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.     

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.     

Patterns of inflammation and the use of reversibility testing in smokers with airway complaints

Background

Although both smoking and respiratory complaints are very common, tools to improve diagnostic accuracy are scarce in primary care. This study aimed to reveal what inflammatory patterns prevail in clinically established diagnosis groups, and what factors are associated with eosinophilia.

Method

Induced sputum and blood plasma of 59 primary care patients with COPD (n = 17), asthma (n = 11), chronic bronchitis (CB, n = 14) and smokers with no respiratory complaints ('healthy smokers', n = 17) were collected, as well as lung function, smoking history and clinical work-up. Patterns of inflammatory markers per clinical diagnosis and factors associated with eosinophilia were analyzed by multiple regression analyses, the differences expressed in odds ratios (OR) with 95% confidence intervals.

Results

Multivariately, COPD was significantly associated with raised plasma-LBP (OR 1.2 [1.04–1.37]) and sTNF-R55 in sputum (OR 1.01 [1.001–1.01]), while HS expressed significantly lowered plasma-LBP (OR 0.8 [0.72–0.95]). Asthma was characterized by higher sputum eosinophilic counts (OR 1.3 [1.05–1.54]), while CB showed a significantly higher proportion of sputum lymphocytic counts (OR 1.5 [1.12–1.9]). Sputum eosinophilia was significantly associated with reversibility after adjusting for smoking, lung function, age, gender and allergy.

Conclusion

Patterns of inflammatory markers in a panel of blood plasma and sputum cells and mediators were discernable in clinical diagnosis groups of respiratory disease. COPD and so-called healthy smokers showed consistent opposite associations with plasma LBP, while chronic bronchitics showed relatively predominant lymphocytic inflammation compared to other diagnosis groups. Only sputum eosinophilia remained significantly associated with reversibility across the spectrum of respiratory disease in smokers with airway complaints.     

Effects of mechanical uncouplers, diacetyl monoxime, and cytochalasin-D on the electrophysiology of perfused mouse hearts

Chemical uncouplers diacetyl monoxime (DAM) and cytochalasin D (cyto-D) are used to abolish cardiac contractions in optical studies, yet alter intracellular Ca(2+) concentration ([Ca(2+)](i)) handling and vulnerability to arrhythmias in a species-dependent manner. The effects of uncouplers were investigated in perfused mouse hearts labeled with rhod-2/AM or 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) to map [Ca(2+)](i) transients (emission wavelength = 585 +/- 20 nm) and action potentials (APs) (emission wavelength > 610 nm; excitation wavelength = 530 +/- 20 nm). Confocal images showed that rhod-2 is primarily in the cytosol. DAM (15 mM) and cyto-D (5 microM) increased AP durations (APD(75) = 20.0 +/- 3 to 46.6 +/- 5 ms and 39.9 +/- 8 ms, respectively, n = 4) and refractory periods (45.14 +/- 12.1 to 82.5 +/- 3.5 ms and 78 +/- 4.24 ms, respectively). Cyto-D reduced conduction velocity by 20% within 5 min and DAM by 10% gradually in 1 h (n = 5 each). Uncouplers did not alter the direction and gradient of repolarization, which progressed from apex to base in 15 +/- 3 ms. Peak systolic [Ca(2+)](i) increased with cyto-D from 743 +/- 47 (n = 8) to 944 +/- 17 nM (n = 3, P = 0.01) but decreased with DAM to 398 +/- 44 nM (n = 3, P < 0.01). Diastolic [Ca(2+)](i) was higher with cyto-D (544 +/- 80 nM, n = 3) and lower with DAM (224 +/- 31, n = 3) compared with controls (257 +/- 30 nM, n = 3). DAM prolonged [Ca(2+)](i) transients at 75% recovery (54.3 +/- 5 to 83.6 +/- 1.9 ms), whereas cyto-D had no effect (58.6 +/- 1.2 ms; n = 3). Burst pacing routinely elicited long-lasting ventricular tachycardia but not fibrillation. Uncouplers flattened the slope of AP restitution kinetic curves and blocked ventricular tachycardia induced by burst pacing.     

Multiple mechanisms of reduced major histocompatibility complex class II expression in endotoxin tolerance

Patients after polytrauma, burns, or septic shock frequently develop a life-threatening immunodeficiency. This state is associated with specific functional alterations of monocytic cells. We previously proposed endotoxin tolerance, the monocyte state after acute response to lipopolysaccharide, as a respective model system. One major feature in both the clinical situation and the in vitro model is the dramatic down-regulation of monocyte major histocompatibility complex (MHC) class II surface expression, which is associated with impaired antigen presentation capacity. This study focused on the mechanisms behind reduced MHC class II expression in endotoxin tolerance. Endotoxin priming provoked a decrease of monocyte intracellular MHC class II. It also led to a reduced expression of the chaperonic invariant chain and to an inhibited synthesis of the major lysosomal enzyme for final cleavage of the invariant chain going along with a relative accumulation of p10. The expression of HLA-DM necessary for loading MHC class II with antigenic peptide was also decreased. Additionally, reduced export of MHC class II alphabeta complexes to the cell surface was observed. The down-regulation of HLA-DR, invariant chain, and HLA-DM was regulated at the mRNA level and may be the consequence of reduced class II transactivator expression observed in this study. The simultaneous interference at different regulatory levels may explain the uniquely strong and long lasting MHC class II down-modulating effect of endotoxin priming compared with transforming growth factor-beta and interleukin-10. These results not only contribute to a better understanding of experimental endotoxin tolerance but may also give rise to new therapeutics for temporary immunodeficiency and, conversely, for MHC class II-dependent diseases such as autoimmunity and transplant rejection.     

Cutting edge: immune cells as sources and targets of the IL-10 family members?

This study investigated the expression of five novel human IL-10-related molecules and their receptors in blood mononuclear cells. IL-19 and IL-20 were found to be preferentially expressed in monocytes. IL-22 and IL-26 (AK155) expression was exclusively detected in T cells, especially upon type 1 polarization, and in NK cells. IL-24 (melanoma differentiation-associated gene 7) expression was restricted to monocytes and T cells. Detection of these molecules in lymphocytes was predominantly linked to cellular activation. Regarding T cells, IL-26 was primarily produced by memory cells, and its expression was independent on costimulation. In contrast to the high expression of receptors for IL-10 homologs in different tissues and cell lines, monocytes and NK, B, and T cells showed clear expression only of IL-10R1, IL-10R2, and IL-20R2. In these cells, IL-20R2 might be part of a still-unknown receptor complex. Therefore, immune cells may represent a major source but a minor target of the novel IL-10 family members.     

Phylogeny of the genus trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species of Trichoderma sect. Pachybasium. To this end, 85 strains-including all the available ex-type strains-were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 including T. polysporum, T. piluliferum, and T. minutisporum, while A2 included T. hamatum, T. pubescens, and T. strigosum in addition to species previously included in sect. Trichoderma (i.e., T. viride, T. atroviride, and T. koningii). The ex-type strain of T. fasciculatum formed a separate branch basal to clade A. Clade B contained the sect. Pachybasium members T. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale, and the anamorphs of Hypocrea semiorbis and H. cf. gelatinosa. Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them and T. longibrachiatum, which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e., T. virens and T. flavofuscum; T. strictipile and H. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g., T. crassum and T. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile, and H. semiorbis; T. hamatum and T. pubescens; and T. viride, T. atroviride, and T. koningii) differences were in the range of 1-3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group of T. harzianum strains (Th2) was strikingly different from that of the ex-type strain of T. harzianum and closely related species and is likely to be a separate species. Copyright 1998 Academic Press.     

Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing.

Transposon-generated mutant N10 of Anabaena sp. strain PCC 7120 has a Het- phenotype (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation reproduced a Het- phenotype, but reconstructions with other insertions at the position of the transposon produced strains that form multiple contiguous heterocysts. Sequence analysis around the site of insertion of the transposon showed that the insertion lies within the 5' end of an 861-bp open reading frame (ORF) (hetN). The product of translation of hetN (HetN) shows extensive similarity to NAD(P)H-dependent oxidoreductases that are involved in biosyntheses of fatty acids, poly-beta-hydroxybutyrate, nod factor, and polyketides. A second, 1,518-bp ORF (hetM) that ends 556 bp 5' from the start of hetN appears to encode a protein that has at least two functional domains: its amino terminus is similar to an acyl carrier protein, while its central portion is similar to domains of proteins that perform reductive reactions. A third, 711-bp ORF (hetI) encoded on the opposite strand ends 42 bp away from the 3' end of hetN. The protein encoded by hetI, HetI, is similar to Sfp from Bacillus subtilis and EntD from Escherichia coli, proteins that are required for the biosynthesis or export of cyclic peptides. Clones from a lambda-EMBL3 library that contain the wild-type DNA for hetN do not complement the hetN::Tn5-1063 mutation in N10. The presence of hetN, as the only ORF, on a replicating plasmid suppresses heterocyst formation in wild-type cells, whereas the additional presence of hetI alleviates this effect.     

Conduction of pEC22, a plasmid coding for MR.EcoT22I, mediated by a resident Tn3-like transposon, Tn5396.

pEC22 is a small plasmid that encodes the restriction-modification system MR.EcoT22I. Restriction and functional analysis of the plasmid identified the positions of genes encoding that system. The plasmid is able to be conducted by conjugal plasmids, a process mediated by a transposon contained within pEC22. This cryptic transposon, called Tn5396, was isolated from pEC22 and partially sequenced. The sequence of Tn5396 is for the most part typical of transposons of the Tn3 family and is most similar to that of Tn1000. The transposon differs from closely related transposons in that it lacks well-conserved sequences in the inverted-repeat region and has an unusually long terminal inverted repeat. Consideration of regions of internal sequence similarity in this and other transposons in the Tn3 family supports a theory of the mechanism by which the ends of Tn3-like transposons may maintain substantial identity between their inverted repeats over the course of evolutionary time.     

Transfer of a genetic marker from a megaplasmid of Anabaena sp. strain PCC 7120 to a megaplasmid of a different Anabaena strain.

The 410-kb alpha megaplasmid of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was found to bear the nucA gene that encodes a sugar-nonspecific nuclease. That gene was mutated by insertion of a cassette that confers resistance to neomycin. The resulting strain, AMP2, was mated with a streptomycin-resistant derivative of Anabaena sp. strain PCC 7118, a strain that does not form heterocysts. Cells resistant to both neomycin and streptomycin that were derived from such matings were found to bear the neomycin resistance cassette of the donor strain in a larger megaplasmid characteristic of the recipient strain and did not form heterocysts. This is the first example of transfer of a genetic marker directly between strains of cyanobacteria in which incontrovertible physical evidence of transfer has been obtained. DNA sequences homologous to the nucA gene were present in 13 heterocyst-forming cyanobacteria that were tested but in none of six diverse unicellular strains that were examined.