Identification of genes controlled by the manganese response regulator, ManR, in the cyanobacterium, Anabaena sp. PCC 7120

A computational search was carried out to identify additional binding sites for the manganese response regulator, ManR, in the genome of Anabaena sp. PCC 7120. This approach predicted ManR binding sites: the promoter regions of the genes of all3575-alr3576 and the gene of alr5134 from Anabaena sp. PCC 7120. Electrophoretic mobility shift assays confirmed that the ManR of Anabaena sp. PCC 7120 specifically bound to the promoter regions of all3575-alr3576 and alr5134.     

Over-expression of BvMTSH,a fusion gene for maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase,enhances drought tolerance in transgenic rice

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli

Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml⁻¹), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.     

Hydrogen metabolism in isolated heterocysts of Anabaena 7120

Isolated heterocysts of Anabaena 7120 evolve H₂ in an ATP-dependent nitrogenase-catalyzed process that is inhibited by N₂ and C₂H₂. Heterocysts have an active uptake hydrogenase that only requires an electron acceptor of positive redox potential, e.g., methylene blue, dichlorophenolindophenol or potassium ferricyanide. O₂ supplied at low partial pressures is a very effective physiological oxidant for H₂ uptake. High concentrations of O₂ are inhibitory to H₂ uptake. The oxyhydrogen reaction in heterocysts appears to be mediated by a cytochrome-cytochrome oxidase system, and it supports ATP synthesis via oxidative phosphorylation. Attempts to demonstrate acetylene reduction in isolated heterocysts employing H₂ as an electron donor were unsuccessful. It is suggested that the uptake hydrogenase functions to conserve reductant that otherwise would be dissipated via nitrogenase-catalyzed H₂ evolution.     

Increased activity of the non-regulated enzymes fructose-1,6-bisphosphate aldolase and triosephosphate isomerase in Anabaena sp. strain PCC 7120 increases photosynthetic yield

Non-regulated enzymes in the Calvin cycle are generally presumed to be less important for the regulation of photosynthetic yield. Here, to investigate the relationship between the activity of non-regulated enzymes and photosynthetic yield, two non-regulated enzymes in the Calvin cycle—a rice cytosolic fructose-1,6-bisphosphate aldolase (FBA) and a spinach chloroplast triosephosphate isomerase (TPI)—were cloned and co-expressed in cells of the cyanobacterium Anabaena sp. strain PCC 7120. The activity of FBA and TPI and the photosynthetic yield reflected by photosynthetic O₂ evolution and cell dry weight were measured and compared between wild-type and transgenic cells. Our results demonstrated that the activity of FBA and TPI were increased in transgenic cells relative to wild-type cells, and that activity was further increased in a transgenic strain harboring two sets of FBA-TPI tandem genes relative to cells containing one copy of the FBA-TPI tandem gene. The increased activity of FBA and TPI in Anabaena sp. strain PCC 7120 increased photosynthetic yield, with increased activity levels correlating closely with the degree of changes in photosynthetic yield. This implies that the photosynthetic yield is limited by the activity of the non-regulated enzymes FBA and TPI, and that the endogenous activity of non-regulated enzymes is not sufficient to increase photosynthetic yield. We discuss the various roles of FBA and TPI, and regulated and non-regulated enzymes, in modulating photosynthetic yield. W. Ma and L. Wei contributed equally to this work.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

The ManR specifically binds to the promoter of a Nramp transporter gene in Anabaena sp. PCC 7120: a novel regulatory DNA motif in cyanobacteria

The Anabaena sp. PCC 7120 ManR and a homologous protein of MntH were identified by BLAST search. Recombinant ManR protein was overexpressed in Escherichia coli and purified by an immobilized metal (Ni) affinity chromatography. Electrophoretic mobility shift assays revealed that ManR specifically bound to the promoter region of the mntH gene. Site-directed mutagenesis experiments demonstrated that the specific recognition site for ManR is TATGAAAAGAATATGAGAA, which is composed of two direct repeats of the consensus sequence (T/A)ATGA(G/A)A(A/G). This is a novel regulatory DNA motif in cyanobacteria, indicating that the expression of mntH was regulated by a two-component Mn(2+)-Sensing System containing ManR in Anabaena sp. PCC 7120. To date, this specific pathway of regulating mntH expression has only been found in cyanobacteria.     

一株海藻糖合成酶产生菌的鉴定及产酶条件优化

目的对一株海洋来源的产海藻糖合成酶菌株进行鉴定及产酶条件的初步优化。方法通过16SrDNA基因序列的同源性分析,对一株来源于东海海水的海藻糖合成酶产生菌进行鉴定,并通过单因素分析初步研究其培养特性和最佳的发酵条件。结果该菌16SrDNA序列与GenBank中已知序列相比,最高相似度为100％,鉴定为假单胞菌属（Pseudomonas）,命名为Pseudomonassp．A50。其最佳碳源和氮源分别为2％麦芽糖和0．5％酵母膏,最佳NaCl浓度为2．5％,在初始pH7．8,接种量1％,装液量125mL／250mL,28℃,130r／min发酵48h,海藻糖合成酶活力达到最高。结论此产海藻糖合成酶菌株为假单胞菌属,优化后,海藻糖合成酶活力达到14．16U／mL。     

Modification of the N-Terminal Nucleotide Sequence of Mature hGM-CSF Results in High Expression in the Foreign Host, Anabaena sp. Strain PCC 7120

Cloning and high foreign expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene were achieved in Anabaena sp. strain PCC 7120 cells. To promote high expression of hGM-CSF in cyanobacterial cells, PCR primers were designed to modify the N-terminal cDNA sequence of mature hGM-CSF, including a GC rich region and some discriminating against codons according to the degeneracy codon rules, selecting for prokaryotic usage codons. The PCR product encoding the modified hGM-CSF was inserted downstream of the promoter, PpsbA of the shuttle vector pRL439, then ligated with pDC-08 to generate the shuttle expression plasmid, pDC-GM1. The resulting shuttle expression plasmid was transferred into the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120 using the tri-parental conjugation transfer method. The results of PCR amplification of wild type and transgenic cells indicated that the hGM-CSF gene was successfully cloned into Anabaena sp. strain PCC 7120 cells. Western blot analysis showed that the protein expression of modified hGM-CSF in transgenic cells harboring pDC-GM1 was 136% higher than that of non-modified hGM-CSF in transgenic cells harboring pDC-GM0. Additionally, there were similar rate of growth and content of Chl a as compared to controls, suggesting that foreign hGM-CSF did not impair the photosynthetic activity of host cells. Taken together, the results indicate that modification of the N-terminal nucleotide sequence of mature hGM-CSF results in high expression in the transgenic cells.     

Localization of a multifunctional chaperonin (GroEL protein) in nitrogen-fixing Anabaena PCC 7120

The occurrence and distribution of a multifunctional chaperonin-60 (cpn60), the GroEL protein, was demonstrated in the cyanobacterium Anabaena PCC 7120 by using a rabbit anti-GroEL (Escherichia coli) antibody. Western-blot analysis showed a distinct cross-reaction with a protein of approx. 65 kilodaltons, analogous to the Mr of the E. coli homologue. Immunocyto-chemical studies of vegetative cells showed that a chaperonin was localized in both vegetative cells and heterocysts. In vegetative cells cpn60 was primarily detected both in the carboxysomes, and in the cytoplasm, though mainly in the thylakoid region of the latter. In heterocysts, specialized cells for nitrogen fixation, the cpn60 label was prominent and was evenly distributed throughout the cell. These results support recent findings that chaperonins are multifunctional proteins, and extend those findings by demonstrating the occurrence of cpn60 in a prokaryotic cyanobacterium and by raising the possibility of the involvement of this chaperonin in the assembly of heterocystous proteins.Abbreviations cpn60 chaperonin-60 - Mr relative molecular mass - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase

This paper questions what types of molecular transformation are involved in the conversion of maltose to trehalose by trehalose synthase from Thermus caldophilus GK24. The reverse reaction pathway has been examined with the aid of alpha,alpha-(2,4,6,6',2',4',6",6"'-(2)H(8))trehalose (1). The mass data of the isolated reaction products clearly indicate that deuterated glucose is confined only to substrate molecules, and thus the reversible enzymatic conversion of trehalose into maltose proceeds through an intramolecular pathway.     

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.     

The function of trehalose biosynthesis in plants

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside) occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant. Until recently, only few plant species, mainly desiccation-tolerant 'resurrection' plants, were considered to synthesise trehalose. Instead of trehalose, most other plants species accumulate sucrose as major transport sugar and during stress. The ability to synthesize sucrose has probably evolved from the cyanobacterial ancestors of plastids and may be linked to photosynthetic function. Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesise trehalose is widely distributed in the plant kingdom. The apparent lack of trehalose accumulation in these plants is probably due to the presence of trehalase activity. After inhibition of trehalase, trehalose synthesis can be detected in Arabidopsis. Since trehalose induces metabolic changes, such as an accumulation of storage carbohydrates, rapid degradation of trehalose may be required to prevent detrimental effects of trehalose on the regulation of plant metabolism. In addition, the precursor of trehalose, trehalose-6-phosphate, is probably involved in the regulation of developmental and metabolic processes in plants.     

Paired cloning vectors for complementation of mutations in the cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain PCC 7120

The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF _A ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.     

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.