Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.     

Chemoheterotrophic Growth of the Cyanobacterium Anabaena sp. Strain PCC 7120 Dependent on a Functional Cytochrome c Oxidase

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium commonly used as a model organism for studying cyanobacterial cell differentiation and nitrogen fixation. For many decades, this cyanobacterium was considered an obligate photo-lithoautotroph. We now discovered that this strain is also capable of mixotrophic, photo-organoheterotrophic, and chemo-organoheterotrophic growth if high concentrations of fructose (at least 50 mM and up to 200 mM) are supplied. Glucose, a substrate used by some facultatively organoheterotrophic cyanobacteria, is not effective in Anabaena sp. PCC 7120. The gtr gene from Synechocystis sp. PCC 6803 encoding a glucose carrier was introduced into Anabaena sp. PCC 7120. Surprisingly, the new strain containing the gtr gene did not grow on glucose but was very sensitive to glucose, with a 5 mM concentration being lethal, whereas the wild-type strain tolerated 200 mM glucose. The Anabaena sp. PCC 7120 strain containing gtr can grow mixotrophically and photo-organoheterotrophically, but not chemo-organoheterotrophically with fructose. Anabaena sp. PCC 7120 contains five respiratory chains ending in five different respiratory terminal oxidases. One of these enzymes is a mitochondrial-type cytochrome c oxidase. As in almost all cyanobacteria, this enzyme is encoded by three adjacent genes called coxBAC1. When this locus was disrupted, the cells lost the capability for chemo-organoheterotrophic growth.     

Genomic analysis of protein kinases,protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.     

Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme

The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.     

Two cAMP receptor proteins with different biochemical properties in the filamentous cyanobacterium Anabaena sp. PCC 7120

Two open reading frames (ORFs), alr0295 and alr2325, are found to encode putative cAMP receptor proteins (CRPs) in the genome of the filamentous cyanobacterium Anabaena sp. PCC 7120. These ORFs were named cAMP receptor protein-like gene A in Anabaena sp. PCC 7120 (ancrpA) and cAMP receptor protein-like gene B in Anabaena sp. PCC 7120 (ancrpB), respectively, and those translated products were investigated. The equilibrium dialysis measurements revealed that AnCrpA bound with cAMP specifically, while AnCrpB bound with both cAMP and cGMP, though the affinity for cGMP was weak. The binding affinity for cAMP of AnCrpA showed the lowest dissociation constant, approximately 0.8 microM, among bacterial CRPs. A gel mobility shift assay elucidated that AnCrpA and AnCrpB formed a complex with the consensus DNA sequence in the presence of cAMP, although AnCrpB did not have ordinary DNA-binding motifs.     

Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.     

From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp. PCC 6803

Activities of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphofructokinase (PFK), enolase, pyruvate kinase (PK) and phosphoenolpyruvate (PEP) carboxylase were determined in extracts of photoautotrophic, mixotrophic, and heterotrophic cultures of Synechocystis sp. PCC 6803. Annotated genomes of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were analyzed for the respective predicted physical properties of each enzyme investigated here. Enzymatic activity was largely unaffected by nutritional mode, with the exception of glucokinase and PK whose activities were significantly elevated in heterotrophic cultures of Synechocystis sp. PCC 6803. PFK activity was insensitive to bacterial PFK-A (allosteric) effectors such as PEP, implying that Synechocystis PFK should be classified as a PFK-B (non-allosteric). Immunoblot and kinetic studies indicated that irrespective of nutritional mode, the Synechocystis PK corresponds to a PK-A (AMP activated) rather than PK-F (fructose-1,6-bisphosphate activated).     

Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacterium Anabaena sp. PCC 7120

A nuclease that could be recovered from the supernatant of cultures, as well as from cell-free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA-containing SDS-polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29,650, presented a presumptive signal peptide in its N-terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.     

Molecular cloning and sequencing of the sodB gene from a heterocystous cyanobacterium Anabaena sp. PCC 7120

Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a heterocystous cyanobacterium Anabaena sp. PCC 7120 was cloned and sequenced. The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa. Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod. Northern blot analysis showed that the sodB gene of Anabaena sp. PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition.     

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.     

NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene.

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.     

Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp. strain PCC 7120 and Rhizobium meliloti.

The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp. strain PCC 7120 and in Rhizobium meliloti, has been determined. This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoxins. Cyanobacteria have not previously been shown to contain bacterial-type ferredoxins. The presence of this gene suggests that nitrogen-fixing cyanobacteria have at least four distinct ferredoxins.     

转TNF-α基因鱼腥藻7120质粒稳定性研究

为了实现转基因鱼腥藻培养生产TMF的目的,讲究了转基因鱼腥藻的稳定性。影印法证实转TNF-α。基因鱼腥藻7120能保持质粒分配稳定性。比较无选择压力下连续传代的转丛因鱼醒藻7120在不同培养基中的生长和外源基因表达,证实没有发生质粒部分缺失,但转基因鱼腥藻在无选择压力下会降低重组质粒拷贝数。在培养过程中,种子培养越含有的新霉素可以保持生产过程质粒稳定,这可以大火减少新霉素用量。     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.     

鱼腥藻7120遗传转化的研究进展

鱼腥藻7120作为模式生物被广泛用于光合、固氮、进化、代谢等基本生命现象的研究。近几年, 对其基因工程的研究使人们看到它在医药、环保、能源等方面的应用潜力, 但表达效率低是其发展的瓶颈。为了提高其表达效率, 研究者从鱼腥藻7120的载体(包括启动子、复制子、选择标记基因等)的改进、目的基因的优化(密码子和SD序列)、宿主的改善、转化方法的改变等方面进行了大量探索, 除了用于功能基因的研究, 已经有几十个外源基因在鱼腥藻7120中表达。除了研究载体, 诱变鱼腥藻7120形成有利于外源基因表达的突变体和摸索转基因蓝藻最佳生长条件和表达条件, 可能是新的发展方向。