The quantitative proteome atlas of a model cyanobacterium.

Cyanobacteria are a group of oxygenic photosynthetic bacteria with great potentials in biotechnological applications and advantages as models for photosynthesis research. The subcellular localizations of the majority of proteins in any cyanobacteria remain undetermined, representing a major challenge in using cyanobacteria for both basic and industrial researches. Here, using label-free quantitative proteomics, we map 2027 proteins of Synechocystis sp. PCC6803, a model cyanobacterium, to different subcellular compartments and generate a proteome atlas with such information. The atlas leads to numerous unexpected but important findings, including the predominant localization of the histidine kinases Hik33 and Hik27 on the thylakoid but not the plasma membrane. Such information completely changes the concept regarding how the two kinases are activated. Together, the atlas provides subcellular localization information for nearly 60% proteome of a model cyanobacterium, and will serve as an important resource for the cyanobacterial research community.

Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications.

BACKGROUND: Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry. RESULTS: In this study, a novel glucoamylase-encoding gene TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 â„ƒ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 â„ƒ. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge-charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification. CONCLUSIONS: We provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of TlGa15B-GA2 make it a good candidate for industrial saccharification applications.

PUL-Mediated Plant Cell Wall Polysaccharide Utilization in the Gut Bacteroidetes.

Plant cell wall polysaccharides (PCWP) are abundantly present in the food of humans and feed of livestock. Mammalians by themselves cannot degrade PCWP but rather depend on microbes resident in the gut intestine for deconstruction. The dominant Bacteroidetes in the gut microbial community are such bacteria with PCWP-degrading ability. The polysaccharide utilization systems (PUL) responsible for PCWP degradation and utilization are a prominent feature of Bacteroidetes. In recent years, there have been tremendous efforts in elucidating how PULs assist Bacteroidetes to assimilate carbon and acquire energy from PCWP. Here, we will review the PUL-mediated plant cell wall polysaccharides utilization in the gut Bacteroidetes focusing on cellulose, xylan, mannan, and pectin utilization and discuss how the mechanisms can be exploited to modulate the gut microbiota.

Comparative Study Reveals Insights of Sheepgrass (Leymus chinensis) Coping With Phosphate-Deprived Stress Condition.

Sheepgrass [Leymus chinensis (Trin.) Tzvel] is a valuable forage plant highly significant to the grassland productivity of Euro-Asia steppes. Growth of above-ground tissues of L. chinensis is the major component contributing to the grass yield. Although it is generally known that this species is sensitive to ecosystem disturbance and adverse environments, detailed information of how L. chinensis coping with various nutrient deficiency especially phosphate deprivation (-Pi) is still limited. Here, we investigated impact of Pi-deprivation on shoot growth and biomass accumulation as well as photosynthetic properties of L. chinensis. Growth inhibition of Pi-deprived seedlings was most obvious and reduction of biomass accumulation and net photosynthetic rate (Pn) was 55.3 and 63.3%, respectively, compared to the control plants grown under Pi-repleted condition. Also, we compared these characters with seedlings subjected to low-Pi stress condition. Pi-deprivation caused 18.5 and 12.3% more reduction of biomass and Pn relative to low-Pi-stressed seedlings, respectively. Further analysis of in vivo chlorophyll fluorescence and thylakoid membrane protein complexes using 2D-BN/SDS-PAGE combined with immunoblot detection demonstrated that among the measured photosynthetic parameters, decrease of ATP synthase activity was most pronounced in Pi-deprived plants. Together with less extent of lipid peroxidation of the thylakoid membranes and increased ROS scavenger enzyme activities in the leaves of Pi-deprived seedlings, we suggest that the decreased activity of ATP synthase in their thylakoids is the major cause of the greater reduction of photosynthetic efficiency than that of low-Pi stressed plants, leading to the least shoot growth and biomass production in L. chinensis.

Combined impact of heat stress and phosphate deficiency on growth and photochemical activity of sheepgrass (Leymus chinensis).

Global climate warming has a crucial impact on many terrestrial ecosystems, including temperate steppe. In addition, phosphate deficiency is known to be the common deficiency in soils worldwide due to the low availability of the phosphate nutrient in the form of inorganic phosphate anions (Pi). Consequently, in the future, land plants are likely to simultaneously encounter heat stress and phosphate deficiency more frequently. Sheepgrass 〔Leymus chinensis (Trin.) Tzvel〕is a dominant perennial forage plant highly significant to grass productivity of Eurasian temperate grasslands. Though effects of environmental stress including drought and Pi starvation have been studied, the combined eff ;ects of phosphate deficiency and heat stress on plant physiology remain largely unclear. Here, we investigated the combined eff ;ects of heat stress and phosphate deficiency on above-ground tissue growth and photochemical properties of L. chinensis using in vivo chlorophyll fluorescence spectroscopy. We observed remarkable phenotypic alterations of reduced shoot growth and considerable leaf chlorosis in L. chinensis seedlings under the combined stress condition. Also, we compared changes in photochemical activity between the control and the corresponding stressed seedlings. Based on chlorophyll fluorescence analysis, impairment of PSI was more severe than that of PSII in the seedlings treated with the combined stress. Compared to the control, PSI and PSII activity decreased up to 35.5% and 30%, respectively, under the combined-stress condition. Moreover, our data show that the decreased photosynthetic activity is not the sum of the single-stressed conditions. These results combined with the distinction of other photochemical parameters indicate that a complex interaction between Pi-deficiency and heat stress may exist in the forage plant.

Slr0151 in Synechocystis sp. PCC 6803 is required for efficient repair of photosystem II under high-light condition.

Cyanobacteria are ancient photosynthetic prokaryotes that have adapted successfully to adverse environments including high-light irradiation. Although it is known that the repair of photodamaged photosystem II (PSII) in the organisms is a highly regulated process, our knowledge of the molecular components that regulate each step of the process is limited. We have previously identified a hypothetical protein Slr0151 in the membrane fractions of cyanobacterium Synechocystis sp. PCC 6803. Here, we report that Slr0151 is involved in PSII repair of the organism. We generated a mutant strain (Δslr0151) lacking the protein Slr0151 and analyzed its characteristics under normal and high-light conditions. Targeted deletion of slr0151 resulted in decreased PSII activity in Synechocystis. Moreover, the mutant exhibited increased photoinhibition due to impairment of PSII repair under high-light condition. Further analysis using in vivo radioactive labeling and 2-D blue native/sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the PSII repair cycle was hindered at the levels of D1 synthesis and disassembly and/or assembly of PSII in the mutant. Protein interaction assays demonstrated that Slr0151 interacts with D1 and CP43 proteins. Taken together, these results indicate that Slr0151 plays an important role in regulating PSII repair in the organism under high-light stress condition.

Different B-type methionine sulfoxide reductases in Chlamydomonas may protect the alga against high-light, sulfur-depletion, or oxidative stress.

The genome of unicellular green alga Chlamydomonas reinhardtii contains four genes encoding B-type methionine sulfoxide reductases, MSRB1.1, MSRB1.2, MSRB2.1, and MSRB2.2, with functions largely unknown. To understand the cell defense system mediated by the methionine sulfoxide reductases in Chlamydomonas, we analyzed expression and physiological roles of the MSRBs under different abiotic stress conditions using immunoblotting and quantitative polymerase chain reaction (PCR) analyses. We showed that the MSRB2.2 protein was accumulated in cells treated with high light (1,300 µE/m² per s), whereas MSRB1.1 was accumulated in the cells under 1 mmol/L H2O2 treatment or sulfur depletion. We observed that the cells with the MSRB2.2 knockdown and overexpression displayed increased and decreased sensitivity to high light, respectively, based on in situ chlorophyll a fluorescence measures. We also observed that the cells with the MSRB1.1 knockdown and overexpression displayed decreased and increased tolerance to sulfur-depletion and oxidative stresses, respectively, based on growth and H2-producing performance. The physiological implications revealed from the experimental data highlight the importance of MSRB2.2 and MSRB1.1 in protecting Chlamydomonas cells against adverse conditions such as high-light, sulfur-depletion, and oxidative stresses.

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 a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803.

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K (m) value of the recombinant protein for glutamine was 26.6 +/- 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na(+). Moreover, the K (m) value was decreased to 12.2 +/- 1.9 mmol/L in the presence of Na(+). These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na(+) through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Deltaslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Deltaslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Deltaslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Deltaslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Deltaslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Deltaslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.

[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.