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.     

Effect of Monochromatic Light on Proton Efflux of the Blue-Green Alga Anabaena variabilis

Light-induced proton efflux of Anabaena variabilis was found to be biphasic, the second phase being inhibited by the ATPase inhibitor nitrofen (2,4-dichloro-1-[4-nitrophenoxy]benzene). The first, fast phase was triggered by monochromatic light of 707 nanometers, whereas the second, slower phase was not. With 707 nanometers, light, respiratory O₂ uptake was inhibited. Using light composed of two wavelengths (616 and 707 nanometers) a marked enhancement of both O₂ evolution as well as the second phase of proton efflux was observed. The first phase was not enhanced. Thus, phase II is driven by both photosystems. As concluded from the action spectrum phase I is markedly determined by photosystem-I activity. Altogether the data show that two different mechanisms of light-induced proton efflux exist on the cytoplasmic membrane of Anabaena, the slower one being dependent on ATP and linear photosynthetic electron flow.     

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.     

Demonstration of a sensory rhodopsin in eubacteria

We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (lambda max 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18 degrees C). Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix-helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.     

Molecular evolution of PAS domain-containing proteins of filamentous cyanobacteria through domain shuffling and domain duplication.

When the entire genome of a filamentous heterocyst-forming N2-fixing cyanobacterium, Anabaena sp. PCC 7120 (Anabaena) was determined in 2001, a large number of PAS domains were detected in signal-transducing proteins. The draft genome sequence is also available for the cyanobacterium, Nostoc punctiforme strain ATCC 29133 (Nostoc), that is closely related to Anabaena. In this study, we extracted all PAS domains from the Nostoc genome sequence and analyzed them together with those of Anabaena. Clustering analysis of all the PAS domains gave many specific pairings, indicative of evolutionary conservations. Ortholog analysis of PAS-containing proteins showed composite multidomain architecture in some cases of conserved domains and domains of disagreement between the two species. Further inspection of the domains of disagreement allowed us to trace them back in evolution. Thus, multidomain proteins could have been generated by duplication or shuffling in these cyanobacteria. The conserved PAS domains in the orthologous proteins were analyzed by structural fitting to the known PAS domains. We detected several subclasses with unique sequence features, which will be the target of experimental analysis.     

Pigment analysis and ammonia excretion in herbicide tolerant cyanobacteria

Isolation of cyanobacteria was attempted from herbicide applied rice soils. The predominant genera were Westiellopsis followed by Anabaena, Nostoc and Oscillatoria. The herbicide tolerance was further tested by growing the cyanobacterial cultures in BG-11 medium supplemented with varying concentrations of the commonly used rice herbicide, viz butachlor under in vitro condition. The chlorophyll-a, phycobiliproteins and ammonia excretion were assessed at periodic intervals. Westiellopsis showed the maximum tolerance followed by Anabaena, Nostoc and Oscillatoria.     

Fate of nitrogen applied as Azolla and blue-green algae (Cyanobacteria) in waterlogged rice soils—a15N tracer study

Summary ¹⁵N tracer was used to detect the extent to which nitrogen of appliedAzolla caroliniana, Anabaena variabilis andNostoc muscorum was available for assimilation by the growing rice plants in pots under 4 cm flood water for 60 days. The rate of release of nitrogen from the above biofertilizers, the amount of nitrogen remaining in the soils and the amount that was lost from the soils during this period were also examined. Previously¹⁵N-labelled biomass of Azolla, Anabaena and Nostoc to provide 40 mg N was mixed thoroughly with 0.5 kg silt loam Bangladesh soil (Sonatola series) in each of three pots used for a single treatment. Each pot received four 16 days old IR8 rice seedlings. A parallet set of experiments was conducted without rice plants.It was found that nitrogen uptake in the rice plants was increased by 91, 176 and 215% on using Azolla, Anabaena and Nostoc which resulted in increased total dry matter yields (shoot plus root) of 74, 105 and 125%, respectively. Of the total¹⁵N applied at the start, 26, 49 and 53% was released from Azolla, Anabaena and Nostoc; about 7, 14 and 13% was lost by denitrification and 74, 51 and 47% remained in the soils as the undecomposed part of the biofertilizers, respeciively, after 60 days. Of 15.76, 22.72 and 25.92 mg N assimilated by the rice plants, 48, 61 and 62% was supplied by Azolla, Anabaena and Nostoc, respectively. The rest was obtained from the soil used.In the absence of the rice plants 30, 43 and 45% of applied¹⁵N of Azolla, Anabaena and Nostoc was released, respectively, in 60 days of which 93–96% was lost as N₂ through denitrification.     

Paracrystalline inclusions in various isolates of the blue-green bacteria Nostoc and Anabaena.

A number of different crystalline inclusions were observed in various isolates of Anabaena and Nostoc. Membrane-limited crystalline bodies were observed in 7 of 20 isolates of Anabaena and 19 of 29 isolates of Nostoc. These are spherical, single membrane-limited bodies from 0.6 to 0.1 micron in diameter. In most of the isolates they contained needle-like crystals 20 A in thickness and up to 80 nm in length. In 9 of the isolates the inclusions contained granular and fibrillar material. The number of bodies per cell varied in the different isolates from only a few, observed in many sections, up to 5 in a single section of A. subtropica (B1618). Crystalloids were observed in the cytoplasm of Anabaena sp. (1551), N. calcicola (B382), Nostoc sp. (588), and N. punctiforme (1629). In Anabaena sp. (1551) the roughly cuboidal inclusions 0.6 micron in diameter were composed of 100 A thick osmiophilic striations spaced to produce a 150 A periodicity. In Nostoc sp. (588) the elongate, 0.1 micron by 2.5 micron, crystalloids were composed of 100 A thick osmiophilic striations spaced to produce a 200 A periodicity. N. punctiforme (1629) and N. calciola (B382) contained intrathylakoidal crystalloids which consisted of short curved segments with 100 A thick osmiophilic striations producing a 200 A periodicity. Granular areas were observed in 2 isolates of Anabaena and 5 of Nostoc. These bodies found in various locations in the cells, were interpreted to be elongate structures 0.2 micron thick, 1.2 micron long and about 5 micron in depth. These inclusions were composed of 15 nm diameter granules which in some section planes appeared in rows spaced 20 nm apart. Spherical bodies up to 0.7 micron in diameter and of medium electron density were observed in 4 isolates of Anabaena and 2 of Nostoc. Convoluted inclusions were found in N. calcicola (B382) and Anabaena sp. (1551). These roughly spherical bodies up to 0.8 micron in diameter contain lighter swirled areas.     

Group 3 sigma factor gene, sigJ, a key regulator of desiccation tolerance, regulates the synthesis of extracellular polysaccharide in cyanobacterium Anabaena sp. strain PCC 7120.

The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.     

Plasmid and chromosomal DNA recovery by electroextraction of cyanobacteria

Abstract High voltage electroporation has been investigated as a method for rapid recovery of plasmid and chromosomal DNA from the cyanobacteria Nostoc PCC 7121, Synechococcus PCC 7002, and Anabaena PCC 7120. Pulses of 18 kV/cm and higher applied to concentrated Nostoc cells carrying a shuttle plasmid (pRL25) resulted in copious release of nucleic acids and phycobiliproteins into the suspending medium. Small portions of these supernatants, when electroporated with Escherichia coli , gave rise to hundreds of E. coli transformants which contained pRL25. Electroporation of Synechococcus carrying plasmid pAQE19 did not cause detectable release of macromolecules but did reveal a low-level, voltage independent 'leakage' of pAQE19 into the medium. Electroextraction of Nostoc or Anabaena followed by addition of E. coli and delivery of a second high-voltage pulse permitted direct, one-cuvette transfer of shuttle plasmids from these cyanobacteria into E. coli . Electroextraction of single cyanobacterial colonies, as shown for Nostoc , also released sufficient chromosomal DNA for amplification of specific sequences by the polymerase chain reaction.     

Dechlorination of lindane by the cyanobacterium Anabaena sp. strain PCC7120 depends on the function of the nir operon.

Nitrate is essential for lindane dechlorination by the cyanobacteria Anabaena sp. strain PCC7120 and Nostoc ellipsosporum, as it is for dechlorination of other organic compounds by heterotrophic microorganisms. Based on analyses of mutants and effects of environmental factors, we conclude that lindane dechlorination by Anabaena sp. requires a functional nir operon that encodes the enzymes for nitrate utilization.     

Distribution of nitrogen-fixing cyanobacteria (Nostocaceae) during rice cultivation in fertilized and unfertilized paddy fields

This study describes the development of nitrogen-fixing cyanobacteria (Nostocaceae) during different stages of rice seedlings plantation in fertilized and unfertilized paddy fields in north Bihar, India. A heterogeneous population of cyanobacteria was randomly harvested around day 20, 40 and 60 of rice seedlings plantation, and the diversity was analyzed. Thirtytwo species (7 genera) were identified from unfertilized fields, of which Anabaena was represented by 12 species, Anabaenopsis and Aulosira by 3 each, Cylindrospermum by 4, Nostoc by 8 and Aphanizomenon and Nodularia by 1 species each. However, fertilized fields contained only 25 species (7 genera), of which 8 each belonged to Nostoc and Anabaena , 3 to Cylindrospermum , 2 to each Anabaenopsis and Aulosira and 1 to each Aphanizomenon and Nodularia . Although Nostoc and Anabaena were dominant in both fertilized and unfertilized paddy fields, a marked decrease in nitrogen-fixing cyanobacteria was recorded from fertilized fields. In both treatments, the diversity of nitrogen-fixing cyanobacteria was at a maximum around day 60 after seedling plantation. The current study concludes that there is a negative effect of nitrogenous fertilizers on the development of heterocystous cyanobacteria in rice fields. It is proposed that early appearing efficient nitrogen-fixers should be used as nitrogen fertilizers in the management for better establishment and exploitation of heterocystous cyanobacteria for sustainable agricultural practices.     

Oxygen-dependent proton efflux in cyanobacteria (blue-green algae).

The oxygen-dependent proton efflux (in the dark) of intact cells of Anabaena variabilis and four other cyanobacteria (blue-green algae) was investigated. In contrast to bacteria and isolated mitochondria, an H+/e ratio (= protons translocated per electron transported) of only 0.23 to 0.35 and a P/e ratio of 0.8 to 1.5 were observed, indicative of respiratory electron transport being localized essentially on the thylakoids, not on the cytoplasmic membrane. Oxygen-induced acidification of the medium was sensitive to cyanide and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Inhibitors such as 2,6-dinitrophenol and vanadate exhibited a significant decrease in the H+/e ratio. After the oxygen pulse, electron transport started immediately, but proton efflux lagged 40 to 60 s behind, a period also needed before maximum ATP pool levels were attained. We suggest that proton efflux in A. variabilis is due to a proton-translocating ATP hydrolase (ATP-consuming ATPase) rather than to respiratory electron transport located on the cytoplasmic membrane.     

Hg2+ and Cd2+ induced inhibition of light induced proton efflux in the cyanobacteriumAnabaena flos-aquae

Light induced proton efflux in intact cells ofAnabaena flos-aquae is inhibited by the heavy metals Hg²⁺ and Cd²⁺. Furthermore, Hg²⁺ and Cd²⁺ reduced the¹⁴CO₂ fixation, oxygen evolution and carbonic anhydrase activity responsible for H⁺ efflux.     

A study of several blue-green algae in the electron microscope

Summary All of the three blue-green algae, Anabaena cylindrica, Mastigocladus laminosus and Nostoc muscorum are characterized by the presence of multi-layered envelopes (sheath, wall and plasma membrane), photosynthetic lamellae and a variety of intracellular granules. Sections of heterocysts of Anabaena cylindrica showed the presence of an internal membrane system as well as lamellae. An unusual feature of the structure of Nostoc muscorum was the presence of densely stained intracellular membranes or lamellae. The results emphasize the variability in appearance of the internal structure of the blue-green algae and point to the need for detailed investigations of the influence of change in physiological environment on the anatomy of these organisms.     

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.     

Cyanobiont diversity within and among cycads of one field site.

Limited diversity was found among cyanobionts from a cultivated population of cycads at a field site in Florida. All isolates were classified as Nostoc but were different from the one Nostoc species found in the soil. These cyanobacteria were root endophytes of several plants of Zamia integrifolia and one of Dioon. The isolates were similar morphologically and in their reactions to four fluorescein isothiocyanate conjugated lectins. Electrophoretic protein profiles and zymograms distinguished one cyanobiont and the soil Nostoc. A tenacious Anabaena epiphyte was also discovered inhabiting the surfaces of root nodules.     

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Abstract The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis . Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis : the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.     

Utilization of hydroxyapatite by Cyanobacteria as their sole source of phosphate and calcium

Two species of Cyanobacteria, (one Nostoc, one Anabaena) are described which are able to grow utilizing hydroxyapatite as their sole source of phosphate and calcium at pH=7.6.     

Molecular genetic and chemotaxonomic characterization of the terrestrial cyanobacterium Nostoc commune and its neighboring species

The phylogeny of the terrestrial cyanobacterium Nostoc commune and its neighboring Nostoc species was studied using molecular genetic and chemotaxonomic approaches. At least eight genotypes of N. commune were characterized by the differences among 16S rRNA gene sequences and the petH gene encoding ferredoxin-NADP? oxidoreductase and by random amplified polymorphic DNA analysis. The genotypes of N. commune were distributed in Japan without regional specificity. The nrtP gene encoding NrtP-type nitrate/nitrite permease was widely distributed in the genus Nostoc, suggesting that the occurrence of the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. The wspA gene encoding a 36-kDa water stress protein was only found in N. commune and Nostoc verrucosum, suggesting that these Nostoc species that form massive colonies with extracellular polysaccharides can be exclusively characterized by the occurrence of the wspA gene. Fifteen species of Nostoc and Anabaena were investigated by comparing their carotenoid composition. Three groups with distinct patterns of carotenoids were related to the phylogenic tree constructed on the basis of 16S rRNA sequences. Nostoc commune and Nostoc punctiforme were clustered in one monophyletic group and characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol glycosides.