Oxylipin formation in Nostoc punctiforme (PCC73102)

The dioxygenation of polyunsaturated fatty acids is mainly catalyzed by members of the lipoxygenase enzyme family in flowering plants and mosses. Lipoxygenase products can be metabolized further and are known as signalling substances that play a role in plant development as well as in plant responses to wounding and pathogen attack. Apart from accumulating data in mammals, flowering and non-flowering plants, information on the relevance of lipid peroxide metabolism in prokaryotic organisms is scarce. Thus we aimed to isolate and analyze lipoxygenases and oxylipin patterns from cyanobacterial origin. DNA isolated from Nostoc punctiforme strain PCC73102 yielded sequences for at least two different lipoxygenases. These have been cloned as cDNAs and named NpLOX1 and NpLOX2. Both proteins were identified as linoleate 13-lipoxygenases by expression in E. coli. NpLOX1 was characterized in more detail: It showed a broad pH optimum ranging from pH 4.5 to pH 8.5 with a maximum at pH 8.0 and alpha-linolenic acid was the preferred substrate. Bacterial extracts contain more 13-lipoxygenase-derived hydroperoxides in wounded than in non-wounded cells with a 30-fold excess of non-esterified over esterified oxylipins. 9-Lipoxygenase-derived derivatives were not detectable. 13-Lipoxygenase-derived hydroperoxides in esterified lipids were present at almost equal amounts compared to non-esterified hydroperoxides in non-wounded cells. These results suggest that 13-lipoxygenases acting on free fatty acids dominate in N. punctiforme strain PCC73102 upon wounding.     

Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.     

Effects of nitrogen source on the synthesis of the UV-screening compound, scytonemin, in the cyanobacterium Nostoc punctiforme PCC 73102

The effects of nitrogen source (N(2), NO(3)(-) and NH(4)(+)) on scytonemin synthesis were investigated in the heterocystous cyanobacterium Nostoc punctiforme PCC 73102. With the required UVA radiation included, Nostoc synthesized three to seven times more scytonemin while fixing nitrogen than when utilizing nitrate or ammonium. A similar increase in scytonemin synthesis occurred when nitrate or ammonium became depleted by growth and Nostoc switched to diazotrophic metabolism with the differentiation of heterocysts. In addition, UVA-exposed cultures grown in medium with both NO(3)(-) and NH(4)(+) synthesized some scytonemin but synthesis increased when NH(4)(+) was depleted and growth had become dependent on NO(3)(-) reduction. Although the mechanism is unclear, these results suggest that the greater the restriction in nitrogen accessibility, the greater the production of scytonemin. Perhaps the entire response may be an interaction between this restriction and a resultant sensitivity to UV radiation that acts as a cue for determining the level of scytonemin synthesis. Scytonemin is a stable UVR screening compound and appears to be synthesized by cyanobacteria as a long-term solution for reducing UVR exposure and damage, but mainly or solely, when metabolic activity is absent. It is likely that during metabolic resurgence, the presence of a dense scytonemin sheath would facilitate the recovery process without the need for active defenses against UV radiation.     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.     

Immunological characterization of hydrogenases in the nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 73102

N₂-fixing Nostoc sp. strain PCC 73102 was examined for the presence of hydrogenases. Native-PAGE/immunoblots demonstrated that two proteins with molecular masses of approximately 200 kDa and 215 kDa are immunologically related to hydrogenases purified from Bradyrhizobium japonicum, Azotobacter vinelandii, Methanosarcina barkeri, and Thiocapsa roseopersicina. SDS-PAGE/immunoblots showed that one polypeptide, with a molecular mass of about 58 kDa, is immunologically related to the hydrogenases purified from all the microorganisms mentioned above. In addition, two polypeptides, with molecular masses of approximately 34 and 70 kDa, are immunologically related to the hydrogenases purified from T. roseopersicina and M. barkeri respectively. Immunogold/transmission electron microscopy showed that the hydrogenase proteins are present in both the heterocysts and the vegetative cells.     

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.     

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.     

Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin

For the heterologous synthesis of keto-carotenoids such as astaxanthin, two carotenoid ketolase genes crtW38 and crtW148, were cloned from the cyanobacterium, Nostoc punctiforme PCC 73102 and functionally characterized. Upon expression in Escherichia coli, both genes mediated the conversion of beta-carotene to canthaxanthin. However in a zeaxanthin-producing E. coli, only the gene product of crtW148 introduced 4-keto groups into the 3,3'-dihydroxy carotenoid zeaxanthin yielding astaxanthin. The gene product of crtW38 was unable to catalyze this reaction. Both ketolases differ in their interaction with a hydroxylase in the biosynthetic pathway from beta-carotene to astaxanthin.     

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.     

Molecular genetics and genomic analysis of scytonemin biosynthesis in Nostoc punctiforme ATCC 29133

The indole-alkaloid scytonemin is the most common and widespread sunscreen among cyanobacteria. Previous research has focused on its nature, distribution, ecology, physiology, and biochemistry, but its molecular genetics have not been explored. In this study, a scytonemin-deficient mutant of the cyanobacterium Nostoc punctiforme ATCC 29133 was obtained by random transposon insertion into open reading frame NpR1273. The absence of scytonemin under conditions of induction by UV irradiation was the single phenotypic difference detected in a comparative analysis of the wild type and the mutant. A cause-effect relationship between the phenotype and the mutation in NpR1273 was demonstrated by constructing a second scytoneminless mutant through directed mutagenesis of that gene. The genomic region flanking the mutation revealed an 18-gene cluster (NpR1276 to NpR1259). Four putative genes in the cluster, NpR1274 to NpR1271, with no previously known functions, are likely to be involved in the assembly of scytonemin. Also in this cluster, there is a redundant set of genes coding for shikimic acid and aromatic amino acid biosynthesis enzymes, leading to the production of tryptophan and tyrosine, which are likely to be biosynthetic precursors of the sunscreen.     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Molecular analysis of genes in Nostoc punctiforme involved in pilus biogenesis and plant infection

Hormogonia are the infective agents in many cyanobacterium-plant symbioses. Pilus-like appendages are expressed on the hormogonium surface, and mutations in pil-like genes altered surface piliation and reduced symbiotic competency. This is the first molecular evidence that pilus biogenesis in a filamentous cyanobacterium requires a type IV pilus system.     

Cellular differentiation in the cyanobacterium Nostoc punctiforme

Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.     

A polyketide-synthase-like gene is involved in the synthesis of heterocyst glycolipids in Nostoc punctiforme strain ATCC 29133

A Tn5-1063-derived mutant of Nostoc punctiforme strain ATCC 29133 was unable to fix N₂ in air although it reduced acetylene in the absence of O₂. Mutant strain UCD 307 formed cells morphologically similar to heterocysts, but it failed to synthesize the characteristic heterocyst glycolipids. Sequence analysis around the site of insertion revealed an ORF of 3,159 base pairs, termed hglE. hglE putatively encodes a 115.4-kDa protein containing two domains with conserved amino acid sequences identified with acyl transferase and the chain length factor variation of β-ketoacyl synthase active sites. These active sites are characteristic of polyketide and fatty acid synthases. The N. punctiforme strain 29133 hglE gene is transcribed only under nitrogen-limiting growth conditions. The hglE gene, or similar sequences, was found in all other heterocyst-forming cyanobacteria surveyed and was absent in unicellular Synechococcus sp. strain PCC 7942. Based on these results, we propose that the synthesis of heterocyst glycolipids follows a pathway characteristic of polyketide synthesis and involves similar large, multienzyme complexes. Received: 4 November 1996 / Accepted: 6 January 1997     

A preliminary investigation of the Nostoc punctiforme proteome

Nostoc punctiforme ATCC 29133 is a filamentous terrestrial cyanobacterium (prokaryote) that expresses several different phenotypes in response to environmental cues. When grown in nitrogen-deficient media the most abundant proteins in addition to phycobiliproteins were superoxide dismutase, ATP synthase, and peptidyl-prolyl cis-trans isomerases. A methylated peptide from an akinete marker protein was also identified, suggesting that methylation could potentially play a regulatory role through signaling. C-phycocyanin alpha-chain was methylated at the C-terminal end of the protein and tandem mass spectrometric data also identified peptides that were deamidated. Since a significant number of putative polyketide/non-ribosomal peptide synthase genes are present in the annotated genome, an analysis of a methanolic extract of whole cells was also performed, and a series of nostopeptolides were identified.     

Partial Purification and Characterization of Ornithine Carbamoyl Transferase (OCT) from the Cyanobacterium Nostoc sp. Strain PCC 73102

Ornithine carbamoyl transferase (OCT) catalyzes the formation of citrulline and orthophosphate from ornithine and carbamoyl phosphate. We have partially purified OCT from the filamentous cyanobacterium Nostoc sp. strain PCC 73102, using ammonium sulfate precipitation (35–55%), a gel-filtration column (Sephacryl S-200), followed by an affinity column (Sepharose-6B-PALO). The partially purified OCT was analyzed on native-PAGE and shown to be an active enzyme with an estimated molecular weight of approximately 80 kDa. The isoelectric point was determined to be about 6.2. Varying the ornithine concentration resulted in a hyperbolic response of the reaction velocity at lower concentrations. Ornithine concentrations above 2 mM inhibited the enzyme. A hyperbolic response of the OCT reaction was observed when increasing the carbamoyl phosphate concentration. From a double reciprocal plot, a saturation concentration of 0.8 mM and a V_max of 0.4 U/mg may be calculated. None of the tested compounds (argininosuccinate, arginine, aspartic acid, urea) had any significant positive effect on the in vitro activity of the partially purified OCT. Moreover, at concentrations higher than 10 mM, all tested compounds had an inhibitory effect. Received: 23 March 1998 / Accepted: 6 May 1998     

The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133

Following exposure to long‐wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole‐alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 cotranscribed genes. A two‐component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator (RR), Npun_F1278, was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain Δ1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, Δ1278 was incapable of producing scytonemin. A phenotypic characterization of Δ1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid‐soluble pigment complement of Δ1278 compared to the wildtype. The mutant, however, had a slower specific growth rate under white light and produced ~2.5‐fold more phycocyanin per cell under UVA than the wildtype. Since Δ1278 does not produce scytonemin, this study demonstrates that the RR gene, Npun_F1278, is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production.