Myxol and 4-ketomyxol 2'-fucosides, not rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and proposal for the biosynthetic pathway of carotenoids

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.     

Myxoxanthophyll in Synechocystis sp. PCC 6803 is myxol 2'-dimethyl-fucoside, (3R,2'S)-myxol 2'-(2,4-di-O-methyl-alpha-L-fucoside), not rhamnoside

We identified the molecular structures of the carotenoids in Synechocystis sp. PCC 6803. Myxoxanthophyll in this cyanobacterium was myxol 2'-dimethyl-fucoside, (3R,2'S)-myxol 2'-(2,4-di-O-methyl-alpha-L-fucoside). The sugar moiety of the pigment was not rhamnose but dimethylated fucose, which has not been reported in carotenoid glycosides. The other carotenoids were beta-carotene, (3R,3'R)-zeaxanthin, echinenone, (3'R)-3'-hydroxyechinenone and deoxymyxol 2'-dimethyl-fucoside, (2'S)-deoxymyxol 2'-(2,4-di-O-methyl-alpha-L-fucoside). Generally, the group of polar carotenoids in cyanobacteria is referred to as myxoxanthophyll, and the structure is considered to be myxol 2'-rhamnoside. Since the name myxoxanthophyll can not specify the sugar moiety and the identification of the sugar moiety is unfeasible in many cyanobacteria, we propose the following naming convention: when the sugar moiety is unknown, the name is myxol glycoside, when known, as in the case of rhamnose and alpha-L-fucose, they should be named myxol 2'-rhamnoside and myxol 2'-alpha-L-fucoside, respectively.     

Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.

The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself.     

The cyanobacterium Anabaena sp. PCC 7120 has two distinct beta-carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis

Two beta-carotene ketolases, CrtW and CrtO, are widely distributed in bacteria, although they show no significant sequence homology with each other. The cyanobacterium Anabaena sp. PCC 7120 was found to have two homologous genes. In the crtW deleted mutant, myxol 2'-fucoside was present, but ketomyxol 2'-fucoside was absent. In the crtO deleted mutant, beta-carotene was accumulated, and the amount of echinenone was decreased. Therefore, CrtW catalyzed myxol 2'-fucoside to ketomyxol 2'-fucoside, and CrtO catalyzed beta-carotene to echinenone. This cyanobacterium was the first species found to have both enzymes, which functioned in two distinct biosynthetic pathways.     

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.     

Tocopherols protect Synechocystis sp. strain PCC 6803 from lipid peroxidation

Tocopherols (vitamin E) are lipid-soluble antioxidants synthesized only by photosynthetic eukaryotes and some cyanobacteria, and have been assumed to play important roles in protecting photosynthetic membranes from oxidative stress. To test this hypothesis, tocopherol-deficient mutants of Synechocystis sp. strain PCC 6803 (slr1736 and slr1737 mutants) were challenged with a series of reactive oxygen species-generating and lipid peroxidation-inducing chemicals in combination with high-light (HL) intensity stress. The tocopherol-deficient mutants and wild type were indistinguishable in their growth responses to HL in the presence and absence of superoxide and singlet oxygen-generating chemicals. However, the mutants showed enhanced sensitivity to linoleic or linolenic acid treatments in combination with HL, consistent with tocopherols playing a crucial role in protecting Synechocystis sp. strain PCC 6803 cells from lipid peroxidation. The tocopherol-deficient mutants were also more susceptible to HL treatment in the presence of sublethal levels of norflurazon, an inhibitor of carotenoid synthesis, suggesting carotenoids and tocopherols functionally interact or have complementary or overlapping roles in protecting Synechocystis sp. strain PCC 6803 from lipid peroxidation and HL stress.     

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.     

Presence of free myxol and 4-hydroxymyxol and absence of myxol glycosides in Anabaena variabilis ATCC 29413, and proposal of a biosynthetic pathway of carotenoids

We identified the molecular structures of all carotenoids in Anabaena variabilis ATCC 29413 (= IAM M-204). The major carotenoids were beta-carotene, echinenone and canthaxanthin. Myxol glycosides were absent, while free forms of myxol and 4-hydroxymyxol were present. The 4-hydroxyl group of the latter was a mixture of (4R) and (4S) configurations, which is a rare mixture in carotenoids. Thus, this strain was the first cyanobacterium found to have free myxol and not myxol glycosides, and seemed to lack the gene for or activity of glycosyl transferase. In another strain of A. variabilis IAM M-3 (= PCC 7118), we recently identified (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, and hence the strain ATCC 29413 might be useful for investigating the characteristics of myxol glycosides in cyanobacteria. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in A. variabilis ATCC 29413, we proposed a biosynthetic pathway of the carotenoids and the corresponding genes and enzymes. The homologous genes were searched by sequence homology only from the functionally confirmed genes.     

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.     

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.     

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.     

Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha

The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.     

Identification and Inactivation of Three Group 2 Sigma Factor Genes in Anabaena sp. Strain PCC 7120

Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth. A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes. luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains, Anabaena sp. strain PCC 7120, Synechococcus sp. strain PCC 7002, and Synechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains, Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.     

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).     

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.     

细胞周期蛋白依赖性激酶抑制剂Roscovitine对3种蓝藻生长和活性的影响

为了明确蓝藻中丝氨酸/苏氨酸激酶的功能是否与调控细胞的生长分裂相关,以丝状鱼腥藻7120、单细胞集胞藻6803和聚球藻7002为对象,利用OD750光吸收测定和MTT方法研究了不同浓度丝氨酸苏氨酸激酶抑制剂roscovitine对其生长和脱氢酶活性的影响。结果表明:4 h roscovitine处理后对鱼腥藻7120和集胞藻6803生长量影响不大,对聚球藻7002的生长有促进作用。4 h roscovitine的处理对鱼腥藻7120有浓度依赖的显著抑制活性,对集胞藻6803的活性无影响,但是却促进聚球藻7002的活性。药物作用4 d后,7120的生长和活性均显著降低,并有浓度效应;6803的生长量较对照减少,但活性变化不明显;聚球藻7002的生长和活性均未受影响。显微观察结果显示,roscovitine对3种细胞形态没有影响,但药物作用4 d后的7120藻丝体较短。结果表明丝氨酸/苏氨酸抑制剂roscovitine影响丝状藻7120的生长和活性。     

Interaction of fructose with the glucose permease of the cyanobacterium Synechocystis sp. strain PCC 6803.

Fructose was bactericidal for the cyanobacterium Synechocystis sp. strain PCC 6803. Each of ten independently isolated fructose-resistant mutants had an alteration of the glucose transport system, measured as uptake of glucose or of 3-O-methyl-D-glucose. In the presence of the analog, the wild-type Synechocystis strain was protected against fructose. Two mutants altered in photoautotrophy were also isolated.     

蓝细菌ORF469的分子克隆和缺失突变工程株的构建

PCR扩增了蓝细菌Synechocystis sp．PCC 6803的ORF469(编码469个氨基酸的开放阅读框),进一步以pUC118为载体将其克隆到E．Coli中,构建了pOQ2质粒。通过DNA体外重组,以红霉素抗性基因取代部分克隆化ORF469片段,又构建丁缺失ORF469片段(保留部分上游和下游序列)的pOQ22质粒。用pOQ22质粒转化Synechocystis sp．PCC 6803野生株细胞,获ORF489缺失突变工程株,它在红霉素抗性培养基上生长正常。对缺失突变工程株DNA的PCR和Southern blot分析证明,Synechocystis sp．PCC 6803的ORF469已被删除。色素测定结果揭示Synechocystis sp．PCC 6803中ORF469表达产物控制细胞内不依赖光的叶绿素生物合成。     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.