Proteomic screening of salt-stress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803

The plasma membrane of a cyanobacterial cell is crucial as barrier against the outer medium. It is also an energy-transducing membrane as well as essential for biogenesis of cyanobacterial photosystems and the endo-membrane system. Previously we have identified 57 different proteins in the plasma membrane of control cells from Synechocystis sp. strain PCC6803. In the present work, proteomic screening of salt-stress proteins in the plasma membrane resulted in identification of 109 proteins corresponding to 66 different gene products. Differential and quantitative analyses of 2-DE profiles of plasma membranes isolated from both control and salt-acclimated cells revealed that twenty proteins were enhanced/induced and five reduced during salt stress. More than half of the enhanced/induced proteins were periplasmic binding proteins of ABC-transporters or hypothetical proteins. Proteins that exhibited the highest enhancement during salt stress include FutA1 (Slr1295) and Vipp1 (Sll0617), which have been suggested to be involved in protection of photosystem II under iron deficiency and in thylakoid membrane formation, respectively. Other salt-stress proteins were regulatory proteins such as PII protein, LrtA, and a protein that belongs to CheY subfamily. The physiological significance of the identified salt-stress proteins in the plasma membrane is discussed integrating our current knowledge on cyanobacterial stress physiology.     

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.     

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.     

Proteomic study of the soluble proteins from the unicellular cyanobacterium Synechocystis sp. PCC6803 using automated matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting

The unicellular cyanobacteria Synechocystis sp. (PCC6803) has become a model organism for a range of biochemical and molecular biology studies aimed at investigating environmental stress responses. In this study the soluble proteins of Synechocystis were analysed using narrow pH range (pH 4.5-5.5) zoom gels, automated matrix-assisted laser desorption/ionization mass spectrometry acquisition, spectral processing and database searching. The work sets the foundation for investigations of proteomic changes following stress treatment. One hundred and ninety-two protein spots were analysed and 105 proteins identified, of these 37 were novel proteins not previously seen on two-dimensional gels. Proteins involved in amino acid biosynthesis, energy metabolism and protein modification were identified using this fully automated procedure demonstrating that automated acquisition and processing will be a useful tool for proteomic analyses on this organism.     

Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803

We investigated the spectrum of secreted proteins in the cyanobacterium Synechocystis, and identified these proteins by amino-terminal sequencing. In total, seven sequences have been determined that corresponded to the proteins Sll0044, Sll1694, Sll1891, Slr0924, Slr0841, Slr0168, and Slr1855. The protein Sll1694 of 18 kDa that formed one of two major bands on SDS-PAGE was identified as cyanobacterial pilin, PilA. The amino-terminal sequence of another protein that formed a second major band was blocked. The analysis of the data revealed that five of seven proteins had distinct putative leader sequences for secretion.     

Proteomics of Synechocystis sp. PCC 6803. Identification of novel integral plasma membrane proteins

The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI-TOF MS. More than half of the proteins were predicted to be integral with 1-12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.     

A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.     

Salt stress and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803.

Acclimation of microorganisms to environmental stress is closely related to the expression of various genes. We report here that salt stress and hyperosmotic stress have different effects on the cytoplasmic volume and gene expression in Synechocystis sp. PCC 6803. DNA microarray analysis indicated that salt stress strongly induced the genes for some ribosomal proteins. Hyperosmotic stress strongly induced the genes for 3-ketoacyl-acyl carrier protein reductase and rare lipoprotein A. Genes whose expression was induced both by salt stress and by hyperosmotic stress included those for heat-shock proteins and the enzymes for the synthesis of glucosylglycerol. We also found that each kind of stress induced a number of genes for proteins of unknown function. Our findings suggest that Synechocystis recognizes salt stress and hyperosmotic stress as different stimuli, although mechanisms common to the responses to each form of stress might also contribute to gene expression.     

An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division

Plastids have evolved from an endosymbiosis between a cyanobacterial symbiont and a eukaryotic host cell. Their division is mediated both by proteins of the host cell and conserved bacterial division proteins. Here, we identified a new component of the plastid division machinery, Arabidopsis thaliana SulA. Disruption of its cyanobacterial homolog (SSulA) in Synechocystis and overexpression of an AtSulA-green fluorescent protein fusion in Arabidopsis demonstrate that these genes are involved in cell and plastid division, respectively. Overexpression of AtSulA inhibits plastid division in planta but rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, demonstrating that its role in plastid division may involve an interaction with AtFtsZ1-1 and AtFtsZ2-1.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Proteome analysis of the responses of Panax ginseng C. A. Meyer leaves to high light: use of electrospray ionization quadrupole-time of flight mass spectrometry and expressed sequence tag data

We performed comparative proteomic analyses in order to understand the physiological responses of ginseng (Panax ginseng C. A. Meyer) to high light (HL). As a first step, we analyzed the proteins expressed in ginseng leaves. Proteins extracted from leaves were separated by two-dimensional polyacrylamide gel electrophoresis. Protein spots were identified by tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS). We used a ginseng expressed sequence tag (EST) database as well as a nonredundant protein database from NCBI to identify proteins. Eighty-one proteins were identified using the nr protein database, 51 of which were also verified from the ginseng EST database. An additional 66 proteins were identified only from the ginseng EST database. Proteins that function in energy metabolism, protein stabilization, and protection against oxidative stress were abundant. To understand the light responses of ginseng leaves, we studied time dependent changes in expressed proteins produced by 0-4 h of HL exposure. Six HL-responsive proteins were identified: three proteins were up-regulated (cytosolic small heat-shock protein, cytosolic ascorbate peroxidase, and putative major latex-like protein) and three proteins were down-regulated (Rieske Fe/S protein, putative 3-beta hydroxysteroid dehydrogenase/isomerase-like protein, and oxygen-evolving enhancer-like protein). Our results show that the ginseng EST database combined with ESI Q-TOF MS analysis can be used to identify ginseng proteins and to elucidate the protective mechanism of ginseng against HL induced damage.     

Correlation between immuno-gold labels and activities of the cytochrome-c oxidase (aa3-type) in membranes of salt stressed cyanobactria

Abstract Anacystis nidulans ( Synechococcus PCC6301) and Synechocystis PCC6803 were grown photoautotrophically in a turbido-statically operated chemostat at a constant cell concentration of 2.0±0.3 μ l packed cell mass per ml in the presence of elevated NaCl concentrations up to 0.5 M ('salt stress'). The impact of salt stress on ccytochrome- c oxidase (EC 1.9.3.1) was` studied on isolated and purified membranes, and by immuno-gold labeling of thin-sectioned whole cells ATPase activities of membranes isolated and separated from cells under varying salt stress were also measured. Anacystis and Synechocystis adapted to the presence of 0.5 M NaCl in the medium with lag phases of 2 days and 2 hours, respectively. Both isolated plasma and thylakoid membranes from salt adapted Synechocystis displayed 5- to 8-times enhanced cytcytochrome- c oxidase activities while in Anacystis the effect was restricted to the plasma membrane. In either case less than proportionately increased counts of immuno-gold labeled cytochrome- c oxidase molecules in the respective membranes were obtained, the additional increment being attributed to the increased lipid content of the membranes from salt-adapted cells, leading to increased specific activities of the enzyme compared to control cells. ATPase activity of plasma membranes from Synechocystis was far more increased than of those from Anacystis while in thylakoid membranes the differentiating effect was less pronounced. Our results are discussed in terms of distinct strategies for salt adaptation in the two cyanobacterial species whereby in Anacystis the plasma membrane-bound respiratory chain and in Synechocystis the plasma membrane-bound ATPase(s) play the major role for plasma membrane energization which, in turn, is necessary for the active exclusion of sodium from the cell interior.     

Cell-line specific chromatin acetylation at the Sox10-Pax3 enhancer site modulates the RET proto-oncogene expression

IscA homologs are known to be involved in iron-sulfur cluster formation in various organisms. Recombinant proteins of two IscA homologs from the cyanobacterium Synechocystis PCC 6803, designated SLR1417 and SLR1565, were purified. The absorption spectrum of purified SLR1565 was typical for [2Fe-2S] cluster-containing proteins, whereas that of SLR1417 predominantly showed the presence of the iron ion alone. In the cyanobacterial cell extracts, only SLR1565 was found to form a complex with a novel prokaryotic HEAT-repeats-containing protein, SLR1098. Thus, the two cyanobacterial IscA protein homologs exist in distinct molecular states, suggesting different cellular roles for these proteins.     

A conserved structure and function of the YidC homologous protein Slr1471 from Synechocystis sp. PCC 6803

In this article, we show that the orf slr1471 from Synechocystis sp. PCC 6803 codes for a functional member of the YidC/Alb3/Oxa1 protein family, and the encoded protein has a transmembrane topology with a common core structure. Using specific antibodies raised against the Synechocystis YidC homologous protein, we further show that the Synechocystis YidC protein appears to be predominantly localized in the cyanobacterial cytoplasmic membrane. The impact of the described findings for synthesis of membrane proteins and for protein sorting within cyanobacterial cells is discussed.     

集胞藻6803藻胆体别藻蓝蛋白多克隆抗体的制备

藻胆体是蓝藻细胞主要的捕光天线色素超分子复合体,主要由核心体和外围的杆两部分组成,核心体主要由别藻蓝蛋白组装而成,参与光能向光合作用反应中心的传递.该研究通过PCR扩增出集胞藻6803别藻蓝蛋白α亚基(ApcA)编码基因apcA,构建表达质粒pET-32a(+)-apcA,并将其转入大肠杆菌BL21(DE3)pLysS菌株中;通过IPTG诱导表达重组蛋白,并利用组氨酸标签将可溶性目的蛋白进行亲和纯化后,免疫日本大耳白兔,从而获得多克隆抗体.间接ELISA法揭示ApcA抗体效价可高达1∶1 025 000;蛋白免疫印迹确定该抗体具有高度特异性.表明该研究成功制备了集胞藻6803藻胆体别藻蓝蛋白多克隆抗体,为进一步研究藻胆体的核心体在光能传递过程中所承担的重要生理角色奠定了生化基础.