Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.Photobiologically produced hydrogen gas (H₂) is a clean energy source with the potential to greatly supplement our use of fossil fuels (39). Whereas coal and oil are limited, cyanobacteria and eukaryotic microalgae can use inexhaustible sunlight as the energy source and water as the electron donor to produce H₂ (42). This gas is generated either by hydrogenases (52) or as an inevitable by-product of N₂ fixation by nitrogenases (49). In contrast to the reaction of hydrogenases which is reversible, nitrogenases catalyze the unidirectional production of H₂, although with substantial energy input in the form of ATP (47). Under optimal N₂-fixing conditions: N₂ + 8 e⁻ + 8 H⁺ + 16 ATP → H₂ + 2 NH₃ + 16 (ADP + P_i), whereas, in the absence of N₂ (e.g., under Ar), all electrons are allocated to proton reduction: 2 e⁻ + 2 H⁺ + 4 ATP → H₂ + 4 (ADP + P_i). Thus, one expects to be able to increase the H₂ production activity of nitrogenase by decreasing the electron allocation to N₂ fixation.Nitrogenases are sensitive to inactivation by O₂; however, N₂-fixing cyanobacteria have developed mechanisms to protect these enzymes from photosynthetically generated oxygen (5). Of particular interest, Anabaena (also known as Nostoc) sp. strain PCC 7120 and some other filamentous cyanobacteria respond to combined-nitrogen deprivation by undergoing differentiation in which a subset of cells become heterocysts that provide a microaerobic environment, allowing nitrogenase to function in aerobic culture conditions. The nitrogenase-related (nif) genes are specifically expressed in heterocysts which lack O₂-evolving photosystem II activity and are surrounded by a thick cell envelope composed of glycolipids and polysaccharides that impede the entry of O₂ (56). Vegetative cells perform oxygenic photosynthesis and fix CO₂. Heterocysts obtain carbohydrates from those cells and, in turn, provide them with fixed nitrogen.The molybdenum-containing nitrogenase of Anabaena sp. strain PCC 7120 consists of the Fe protein (encoded by nifH) and the MoFe protein (encoded by nifD and nifK). As in other organisms, the Fe protein is a homodimer containing a single [4Fe-4S] cluster and functions as an ATP-dependent electron donor to the MoFe protein. The latter is an α₂β₂ heterotetramer with each nifD-encoded α subunit coordinating the FeMo cofactor (FeMo-co; MoFe₇S₉X-homocitrate) that binds and reduces substrate, while α plus the nifK-encoded β subunits coordinate the [8Fe-7S] P-cluster (14). Additional nif genes are required for the biosynthesis of the metal clusters and maturation of the enzyme (40). The major nif gene cluster of Anabaena sp. strain PCC 7120 undergoes two rearrangements in the heterocyst to yield nifB-fdxN-nifSUHDK-(1 ORF)-nifENX-(2 ORFs)-nifW-hesAB-fdxH (19).One approach to increase H₂ production by nitrogenase is to enhance the electron flux to proton reduction and away from N₂ reduction. Although replacement of N₂ by Ar is effective for increasing H₂ production, this approach increases the operational cost for large-scale generation of H₂. Mutagenesis offers an alternative mechanism to overcome N₂ competition. The amino acid sequences of the MoFe α subunit are highly conserved among different phyla (18). The V75I substitution in the suspected gas channel of NifD2 of Anabaena variabilis (equivalent to V70 in A. vinelandii) resulted in greatly diminished N₂ fixation, while allowing for H₂ production rates (under N₂) that were similar to those of wild-type cells under Ar (55). Significantly, however, the nonheterocyst nitrogenase of this strain, which is expressed mainly in vegetative cells under anaerobic conditions, is incompatible with O₂-evolving photosynthesis and thus requires continuous anaerobic conditions along with a supply of exogenous reducing sugars for H₂ production. Substitutions of selected amino acids in the vicinity of the FeMo-co active site within Azotobacter vinelandii nitrogenase were shown to eliminate or greatly diminish N₂ fixation while, in some cases, allowing for effective proton reduction (2, 10, 17, 27, 36, 44, 45, 48). Therefore, certain amino acid exchanges near FeMo-co might produce variant MoFe proteins in heterocyst-forming Anabaena that redirect the electron flux through the enzyme preferentially to proton reduction so as to synthesize more H₂ in the presence of N₂ in an aerobic environment.To examine whether Anabaena sp. strain PCC 7120 nitrogenase can be modified to increase photobiological H₂ production by effecting such a redirection, we evaluated in vivo H₂ production and acetylene reduction rates of a series of cyanobacterial nifD site-directed mutants. We mutated six NifD residues (Fig. ​(Fig.1)1) predicted to lie within 5 Å of FeMo-co to create 49 variants using an Anabaena ΔNifΔHup (previously denoted ΔhupL) parental strain that lacks both an intact nifD and an uptake hydrogenase (34). In an atmosphere containing N₂ and O₂, several mutants exhibited significantly enhanced rates of in vivo H₂ production and accumulated high levels of H₂ compared to the reference strains.Open in a separate windowFIG. 1.Side-on (left) and Mo end-on (right) views of the predicted active site for nitrogenase of Anabaena sp. strain PCC 7120. The FeMo-co cluster, a [7Fe-8S-Mo-X-homocitrate] complex, where X is a central unidentified light atom (N, C, or O), and its two coordinating residues (C282 and H449) are shown in a ball-and-stick representation. Water molecules near the FeMo-co are indicated by isolated spheres in red. The side chains of the residues targeted for mutagenesis—Q193, H197, Y236, R284, S285, and F388—are shown in stick representation. Residues V362 through P367 are represented by lines. The Anabaena residues were mapped onto the corresponding residues from the crystal structure of the A. vinelandii enzyme (PDB file 1M1N). The figure was generated by using PyMOL (www.pymol.org/), with the following color scheme: Fe, orange; S, yellow; C, gray; N and central atom X, blue; O, red; and Mo, pink.     

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.     

鱼腥蓝细菌PCC7120铁吸收机制

铁离子是鱼腥蓝细菌PCC7120进行呼吸作用、光合作用和固氮作用中相关酶的重要辅基之一,缺铁将严重影响蓝细菌的生存.富氧的生态环境中铁通常以不溶的Fe3+形式存在,不易被细胞吸收利用.低铁条件下,鱼腥蓝细菌PCC7120分泌能螯合铁离子的嗜铁素,通过外膜上相应的转运体将嗜铁素-铁复合物转运到细胞内.综述了近年来在嗜铁素的种类及其生物合成途径、铁吸收系统的组成和功能等方面的最新进展,分析了铁吸收系统的调控机制,为进一步开展鱼腥蓝细菌铁吸收机制的研究提供依据.     

Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels.     

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.     

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。     

A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.     

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.     

Expression of Shewanella oneidensis MR-1 [FeFe]-Hydrogenase Genes in Anabaena sp. Strain PCC 7120

H₂ generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H₂ from water and sunlight using the cells'' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O₂ sensitive. Certain filamentous cyanobacteria protect nitrogenase from O₂ by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P_hetN. Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O₂.     

Detection of Glutamate Synthase in Heterocysts of Anabaena sp. Strain 7120

The formation of [¹⁴C]glutamate from [¹⁴C]glutamine in the presence of α-oxoglutarate was observed in extracts of heterocysts of Anabaena sp. strain 7120 under conditions that indicate the operation of glutamate synthase.     

Identification of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120 with different cleavage activities

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes that oxidatively cleave carotenoids into apocarotenoids. Dioxygenases have been identified in plants and animals and produce a wide variety of cleavage products. Despite what is known about apocarotenoids in higher organisms, very little is known about apocarotenoids and CCDs in microorganisms. This study surveyed cleavage activities of ten putative carotenoid cleavage dioxygenases from five different cyanobacteria in recombinant Escherichia coli cells producing different carotenoid substrates. Three CCD homologs identified in Nostoc sp. PCC 7120 were purified, and their cleavage activities were investigated. Two of the three enzymes showed cleavage of beta,beta-carotene at the 9,10 and 15,15' positions, respectively. The third enzyme did not cleave full-length carotenoids but cleaved the apocarotenoid beta-apo-8'-carotenal at the 9,10 position. 9,10-Apocarotenoid cleavage specificity has previously not been described. The diversity of carotenoid cleavage activities identified in one cyanobacteria suggests that CCDs not only facilitate the degradation of photosynthetic pigments but generate apocarotenals with yet to be determined biological roles in microorganisms.     

Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms

Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.     

The Insertion Sequences of Anabaena sp. Strain PCC 7120 and Their Effects on Its Open Reading Frames

Anabaena sp. strain PCC 7120, widely studied, has 145 annotated transposase genes that are part of transposable elements called insertion sequences (ISs). To determine the entirety of the ISs, we aligned transposase genes and their flanking regions; identified the ISs'' possible terminal inverted repeats, usually flanked by direct repeats; and compared IS-interrupted sequences with homologous sequences. We thereby determined both ends of 87 ISs bearing 110 transposase genes in eight IS families (http://www-is.biotoul.fr/) and in a cluster of unclassified ISs, and of hitherto unknown miniature inverted-repeat transposable elements. Open reading frames were then identified to which ISs contributed and others—some encoding proteins of predictable function, including protein kinases, and restriction endonucleases—that were interrupted by ISs. Anabaena sp. ISs were often more closely related to exogenous than to other endogenous ISs, suggesting that numerous variant ISs were not degraded within PCC 7120 but transferred from without. This observation leads to the expectation that further sequencing projects will extend this and similar analyses. We also propose an adaptive role for poly(A) sequences in ISs.Insertion sequences (ISs) are transposable elements found in prokaryotic and eukaryotic genomes (17). A fully functional bacterial IS comprises one or more transposase genes, ends that are often inverted repeats (IRs), and, between the transposase genes and the ends, sequences termed linkers (32). Diverse bacterial ISs have been classified, and a searchable database of ISs has been constructed (ISfinder [http://www-is.biotoul.fr/]) (28). Miniature inverted-repeat transposable elements (MITEs) and even smaller mobile elements lack their own transposases and are also found in Anabaena spp. (11, 12, 33).Anabaena sp. strain PCC 7120 (also known as Nostoc sp. [25], here denoted Anabaena sp.) is widely used to study the patterned differentiation of dinitrogen-fixing cells called heterocysts. Transposition of ISs in Anabaena sp. has been documented (1, 7-9). We earlier reported, with few details, three genes that are intercepted by ISs in Anabaena sp. (23). We here describe the approach more extensively, organize the ISs of Anabaena sp., and present our efforts to identify Anabaena sp. open reading frames (ORFs) interrupted or contributed to by ISs.     

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.