中国淡水水体常见束丝藻种类的形态及生理特性研究

束丝藻(Aphanizomenon Morr. ex Born. et Flah)是我国水华蓝藻的重要种类, 由其产生的束丝藻水华已经引起了许多的环境问题。水华束丝藻、柔细束丝藻和依沙束丝藻是我国淡水水体常见的三种束丝藻种类,然而, 国内外对它们的生理学研究却相对较少。基此, 文章对水华束丝藻、柔细束丝藻和依沙束丝藻的形态特性、色素含量、生长及光合作用进行了比较研究, 结果表明丝状体的营养细胞、异形胞和厚壁孢子的长宽比具有一定的差异性, 揭示了厚壁孢子的长宽比可作为三种束丝藻分类的一个参数; 同时, 种间的差异性也体现在三种束丝藻的生理特性上, 相比水华束丝藻和依沙束丝藻, 柔细束丝藻的藻蓝素含量较高, 而叶绿素a、类胡萝卜素含量、最大光合作用(Pm)、表观光合作用效率(α)和最大电子传递速率(ETRmax)显著偏低。此外, 水华束丝藻和依沙束丝藻生理特性基本一致, 表明了形态不同的三种束丝藻在生理上可被分为两种类型, 暗示了依沙束丝藻可能具有像水华束丝藻一样形成水华的生理潜能且由于它的产毒性所以应该倍加关注       

Nitrogen starvation mediated by DL-7-azatryptophan in the cyanobacterium Anabaena sp. strain CA.

The addition of DL-7-azatryptophan (AZAT), a tryptophan analog, to continuous cultures of Anabaena sp. strain CA grown with 10 mM nitrate as the nitrogen source resulted in the differentiation of heterocysts. Analysis of the intracellular amino acid pools of Anabaena sp. strain CA after the addition of AZAT showed a marked decline in the intracellular glutamate pool and a slight increase in the levels of glutamine. The in vitro activity of glutamate synthase, the second enzyme involved in primary ammonia assimilation in Anabaena spp., was partially inhibited by the presence of AZAT at concentrations which are effective in triggering heterocyst formation (15% inhibition at 10 microM AZAT and up to 85% inhibition at 1.0 mM AZAT). Azaserine, a glutamine analog and potent glutamate synthase inhibitor, had no effect on the triggering of heterocyst development from undifferentiated batch and continuous cultures of Anabaena sp. strain CA. However, the presence of 1.0 microM azaserine significantly decreased the intracellular glutamate pool and increased the glutamine pool. The addition of AZAT also caused a decrease in the C-phycocyanin content of Anabaena sp. strain CA as a result of its proteolytic degradation. AZAT also had an inhibitory effect on the nitrogenase activity of Anabaena sp. strain CA. All these results suggest that AZAT causes a general nitrogen starvation of Anabaena sp. strain CA filaments, triggering heterocyst synthesis.     

洱海螺旋鱼腥藻生长生理特性的初步研究

近年来,洱海正处于中营养水平向富营养湖泊的过渡阶段,蓝藻水华也频繁发生。本文作者在洱海大规模水华暴发期间,分离、纯化了水华优势种螺旋鱼腥藻,并对其生长生理特性进行了初步研究,以期为探讨洱海鱼腥藻水华发生的环境影响因素提供基础的参考资料。实验结果表明,氮含量1.5mmol/L、磷含量12µmol/L、光照强度30E/ m·s、pH值8－10及温度25℃时,螺旋鱼腥藻生长状况最好,生物量及相对生长速率较高。不同氮、磷浓度下的氮磷代谢活性表明：氮浓度在0－0.36mmol/L时,硝酸还原酶活性随着氮浓度的增加而增强;氮浓度在0.36－6 mmol/L时,酶活性由其生长状况决定,生长越好,酶活性越高。碱性磷酸酶受磷影响较大,随着磷浓度的增加其活性逐渐减弱,磷充足时,氮对其活性并无显著影响。此外,洱海螺旋鱼腥藻可在低的氮、磷浓度下生长,这与其氮磷代谢活性的调节作用有关。     

Two virus-like particles that cause lytic infections in freshwater cyanobacteria

Dear Editor, We report the results of the preliminary isolation of two virus-like particles (VLPs) that are infectious to freshwater cyanobacteria from Lake Donghu,the largest urban lake in China,located in Wuhan City,Hubei Province.By light and transmission electron microscopy,we observed VLPs causing lytic infections in freshwater bloom-forming cyanobacteria,and we detected their infections by exposing the VLPs to 12 cyanobacterial strains,including Microcystis aeruginosa HAB 1801 and Anabaena spiroides HAB 1211.They were termed ‘Anabaena spiroides geminivirus-like (AsGV-L)' particles and ‘Microcystis aeruginosa corticoviruslike (MaCV-L)' particles,based on their ultrastructural morphological characteristics and host specificities.     

Phylogenetic comparison among the heterocystous cyanobacteria based on a polyphasic approach

Phylogenetic comparison has been done among the selected heterocystous cyanobacteria belonging to the sections IV and V. The hierarchical cluster analysis based on antibiotics sensitivity showed a distant relationship between the members of Nostocales and Stigonematales. Thus, multiple antibiotic resistance pattern used as marker provide easy, fast, and reliable method for strain discrimination and genetic variability. However, morphological, physiological (both based on principal component analysis) and biochemical analysis grouped true branching cyanobacteria along with the members of section IV. Molecular analysis based on 16S rRNA gene sequences revealed that Hapalosiphon welwitschii and Westiellopsis sp. were grouped in cluster I whereas Scytonema bohnerii, a false branching genera showed a close proximity with Calothrix brevissima in cluster II. Cluster III of clade 2 included Nostoc calcicola and Anabaena oryzae which proved the heterogeneity at the generic level. Cluster IV the largest group of clade 2 based on 16S rRNA gene sequences includes six strains of the genera Nostoc, Anabaena, and Cylindrospermum showing ambiguous evolutionary relationship. In cluster IV, Anabaena sp. and Anabaena doliolum were phylogenetically linked by sharing 99% sequence similarity. Probably, they were of the same genetic makeup but appear differently under the diverse physiological conditions. Section IV showed polyphyletic origin whereas section V showed monophyletic origin. Results suggested that either morphological or physiological or biochemical or molecular attribute is not sufficient to provide true diversity and phylogeny of the cyanobacteria at the generic level and thus, a polyphasic approach would be more appropriate and reliable.     

六种固氮蓝藻提取液对玉米的促长作用和提取液成分比较

本文用六种固氮蓝藻的提取液处理玉米种子,同时对其提取液氨基酸组成和碳水化合物与维生素Ｂ１２的含量进行了分析。结果表明固氮鱼腥藻ＨＢ６８６（ＡｎａｂａｅｎａａｚｏｔｉｃａＨＢ６８６）、球孢鱼腥藻ＨＢ１０１７（Ａ．ｓｐｈａｅｒｉｃａＨＢ１０１７）、多变鱼腥藻ＨＢ１０５８（Ａ．ｖａｒｉａｂｉｌｉｓＨＢ１０５８）和小单歧藻ＨＢＴＴ（ＴｏｌｙｐｏｔｈｒｉｘｔｅｎｕｉｓＨＢＴＴ）的提取液中氨基酸、碳水化合物和维生素Ｂ１２的含量高于鱼腥藻ＳＰ．ＨＢ１０４２（Ａｎａｂａｅｎａｓｐ．ＨＢ１０４２）和繁育管链藻ＨＢ３８（ＡｕｌｏｓｉｒａｆｅｒｔｉｌｉｓｓｉｍａＨＢ３８）。同时,促进玉米种子萌发和幼苗生长的效果前四种藻较好,后两种则较差。     

鱼腥藻PCC 7120乙酰辅酶A合成酶All0769的缺失降低异形胞频率

研究鉴定了All0769为鱼腥藻PCC 7120中乙酰辅酶A合成酶,通过CRISPR/Cpf1系统敲除鱼腥藻PCC7120中的乙酰辅酶A合成酶(由all0769编码),探究了乙酰辅酶A合成酶在异形胞分化中的调控机制。结果所示:All0769能在体外反应中催化乙酰辅酶A的生成。在供氮环境下,敲除all0769会影响藻细胞生长速率。而无论环境中是否存在化合氮,Δall0769突变株的乙酰辅酶A和α-酮戊二酸含量均显著减少。在供氮环境下,Δall0769突变株中检测到(26.17±1.55) nmol/mg protein的乙酰辅酶A,而在野生型中检测出(43.04±1.09) nmol/mg的乙酰辅酶A。Δall0769突变株的α-酮戊二酸[(1.41±0.24) nmol/mg protein]低于野生型的α-酮戊二酸[(2.13±0.05) nmol/mg protein]。在缺氮环境下,Δall0769突变株中检测到(10.00±2.81) nmol/mg protein的乙酰辅酶A,而在野生型中检测出(29.82±4.04) nmol/mg protein的乙酰辅酶A。Δall07...     

Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from Anabaena sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant. The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR. The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.     

Septum-localized protein required for filament integrity and diazotrophy in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.     

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。     

Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential

Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields.     

The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.     

Nitrogenase activity of blue-green algae on seasonally flooded soils in Bangladesh

Blue-green algal communities formed an extensive cover on soils at five deepwater rice-growing locations in Bangladesh during the month before the arrival of floodwater. The principal taxa wereAnabaena, Cylindrospermum, Lyngbya, Microcoleus, Nostoc, Prophyrosiphon notarisii, Scytonema mirabile andTolypothrix byssoidea. One of two locations studied after the floodwaters had receded also had an extensive cover, mainlyScytonema mirabile. Nitrogenase activity assayed at mid-day was from one to three orders of magnitude higher per unit area of community than bare soil. Nostoc showed higher activity than Tolypothrix, whether expressed per unit area or biomass. Whole field estimates of N₂ fixed by blue-green algal communities during the pre-flood period ranged from 1.0 to 10.2 kg N ha^–1. Much of this is probably not recycled until floodwaters cover the fields. However N₂ fixed after floodwaters have receded is probably recycled rapidly due to ploughing.     

Comparison of the light-limited growth of the nitrogen-fixing cyanobacteria Anabaena and Aphanizomenon

The effect of simultaneous N₂ fixation and light limitation on the growth of two strains of Anabaena sp. Bory de St. Vincent and Aphanizomenon flos-aquae (L.) Ralfs was investigated using continuous cultures. Under severely light-limited conditions, Aphanizomenon showed a broader absorption spectrum (due to the presence of phycoerythrin), a higher maximum efficiency of photosynthesis, a higher steady-state N₂ fixation activity and a higher growth affinity for light than did Anabaena . On the other hand, under light saturation, Anabaena showed a higher maximum rate of O₂ production and a higher maximum specific growth rate than Aphanizomenon . These monoculture results characterize Anabaena and Aphanizomenon , in relative terms, as a 'sun' and a 'shade' species respectively, and are in accordance with field observations. The difference between the two species in their acclimatory response is discussed in terms of a species-specific alteration of the PSI:PSII stoichiometry. Besides the species-specific modulation of the accessory pigments, such an acclimation would provide a biochemical basis for the observed physiological differences. The monoculture results were used to differentiate the niches of the two species and suggested that Aphanizomenon would competitively displace Anabaena under N₂-fixing, light-limited conditions. However, when both species were grown together, Anabaena became dominant and seemed to be the superior competitor for light. In order to explain this finding, the possible effects of release of allelopathic compounds, or dynamic aspects of light supply, are discussed.     

Anabaena sp. strain PCC 7120 hetY gene influences heterocyst development

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.     

Cyanobacterial Microbiotic Crusts in Eroded Soils of a Tropical Dry Forest in the Baja California Peninsula,Mexico

This study deals with the cyanobacterial composition, and the nitrogen fixation of four members, of cryptobiotic crusts collected from eroded soils in a transitional area between arid and tropical climatic environments. Identification was based on microscopic analyses. Morphotypes were identified directly from reactivated natural crusts and from cultured strains. The identified morphotypes were Scytonema cf. ocellatum, Scytonema sp., Microcoleus cf. paludosus, M. cf. sociatus, Calothrix cf. elenkinii, C. cf. marchica, Nostoc cf. microscopicum, and Phormidium sp. Results show that the cyanobacterial composition of the microbiotic crusts studied is different from those in warmer and cooler deserts, particularly the absence of Microcoleus cf. vaginatus and Nostoc cf. commune in our samples. Such differences could be caused by the transitional character of the area. The results of the acetylene reduction assay show that the capacity of nitrogen fixation of some morphotypes is limited to the heterocyst-forming morphotypes.