pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Construction and analysis of a high-CO2-requiring mutant of the cyanobacterium Anabaena sp strain PCC7120

A mutant of Anabaena sp. strain PCC7120 requiring high CO2 was generated using Tn5 mutagenesis. This is the first data for a filamentous cyanobacterium. The mutant was capable of growing at 5% CO2, but incapable of growing at air levels of CO2. Southern hybridization analysis indicated that the Anabaena genome was inserted by the transposon at one site. The apparent photosynthetic affinity of the mutant to external dissolved inorganic carbon (DIC) was about 300 times lower that of the wild type (WT), and the medium alkalization rate as well as the carboxysomal carbonic anhydrase activity of the mutant was also lower than those of the WT. When the mutant was transferred from the culture medium bubbled with 5% CO2 to higher DIC (8.4% CO2) or 1% CO2, it showed similar responses to the WT. However, aberrant carboxysomes were found in the mutant cells through ultrastructural analysis, indicating it was most probably the wrong organization of the carboxysomes that eventually led to the inefficient operation of carboxysomal carbonic anhydrase and the subsequent defectiveness of the mutant in utilizing DIC.

An inducible CO2 concentrating mechanism in cyanobacterium Anabaena sp strain PCC7120

In order to define its characteristics of the photosynthetic utilization of CO2 and HCO3- when the ambient inorganic carbon changed, HCG (High-CO2-Growing Cells) of cyanobacterium Anabaena sp. strain PCC7120 were prepared. The growth rate of HCG was higher than that of LCG (low-CO2-growing cells, i.e. air-growing cells). When the HCG cells were transferred from 5% CO2 to air levels of CO2 , a series of changes took place: its carbonic anhydrase activity as well as its photosynthetic affinity to the external inorganic carbon significantly increased; the number of the carboxysomes, which is one of the most important components of CCM in cyanobacteria also increased. These facts indicated that the CCM activity of Anabaena PCC 7120 was induced. When the pH in the medium increased from 6 to 9, the photosynthetic affinity to external inorganic carbon of both HCG and LCG declined, while the apparent photosynthetic affinity to external CO2 increased. In the light of these findings, this inducible CCM in cyanobacteria provided a good model for the study of the photosynthetic Ci utilization in the phototrophic microoganisms.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120

From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium Anabaena PCC 7120 has been deduced. Anabaena has nine DNA MTases. Four are associated with Type II restriction enzymes (AvaI, AvaII, AvaIII and the newly recognized inactive AvaIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AvaVI, DmtB/M.AvaVII, DmtC/M.AvaVIII and DmtD/M.AvaIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AvaV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.

A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp strain PCC 7120

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Regulation by hetC of genes required for heterocyst differentiation and cell division in Anabaena sp strain PCC 7120

Unlike those of the wild-type strain, proheterocysts of the Anabaena sp. strain PCC 7120 hetC strain keep dividing. ftsZ, the most critical cell division gene, is up-regulated in hetC proheterocysts. Heterocyst differentiation genes hglD, hglE, patB, nijB, and xisA are no longer expressed in the hetC mutant. hetC also regulates the expression of patA, a pattern formation gene.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions

Heterocyst differentiation in the filamentous cyanobacterium Anabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petE∷hetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.

A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium.

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may fix nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Using a lacZ reporter with a fluorescent beta-galactoside substrate for in situ localization of gene expression, we have shown that the two clusters of nif genes are expressed independently. One nitrogenase functions only in heterocysts under either aerobic or anaerobic growth conditions, whereas the second nitrogenase functions only under anaerobic conditions in vegetative cells and heterocysts. Differences between the two nif clusters suggest that the nitrogenase that is expressed in heterocysts is developmentally regulated while the other is regulated by environmental factors.

蓝藻磷脂磷酸酶基因的缺失对光合作用的影响

磷脂酰甘油(phosphatidylglycerol, PG)是类囊体膜(也叫光合膜)中唯一的一种磷脂。在蓝藻中，PG的合成途径为：磷脂酸(phosphatidic acid, PA)胞嘧啶双磷酸-二酰基甘油 (cytidine diphosphate diacylglycerol, CDP-DAG) 磷酸磷脂酰甘油 (phosphatidylglycerol phosphate, PGP)PG。其中最后一步反应是由PGP去磷酸化而生成PG，催化该反应的是PGP磷酸酶。然而迄今为止，PGP磷酸酶还没有在蓝藻和高等植物中得到克隆和鉴定。本工作在鱼腥藻Anabaena sp. PCC7120中通过将一个可能编码PGP磷酸酶的基因(alr1715)进行突变，获得缺失PG的突变体。与野生型相比，该突变体PG的含量降低了30%左右。突变后的蓝藻藻丝发黄、生长缓慢，叶绿素含量降低。整体细胞的光合作用活性、光系统II(photosystem II，PSII)的放氧活性以及PSII反应中心的光能转化效率显著下降，传递给PSII的激发能减少。

Studies on temporal and spatial variations of phytoplankton in Lake Chaohu

Temporal and spatial variations of the phytoplankton assemblage in Lake Chaohu, a large shallow eutrophic lake in China, were studied from September 2002 to August 2003. A total of 191 phytoplankton species was identified, among which Chlorophytes (101) ranked the first, followed by Cyanophytes (46) and Bacillariophytes (28). On average over the entire lake, the maximum total algal biomass appeared in June (19.70 mg/L) with a minimum (5.05 mg/ L) in November. In terms of annual mean biomass, cyanobacteria contributed 45.43% to total algal biomass, followed by Chlorophytes (27.14%), and Bacillariophytes (20.6%). When nitrate (NO3-N) and ammonium (NH4-N) concentrations dropped in spring, fixing-nitrogen cyanobacterium (Anabaena) developed quickly and ranked the first in terms of biomass in summer. It is likely that dominance of zooplanktivorous fish and small crustacean zooplankton favored the development of the inedible filamentous or colony forming cyanobacteria. The persistent dominance of cyanobacteria throughout all seasons may indicate a new tendency of the response of phytoplankton to eutrophication in Lake Chaohu.

ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites1

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706–24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107–24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.