously identified dozens of salt-responsive proteins in the plasma membrane of Synechocystis sp. PCC 6803. Five histidine kinases (Hiks) including Hik33 were also proposed to be involved in the perception of salt stress in Synechocystis. In this study, we analyzed proteomic profiles of the plasma membrane from a hik33-knockout mutant (ΔHik33) under normal and salt-stress conditions. Using 2D-DIGE followed by mass spectrometry analysis, we identified 26 differentially expressed proteins in ΔHik33 mutant cells. Major changes, due to the Hik33 mutation, included the substrate-binding proteins of ABC transporters, such as GgtB and FutA1, regulatory proteins including MorR and Rre13, as well as several hypothetical proteins. Under salt-stress conditions, the Hik33 mutation reduced levels of 7 additional proteins, such as NrtA, nitrate/sulfonate/bicarbonate-binding protein and LexA, and enhanced levels of 9 additional proteins including SphX. These observations suggest a substantial rearrangement in the plasma membrane proteome of Synechocystis due to the loss of hik33. Furthermore, a comprehensive molecular network was revealed in ΔHik33 mutant coping with salt stress.

Cd-induced changes in leaf proteome of the hyperaccumulator plant Phytolacca americana.

Cadmium (Cd) is highly toxic to all organisms. Soil contamination by Cd has become an increasing problem worldwide due to the intensive use of Cd-containing phosphate fertilizers and industrial zinc mining. Phytolacca americana L. is a Cd hyperaccumulator plant that can grow in Cd-polluted areas. However, the molecular basis for its remarkable Cd resistance is not known. In this study, the effects of Cd exposure on protein expression patterns in P.americana was investigated by 2-dimensional gel electrophoresis (2-DE). 2-DE profiles of leaf proteins from both control and Cd-treated (400µM, 48h) seedlings were compared quantitatively using ImageMaster software. In total, 32 differentially expressed protein spots were identified using MALDI-TOF/TOF mass spectrometry coupled to protein database search, corresponding to 25 unique gene products. Of those 14 were enhanced/induced while 11 reduced under Cd treatment. The alteration pattern of protein expression was verified for several key proteins involved in distinct metabolic pathways by immuno-blot analysis. Major changes were found for the proteins involved in photosynthetic pathways as well as in the sulfur- and GSH-related metabolisms. One-third of the up-regulated proteins were attributed to transcription, translation and molecular chaperones including a protein belonging to the calreticulin family. Other proteins include antioxidative enzymes such as 2-cys-peroxidase and oxidoreductases. The results of this proteomic analysis provide the first and primary information regarding the molecular basis of Cd hypertolerance in P. americana.

Proteomic analysis of plasma membranes of cyanobacterium Synechocystis sp. Strain PCC 6803 in response to high pH stress.

Cyanobacteria are unique prokaryotes possessing plasma-, outer- and thylakoid membranes. The plasma membrane of a cyanobacterial cell serves as a crucial barrier against its environment and is essential for biogenesis of cyanobacterial photosystems. Previously, we have identified 79 different proteins in the plasma membrane of Synechocystis sp. Strain PCC 6803 based on 2D- and 1D- gels and MALDI-TOF MS. In this work, we have performed a proteomic study screening for high-pH-stress proteins in Synechocystis. 2-D gel profiles of plasma membranes isolated from both control and high pH-treated cells were constructed and compared quantitatively based on different protein staining methods including DIGE analysis. A total of 55 differentially expressed protein spots were identified using MALDI-TOF MS and MALDI-TOF/TOF MS, corresponding to 39 gene products. Twenty-five proteins were enhanced/induced and 14 reduced by high pH. One-third of the enhanced/induced proteins were transport and binding proteins of ABC transporters including 3 phosphate transport proteins. Other proteins include MinD involved in cell division, Cya2 in signaling and proteins involved in photosynthesis and respiration. Furthermore, among these proteins regulated by high pH, eight were found to be hypothetical proteins. Functional significance of the high-pH-stress proteins is discussed integrating current knowledge on cyanobacterial cell physiology.

Characterization of two glutaminases from the filamentous cyanobacterium Anabaena sp. PCC 7120.

The Anabaena genome contains two ORFs that appear to encode glutaminases. The genes were expressed as histidine-tagged fusion proteins in Escherichia coli. The purified proteins possessed glutaminase activity using l-glutamine as the substrate, but differed in biochemical properties. All2934 showed an optimal activity at 20 degrees C and pH 6.0, with a higher affinity for l-glutamine than All4774, which had optimal activity at 37 degrees C and pH 7.5. Remarkably, the glutaminase activity of All2934 was phosphate dependent, while All4774 was phosphate independent. The expression of all2934 and all4774 was analyzed using semi-quantitative reverse transcriptase-PCR. The expression level of all2934 was much higher than that of all4774 under normal and nitrogen-depletion conditions, indicating that All2934 may play an important role in metabolizing glutamine in Anabaena.

[Improvement of the thermostability of xylanase by N-terminus replacement].

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNB's after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.

[Hydrophobic interaction between beta-sheet B1 and B2 in xylanase XYNB influencing the enzyme thermostability].

A homology modeling of xylanase XYNB from Streptomyces olivaceoviridis A1 was made by Swiss-Model. The hydrophobic Interaction between beta-sheet B1 and B2 in the tertiary structure model of XYNB was compared with other thermophilic xylanase. A T11Y mutation was introduced in XYNB by site-dirrected mutagenesis to improve the thermostability of the enzyme. The XYNB and mutant xylanase (XYNB') expressed in Pichia pastoris were purified and their enzymatic properties were determined. The result revealed that the thermostability of XYNB' was obviously higher than that of XYNB. The optimal temperature of XYNB' for its activity was 60 degrees C, similar to XYNB. But, compare to XYNB, the optimal pH value, the Km value and the specific activity of XYNB' had also been changed. The research results suggested that the aromatic interaction between beta-sheet B1 and B2 in xylanase should increase enzyme thermostability. The mutant xylanase XYNB' is a good material for further research in the relationship between structure and function of xylanase.

[Recent advances in structures and relative enzyme properties of xylanase].

Xylanase can hydrolyze xylans into xylooligosaccharides and D-xylose, and has great prospect for applications in feed industry, paper and pulp industry, food industry and environment science. The study of xylanase had been started in 1960's. With the development and application of the new technologies, such as molecular biology, structural biology and protein engineering, many progresses have been made in the research of structures and functions of xylanase. This paper reviews the research progress and trend in the structure correlating with the important properties of xylanase. Analyses of three-dimensional structures and properties of mutants have revealed that glutamine and aspartic acid residues are involved in the catalytic mechanism. The thermostability of xylanase correlated with many factors, such as disulfide bridges, salt bridges, aromatic interactions, cotent of arginine and proline, and some multidomain xylanase have thermostability domains in N or C terminal. But no single mechanism is responsible for the remarkable stability of xylanase. The isoelectic points and reaction pH of xylanase are influenced by hydrophobicity and content of electric charges. Many researches had demonstrated that aromatic amino acid, histidine, and tryptophan play an important role in improving enzyme-substrate affinity. The researches of structures and functions of xylanase are of great significance in understanding the catalytic mechanism and directing the improvement of xylanase properties to meet the application requirement.