Genetic analysis of the het and nif genes in the blue-green alga Nostoc muscorum

Summary Two multiply marked complementary strains namely Het ⁺ Nif⁺ Str-R and Het ^- Nif^- Ery-R MSO-R were constructed and crossed under conditions counterselective for the Het ⁺ Nif⁺ Str-R parent and selective only for recombinants of Str-R and Ery-R or Str-R and MSO-R constitution. The results of the recombinant analysis with regard to the selected and unselected markers suggested that the Het ^- Nif^- Ery-R MSO-R parent acted as a recipient and the Het ⁺ Nif⁺ Str-R parent as donor of the genetic markers in the cross. The joint inheritance of Het ⁺ and Nif ⁺ unselected markers among the recombinants was found to occur more frequently than the inheritance of the Het ⁺ or Nif ⁺ markers alone. The observed joint inheritance of Het ⁺ and Nif ⁺ markers among the recombinants probably results from the inheritance of the regulatory gene(s) required for the activation of latent het and nif genes. This interpretation is fully supported by (a) the frequency distribution of unselected Het ⁺ and Nif ⁺ markers and (b) the reversion frequency of Het ^- Nif ^- strains to Het ⁺ Nif⁺ prototrophy. Accordingly the apparent close genetic linkage of het and nif genes is not due to their organization in a single operon but to their common regulation by regulatory gene(s) of a positive control nature. The Het ⁺ Nif⁺ wild type, mutant, revertant, and recombinant strains all appear similar in their NO ₃ ^- repression of both heterocyst and nitrogenase. The Het ⁺ Nif^- and Het ^- Nif⁺ recominants also show similar NO ₃ ^- repression of their heterocyst and nitrogenase respectively. The presence of only microaerobic acetylene reducing activity in Het ^- Nif⁺ recombinants clearly indicates the heterocyst to be an organ for protection of nitrogenase against oxygen toxicity.Abbreviations CFU Colony forming units - Ery erythromycin - Ery-R erythromycin resistance - het genotypic designation of genes required for heterocyst differentiation - Het phenotype designation of genes required for heterocyst differentiation - MSO l-Methionine-dl-sulfoximine - MSO-R MSO-resistance - N₂ medium Chu 10 medium without combined nitrogen - NH ₄ ⁺ medium basic mineral medium with ammonium nitrogen - nif genotype designation of genes required for N₂ fixation - Nif phenotype designation of genes required for N₂ fixation - NO ₃ ^- medium Chu 10 medium supplemented with KNO₃ - NTG N-methyl-N-nitro-N-nitrosoguanidine - r gene(s) regulatory gene(s) - Str streptomycin - Str-R streptomycin resistance - Str-S streptomycin sensitive     

发状念珠藻醛酮还原酶基因NfAKR的克隆与表达

以发状念珠藻细胞为试材,采用PCR技术克隆了醛酮还原酶基因的开放阅读框(ORF)序列,命名为NfAKR。对基因序列特征进行了生物信息学分析,根据其编码氨基酸序列预测了NfAKR蛋白的三维结构,同时探讨了PEG-6000胁迫下NfAKR的表达特性。结果表明:NfAKR基因的编码序列长912bp,编码304个氨基酸,预测其编码蛋白的相对分子量为33.51kD,理论等电点为4.94,具有醛酮还原酶超家族保守结构域。NfAKR蛋白主要由10个α-螺旋和11β-折叠组成,中间形成一个疏水穴,作为酶的催化活性中心。NfAKR与点形念珠藻处在同一进化枝上,具有较近的亲缘关系。qRT-PCR分析显示,PEG-6000胁迫下NfAKR基因上调表达,当PEG-6000浓度为8%时,其相对表达量为5.66并达到峰值。依据NfAKR基因响应干旱胁迫上调表达的特性,推测醛酮还原酶可能参与发状念珠藻抵御干旱胁迫过程。     

发状念珠藻干旱胁迫响应基因NfGR的克隆与鉴定

该研究采用PCR技术,从发状念珠藻细胞中克隆了谷胱甘肽还原酶(glutathione reductase,GR)基因,命名为NfGR,其开放阅读框长1 374bp,编码458个氨基酸,蛋白相对分子量为49.42kD,理论等电点为5.49。氨基酸序列分析表明,NfGR蛋白具有NADPH结合位点超家族(NADB-Rossmann superfamily)和吡啶氧化还原酶二聚体超家族(Pyr_redox_dim superfamily)2个结构域,与点形念珠藻(Nostoc punctiforme)的相似性达93%。系统进化树分析表明,NfGR与点形念珠藻处在同一进化枝上,亲缘关系较近。qRT-PCR表达分析表明,在不同浓度PEG-6000处理下,NfGR基因均保持上调表达,其中,PEG-6000浓度为8%时,NfGR基因的相对表达量达到峰值(32.69)。研究推测,谷胱甘肽还原酶可能参与了发状念珠藻对干旱胁迫过程的响应。     

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.     

三孢布拉霉crgA启动子的克隆和活性分析

【背景】CrgA是三孢布拉霉(Blakesleatrispora,Bt)中调控类胡萝卜素合成的关键负调控因子,其表达水平会影响类胡萝卜素的合成。【目的】克隆三孢布拉霉crgA启动子并分析其活性,为进一步解析CrgA表达调控机制奠定基础。【方法】通过综合微生物基因组(integrated microbial genomes, IMG)数据库提供的基因组序列,克隆crgA翻译起始位点上游2 000 bp序列,分析其顺式调控元件和转录起始区域预测,通过RT-qPCR分析不同光照时间对三孢布拉霉crgA相对转录水平的影响;构建4个不同长度的crgA启动子截短序列驱动的GUS-mGFP5重组表达载体p1303-procrgAF、F1、F2和F3,利用农杆菌侵染整合到三孢布拉霉基因组中,在黑暗和光照条件下测定β-D-葡萄糖苷酸酶(β-D-glucuronidase,GUS)酶活性并观察荧光信号。【结果】crgA启动子不仅包含基础的TATA-box和CAAT-box元件,还包括多个与光响应相关的元件。观察荧光结果显示CaMV35S和构建的4个突变启动子均能在三孢布拉霉体内驱动下游基因表达,检测GUS...     

Characterization of the thermophilic isoamylase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Isoamylase catalyzes the hydrolysis of -1,6-glucosidic linkages of starch and related polysaccharides. In this study, the treX gene (GenBank accession no. AE006815 REGION: 9279 … 11435) encoding the thermophilic isoamylase was PCR-cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7lac promoter. Both wild-type and His-tagged isoamylases were expressed in Escherichia coli. The wild-type isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4 °C. They had an apparent optimal pH of 5 and an optimal temperature at 75 °C. The enzyme activities remained unchanged after a 2 h incubation at 80 and 75 °C for the wild-type and His-tagged enzymes, respectively. These thermophilic isoamylases showed a potential to be used in industry to degrade the branching points of starch at a high temperature.     

Light intensity determines the extent of mercury toxicity in the cyanobacterium Nostoc muscorum

The extent of mercury (Hg) toxicity in the heterocystous cyanobacterium Nostoc muscorum grown for 72 h in three different light intensities was tested for various physiological parameters viz. growth, pigment contents, photosynthesis, respiration, reactive oxygen species (ROS), malondialdehyde formation and antioxidants. A general reduction in growth and pigments, whole cell O₂-evolution, photosynthetic electron transport activities and ¹⁴CO₂-fixation was observed in a metal concentration–dependent manner, and this effect was more pronounced in high light (130 μmol photon m⁻² s⁻¹)–exposed cells as compared to low (10 μmol photon m⁻² s⁻¹) and normal (70 μmol photon m⁻² s⁻¹) light intensity–exposed cells; however, carotenoids and respiration showed reverse trend. Among photosynthetic electron transport activities, whole chain activity was found to be most sensitive in comparison with photosystem II (PS II) and photosystem I (PS I). Comparing the different photosynthetic processes, ¹⁴CO₂-fixation was most affected in cyanobacterial cells when exposed to Hg and different light intensities. After application of various exogenous electron donors, diphenyl carbazide was found to be more effective to restore PS II activity, suggesting that site of damage lies in between oxygen evolving complex and PS II. Level of oxidative stress (superoxide radical and lipid peroxidation) was maximum at 3.0 μM of Hg when coupled with high light intensity (except hydrogen peroxide). A dose-dependent increase in enzymatic antioxidants such as superoxide dismutase, peroxidase and catalase as well as non-enzymatic antioxidants such as proline, ascorbate, cysteine (except under high light intensity) and non-protein thiols [NP-SH] was observed, which further increased with the increase in light intensity. It was noticed that Hg intoxicates N. muscorum through ROS production, which is aggravated along with the increase in light intensity. Overall results suggest that the severity of the metal stress does increase with Hg concentrations but when coupled with light, it was the light intensity that determines the extent of Hg toxicity.     

Histidine sensitive strain of the blue-green alga Nostoc muscorum: possible nif-his interactions

Summary A variant strain of the nitrogen-fixing bluegreen alga Nostoc muscorum, displaying impairment in its elemental nitrogen dependent growth and complete inhibition of growth by L-histidine in otherwise nitrogen-free medium, has been isolated and characterized for its response to L-glutamic acid and L-glutamine in presence of other inorganic nitrogen sources. A model based on the possibility of nif-his interaction has been proposed to account for the observed behaviour of the strain. It is inferred that the two sets of genes may occupy neighbouring positions on the blue-green algal genome.     

Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Genetic control of ammonium transport in nitrogen fixing cyanobacterium Nostoc muscorum

Summary Ammonium (NH ₄ ⁺ ) transport was investigated in Nostoc muscorum ISU (wild type) and spontaneous mutants resistant to cyanophage N-1 (Nm/N-1), streptomycin (Nm/Sm) and methylamine (Nm/MA). N₂-fixing wild-type cells transported NH ₄ ⁺ via two transport systems: the high-affinity (K _m 11 M) and low-affinity (K _m 66 M), which formed 10 and 50-fold concentration gradients, respectively. The high-affinity system of Nm/MA (K _m 11 M) was similar to the wild type but the low-affinity system had reduced affinity for NH ₄ ⁺ (K _m 125 M), while Nm/N-1 and Nm/Sm mutants had only a high-affinity transport system (K _m 20 and 28 M, respectively). The growth of mutant Nm/N-1 was more sensitive to 1 mM NH ₄ ⁺ or methylamine than other strains, and also glutamine-synthetase activity was most reduced in NH ₄ ⁺ -grown cells. l-methionine-d, l-sulfoximine (20 M) treatment of N₂-grown Nm/N-1 cells resulted in a higher rate of NH ₄ ⁺ efflux. The apparent alterations in kinetic constants of NH ₄ ⁺ transport in mutants and glutamine synthetase activity suggested that NH ₄ ⁺ in N. muscorum is transported by specific carrier(s) and the transport is genetically controlled.     

An ATP-grasp ligase involved in the last biosynthetic step of the iminomycosporine shinorine in Nostoc punctiforme ATCC 29133

We investigated the genetic basis for mycosporine sunscreen biosynthesis by the cyanobacterium Nostoc punctiforme ATCC 29133. Heterologous expression in Escherichia coli of three contiguous N. punctiforme genes (NpR5600, NpR5599, and NpR5598, here named mysA, mysB, and mysC, respectively) led to the production of mycosporine-glycine, an oxomycosporine. Additional expression of gene NpF5597 (mysD) led to the conversion of mycosporine-glycine into iminomycosporines (preferentially shinorine but also others like mycosporine-2-glycine and porphyra-334). This represents a new mode of enzymatic synthesis for iminomycosporines, one that differs in genetic origin, mechanism, and apparent substrate specificity from that known in Anabaena variabilis ATCC 29413. These results add to the emerging profile of the protein family of ATP-dependent ligases, to which the mysC product belongs, as important condensation enzymes in microbial secondary metabolism.     

A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133

A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H₂O₂ could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

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     

不同失水状态发菜类囊体膜蛋白的分离及鉴定

采用液氮研磨与超声波破碎相结合的方法,破碎不同失水状态的发菜藻体和细胞,用差速离心法制备发菜类囊体膜粗制品,蔗糖密度梯度高速离心纯化类囊体膜,SDS-PAGE电泳分离类囊体膜蛋白,并对膜蛋白进行了MALDI-TOF-TOF/MS质谱分析和鉴定。结果表明:(1)充分吸胀4h后失水6h的发菜(含水量51.2%)和失水24h的发菜(含水量14.9%),经过多步差速离心后再进行蔗糖密度梯度高速离心,可得到纯化的类囊体膜。(2)发菜类囊体膜蛋白SDS-PAGE电泳分离到14个条带,共鉴定出8种蛋白,根据其功能可分为4类——光合作用相关蛋白(光系统Ⅱ锰稳定蛋白PsbO,F1F0 ATP合成酶α亚基和β亚基)、结构域蛋白、选择性通道蛋白OprB、未知蛋白(hypothetical protein Npun_R1321、Npun_R3785、N9414_02186),它们在发菜的光合作用中具有重要作用。