Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.     

Boron Protection for O(2) Diffusion in Heterocysts of Anabaena sp. PCC 7119

The effect of boron on nitrogenase activity has been studied. When cells were dependent on N₂ fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization.     

The conformational stability and thermodynamics of Fur A (ferric uptake regulator) from Anabaena sp. PCC 7119

Fur (ferric uptake regulator) is a key bacterial protein that regulates iron acquisition and its storage, and modulates the expression of genes involved in the response to different environmental stresses. Although the protein is involved in several regulation mechanisms, and members of the Fur family have been identified in pathogen organisms, the stability and thermodynamic characterization of a Fur protein have not been described. In this work, the stability, thermodynamics and structure of the functional dimeric Fur A from Anabaena sp. PCC 7119 were studied by using computational methods and different biophysical techniques, namely, circular dichroism, fluorescence, Fourier-transform infrared, and nuclear magnetic resonance spectroscopies. The structure, as monitored by circular dichroism and Fourier-transform infrared, was composed of a 40% of alpha-helix. Chemical-denaturation experiments indicated that Fur A folded via a two-state mechanism, but its conformational stability was small with a value of DeltaG = 5.3 +/- 0.3 kcal mol(-1) at 298 K. Conversely, Fur A was thermally a highly stable protein. The high melting temperature (Tm = 352 +/- 5 K), despite its moderate conformational stability, can be ascribed to its low heat capacity change upon unfolding, DeltaCp, which had a value of 0.8 +/- 0.1 kcal mol(-1) K(-1). This small value is probably due to burial of polar residues in the Fur A structure. This feature can be used for the design of mutants of Fur A with impaired DNA-binding properties.     

Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.     

Effect of Sodium Deficiency on Ammonium-Grown Cells of Anabaena PCC 7119

This study examined how the absence of sodium affects growth,photosynthesis, photosynthetic pigments and superoxide dismutase(SOD) activity of ammonium-grown cells of AnabaenaPCC 7119 culturedunder low CO₂ levels (air) and alkaline pH. The growth rateswereseverely inhibited. After 24-h culture, photoreduction ofdioxygen was observed which, however, did not induce any photooxidationsuch as that found in sodium-deficient cells grown on nitratesor dinitrogen [Sanchez Maeso et al. (1987) Plant Physiol. 85:585–587]. In fact, SOD increasedsignificantly with sodiumdeficiency. No change was observed in the contents of photosyntheticpigments. (Received February 8, 1988; Accepted September 16, 1988)     

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

 A soluble monoheme c–type cytochrome c ₆ has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP⁺reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c ₆ cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c ₆ at pH 6.7 was determined to be 338 mV and is pH dependent, with pK _a ^ox=8.4±0.1 and pK _a ^red≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and ¹H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pK _a of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c ₆ is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation. Received: 17 May 1996 / Accepted: 13 January 1997     

CO2对转基因鱼腥藻生长的影响

为了进一步探索转基因鱼腥藻高密度培养的方法,在小型气升式反应器中研究了CO2对转基因鱼腥藻7120培养的影响。结果发现转基因鱼腥藻培养过程中通入5％ CO2能促进藻细胞生长,12d生物量提高7．44％,由于光照限制,不能大幅提高15d收获生物量,但生长周期能缩短近20％;而高浓度(10％)的CO2抑制转基因鱼腥藻的生长。CO2是通过调节pH值和影响碳源利用来影响藻细胞生长的,合适浓度的CO2有利于转基因鱼腥藻的培养。     

Anabaena sp. PCC 7119 flavodoxin as electron carrier from photosystem I to ferredoxin-NADP+ reductase. Role of Trp(57) and Tyr(94)

The influence of the amino acid residues sandwiching the flavin ring in flavodoxin (Fld) from the cyanobacterium Anabaena sp. PCC 7119 in complex formation and electron transfer (ET) with its natural partners, photosystem I (PSI) and ferredoxin-NADP(+) reductase (FNR), was examined in mutants of the key residues Trp(57) and Tyr(94). The mutants' ability to form complexes with either FNR or PSI is similar to that of wild-type Fld. However, some of the mutants exhibit altered kinetic properties in their ET processes that can be explained in terms of altered flavin accessibility and/or thermodynamic parameters. The most noticeable alteration is produced upon replacement of Tyr(94) by alanine. In this mutant, the processes that involve the transfer of one electron from either PSI or FNR are clearly accelerated, which might be attributable to a larger accessibility of the flavin to the reductant. However, when the opposite ET flow is analyzed with FNR, the reduced Y94A mutant transfers electrons to FNR slightly more slowly than wild type. This can be explained thermodynamically from a decrease in driving force due to the significant shift of 137 mV in the reduction potential value for the semiquinone/hydroquinone couple (E(1)) of Y94A, relative to wild type (Lostao, A., Gómez-Moreno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemistry 36, 14334-14344). The behavior of the rest of the mutants can be explained in the same way. Overall, our data indicate that Trp(57) and Tyr(94) do not play any active role in flavodoxin redox reactions providing a path for the electrons but are rather involved in setting an appropriate structural and electronic environment that modulates in vivo ET from PSI to FNR while providing a tight FMN binding.     

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

Purification and molecular properties of nitrite reductase from Anabaena sp. 7119

Ferredoxin-nitrite reductase (EC 1.7.7.1.) from the cyanobacteria Anabaena sp. 7119 has been purified 763-fold with a specific activity of 21.5 units/nig protein (0.358 μkatals/mg). The enzyme has a molecular mass of 52,000 daltons with a Stokes radius of 3.09 nm and a sedimentation coefficient of 4.07 S. The cellular level of nitrite reductase activity gradually increases in response to the addition of increasing amounts of iron to the culture medium.
When partially purified nitrite reductase preparations are subjected to sucrose-density-gradient centrifugation there is a dose correspondence between nitrite reductase activity and absorbance at 400 nm. This suggests the association of a heme chromophore with the enzyme. Furthermore, the presence of an iron-sulfur center is suggested by a close association of acid-labile sulfide with nitrite reductase activity. Carbon monoxide inhibits nitrite reductase activity. The nature and kinetics of this reaction are comparable to other siroheme-containing nitrite reductases.     

环境因子对转基因鱼腥藻培养的影响

摇瓶中对转TNF-α基因鱼腥藻7120（Anabaenasp.PCC7120,pDC-TNF)混合培养条件进行了研究,在含蔗糖9g/L,NaNO32.25g/L的BG-11培养基混合培养时,得到最适培养条件接种量5%,光照强度为1000Lux,光/暗周期（光照时间/黑暗时间）12h/12h,温度25-30℃,自然初始pH值,100mL摇瓶装液量40mL,转基因鱼腥藻15d生物量可达到3g/L以上,可溶性蛋白含量接近30%,TNF表达水平大于22%,与自养相比,生物量增加82.06%,表达水平提高38.79%,证明混合营养型培养是转rhTNF-α基因鱼腥藻7120实现高密度,高表达培养的途径。     

CW-EPR and ENDOR study of cytochrome c6 from Anabaena PCC 7119

The detailed analysis of the continuous-wave electron paramagnetic resonance and electron nuclear double resonance measurements on cytochrome c(6) from Anabaena PCC7119 reveals several electronic and structural properties of this hemeprotein. The oxidized protein shows two forms that differ in the arrangement of the residues that act as heme axial ligands. Information about the orientation of these residues is obtained for one of the forms, which turns out to differ from that found in the reduced protein from x-ray experiments. The biological significance of these results is discussed.     

Homology of the N-terminal domain of the petH gene product from Anabaena sp. PCC 7119 to the CpcD phycobilisome linker polypeptide

The complete nucleotide sequence of the petH gene encoding ferredoxin-NADP⁺ reductase from the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7119 has been determined. The encoded polypeptide is 136 amino acids longer than the enzyme obtained after purification to homogeneity. The extended N-terminal domain consists of 80 amino acids which shows homology to the CpcD phycobilisome linker polypeptide, through which FNR might be anchored to the thylakoid-bound phycobilisomes. A 56 amino acid interdomain fragment is found which could be a target for proteolysis.     

转基因鱼腥藻7120适宜生长条件的初步研究

以前的研究报导了将人肿瘤坏死因子基因(TNF-α)成功转入鱼腥藻7120中,这项研究在实验室小规模范围内探讨了其转TNF-α基因鱼腥藻7120的适宜生长条件。结果表明,转基因鱼腥藻7120最适生长温度在25～30℃,最适pH7.0～7.5。不同光照强度下的净光合放氧速率测定结果表明,转基因鱼腥藻7120与野生型具有一致的光饱和点和光补偿点,且适合在10～700μE.m-2.s-1下生长。外加有机碳源(4g/L葡萄糖)一定程度上可以增加转基因鱼腥藻7120生长速度。对不同藻龄转基因鱼腥藻7120中TNF-α的累积量的检测发现:生长8d左右时转基因鱼腥藻7120中TNF-α累积量达到最大值。这将为产业化生产、适时收获转基因蓝藻提供理论指导,同时讨论了葡萄糖对转基因鱼腥藻7120代谢的调节。     

植物生长调节剂对转基因鱼腥藻7120生长与外源基因表达的影响

适宜浓度的IBA、6-BA、赤霉酸和氯化胆碱均促进转基因鱼腥藻7120生长,提高其生物量,不影响外源基因表达,但从总体上讲高浓度植物生长调节剂则抑制藻细胞生长,降低外源基因表达水平.这些植物生长调节剂混合施用促进转基因鱼腥藻生长效果不如单一因子好,外源基因表达水平也略有下降.     

The primary structure of thioredoxin from the filamentous cyanobacterium Anabaena sp. 7119

Thioredoxin from the cyanobacterium Anabaena 7119 serves as electron donor to ribonucleotide reductase and as a protein disulfide reductase. This small, heat-stable protein was found to have structural and functional similarities to thioredoxins from both bacterial and mammalian sources. We here report the complete primary structure of Anabaena thioredoxin. The structure was determined by analysis of peptides obtained after cleavage with cyanogen bromide, Staphylococcus aureus protease, and trypsin. The protein consists of 106 residues with the following amino acid sequence: Ser-Ala-Ala-Ala-Gln-Val-Thr-Asp- Ser-Thr-Phe-Lys-Gln-Glu-Val-Leu-Asp-Ser-Asp-Val-Pro-Val-leu-Val-Asp-Phe- Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Arg-Met-Val-Ala-Pro-Val-Val-Asp-Glu- Ile-Ala-Gln-Gln-Tyr-Glu-Gly-Lys-Ile-Lys-Val-Val-Lys-Val-Asn-Thr-Asp- Glu-Asn-Pro-Gln-Val-Ala-Ser-Gln-Tyr-Gly-Ile-Arg-Ser-Ile-Pro-Thr-Leu- Met-Ile-Phe-Lys-Gly-Gly-Gln-Lys-Val-Asp-Met-Val-Val-Gly-Ala-Val-Pro- Lys-Thr-Thr-Leu-Ser-Gln-Thr-Leu-Glu-Lys-His-Leu. The sequence of Anabaena thioredoxin shows a definite homology to the protein from Escherichia coli, with 49% residue identities occurring in the proteins when aligned at the active site disulfide.     

Purification and properties of glutathione reductase from the cyanobacterium Anabaena sp. strain 7119.

An NADPH-glutathione reductase (EC 1.6.4.2) has been purified 6,000-fold to electrophoretic homogeneity from the filamentous cyanobacterium Anabaena sp. strain 7119. The purified enzyme exhibits a specific activity of 249 U/mg and is characterized by being a dimeric flavin adenine dinucleotide-containing protein with a ratio of absorbance at 280 nm to absorbance at 462 nm of 5.8, a native molecular weight of 104,000, a Stokes radius of 4.13 nm, and a pI of 4.02. The enzyme activity is inhibited by sulfhydryl reagents and heavy-metal ions, especially in the presence of NADPH, with oxidized glutathione behaving as a protective agent. As is the case with the same enzyme from other sources, the kinetic data are consistent with a branched mechanism. Nevertheless, the cyanobacterial enzyme presents three distinctive features with respect to that isolated from non-photosynthetic organisms: (i) absolute specificity for NADPH, (ii) an alkaline optimum pH value of ca. 9.0, and (iii) strong acidic character of the protein, as estimated by column chromatofocusing. The kinetic parameters are very similar to those found for the chloroplast enzyme, but the molecular weight is lower, being comparable to that of non-photosynthetic microorganisms. A protective function, analogous to that assigned to the chloroplast enzyme, is suggested.