鱼腥藻(Anabaena 7120)DNA的转化作用

用化学方法从固氮蓝藻鱼腥藻(Anabaena 7120)细胞中有效地提取了DNA,以此为供体DNA,用它的氧敏感固氮突变种鱼腥藻一1(Anabaena-l)为受体进行转化实验。在大量的转化实验中,仅有两次获得转化后的突变种在空气中、在无氮培养基上能生长,其转化频率为10~(-6)—10~(-5)。转化子在有氧条件下的乙炔还原活力相当于野生种。它表明突变种的除氧系统通过转化而得到恢复。推测鱼腥藻7120突变种可能具有吸收和整合外源DNA的能力。对丝状蓝藻转化困难的原因进行了探讨,结果表明受体藻胞外DNA酶活

鱼腥藻(Anabaena)对氧敏感的固氮突变种

用紫外线和亚硝基胍,诱变筛选出两株具异形胞而不能在空气中固定分子氮的鱼腥藻Anabaena)突变种。突变表型是稳定的。在微氧条件下,能固定分子氮而生长,但固氮酶活很弱。酶活的高低与藻蓝素含量和光照强度成反相关。突变种细胞的固氮酶对氧非常敏感,反应气相中加入1%的氧对乙炔还原活力已有明显抑制,20%的氧完全阻抑固氮作用。除去氧后固氮酶重新合成,其过程受 NH_4~+和氯霉素阻遏。通过氮饥饿使藻蓝素含量降低,或降低光照强度,或加入二氯苯基甲基脲抑制光合放氧时,均可显著地提高突变种的固氮酶活力。突变种细胞还原氯

Outer membrane proteins induced by iron deficiency in Anabaena sp PCC 7120

Iron deficiency can induce cyanobacteria to synthesize siderophore receptor proteins on the outer membrane to enhance the uptake of iron. In this study, an outer membrane of high purity was prepared from Anabaena sp. PCC 7120 based on aqueous polymer two-phase partitioning and discontinuous sucrose density ultra-centrifugation, and the induction of outer membrane proteins by iron deficiency was investigated using 2-D gel electrophoresis. At least. five outer membrane proteins were newly synthesized or significantly up-regulated in cells transferred to iron-deficient conditions, which were all identified to be siderophore receptor proteins according to MALDI-TOF-MS analyses. Bacterial luciferase reporter genes luxAB were employed to monitor the transcription of the encoding genes. The genes were induced by iron deficiency at the transcriptional level in different responsive modes. Luciferase activity expressed from an iron-regulated promoter may be used as a bioreporter for utilizable iron in natural water samples. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Mouse Toxicity of Anabaena flos-aquae from Lake Dianchi, China

Some species of the genera Anabaena can produce various kinds of cyanotoxins, which may pose risks to environment and human health. Anabaena has frequently been observed in eutrophic freshwater of China in recent years, but its toxicity has been reported only in a few studies. In the present study, the toxicity of an Anabaena flos-aquae strain isolated from Lake Dianchi was investigated. Acute toxicity testing was performed by mouse bioassay using crude extracts from the lyophilized cultures. The mice exposed to crude extracts showed visible symptoms of toxicity and died within 10-24 h of the injection. Serum biochemical parameters were evaluated by the use of commercial diagnostic kits. Significant alterations were found in the serum biochemical parameters: alkaline phosphatase (AKP), gamma-glutamyl transpepticlase (gamma-GT), aspartate amino transferase (AST), alanine amino transferase (ALT), AST/ALT ratio, total protein content, albumin content, albumin/globulin (A/G) ratio, blood urea nitrogen (BUN), serum creatinine (Ssr), and total antioxidative capacity (T-AOC). Histopathological observations were carried out with hematoxylin and eosin (HE) stain under light microscope. Severe lesions were seen in the livers, kidneys, and lungs of the mice injected with crude extracts. The alterations of biochemical parameters were in a dose-dependent manner, and the severities of histological lesions were in the same manner. Based on biochemical and histological studies, this research firstly shows the presence of toxin-producing Anabaena species in Lake Dianchi and the toxic effects of its crude extracts on mammals. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 10-18, 2009.

Morphological characteristics and phylogenetic relationship of Anabaena species from Lakes Dianchi and Erhai, China

Although Anabaena is one of the most prevalent planktonic freshwater genus in China, there are few taxonomic reports of Anabaena strains by morphology and genetics. In this study, morphological characteristics and phylogenetic relationships of seven Anabaena strains isolated from two plateau lakes, Lakes Dianchi and Erhai, were investigated. Morphological characteristics such as morphology of filament, cellular shapes and sizes, relative position of heterocytes and akinetes, and presence or absence of aerotopes, were described for these seven strains. Phylogenetic relationships were determined by constructing 16S rRNA gene tree using the neighbor-joining algorithm. The seven strains were morphologically identified as three groups, and phylogenetic analysis based on 16S rRNA gene sequences also showed that these seven strains were in three groups. Strains EH-2, EH-3, and EH-4 were in group A belonging to the Anabaena circinalis and A. crassa group, and strains DC-1, DC-2, and EH-1 were in group B and identified as A. flos-aquae. Strain DC-3 without aerotopes was significantly different from the other isolated strains and was determined as A. cylindrica.

A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp strain PCC 7120

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp strain PCC 7120

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.

Regulation by hetC of genes required for heterocyst differentiation and cell division in Anabaena sp strain PCC 7120

Unlike those of the wild-type strain, proheterocysts of the Anabaena sp. strain PCC 7120 hetC strain keep dividing. ftsZ, the most critical cell division gene, is up-regulated in hetC proheterocysts. Heterocyst differentiation genes hglD, hglE, patB, nijB, and xisA are no longer expressed in the hetC mutant. hetC also regulates the expression of patA, a pattern formation gene.

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp PCC 7120 (Cyanobacterium) under simulated microgravity

It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity. (C) 2004 Elsevier Ltd. All rights reserved.

alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp PCC 7120

Anabaena sp. PCC; 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Construction and analysis of a high-CO2-requiring mutant of the cyanobacterium Anabaena sp strain PCC7120

A mutant of Anabaena sp. strain PCC7120 requiring high CO2 was generated using Tn5 mutagenesis. This is the first data for a filamentous cyanobacterium. The mutant was capable of growing at 5% CO2, but incapable of growing at air levels of CO2. Southern hybridization analysis indicated that the Anabaena genome was inserted by the transposon at one site. The apparent photosynthetic affinity of the mutant to external dissolved inorganic carbon (DIC) was about 300 times lower that of the wild type (WT), and the medium alkalization rate as well as the carboxysomal carbonic anhydrase activity of the mutant was also lower than those of the WT. When the mutant was transferred from the culture medium bubbled with 5% CO2 to higher DIC (8.4% CO2) or 1% CO2, it showed similar responses to the WT. However, aberrant carboxysomes were found in the mutant cells through ultrastructural analysis, indicating it was most probably the wrong organization of the carboxysomes that eventually led to the inefficient operation of carboxysomal carbonic anhydrase and the subsequent defectiveness of the mutant in utilizing DIC.

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

An inducible CO2 concentrating mechanism in cyanobacterium Anabaena sp strain PCC7120

In order to define its characteristics of the photosynthetic utilization of CO2 and HCO3- when the ambient inorganic carbon changed, HCG (High-CO2-Growing Cells) of cyanobacterium Anabaena sp. strain PCC7120 were prepared. The growth rate of HCG was higher than that of LCG (low-CO2-growing cells, i.e. air-growing cells). When the HCG cells were transferred from 5% CO2 to air levels of CO2 , a series of changes took place: its carbonic anhydrase activity as well as its photosynthetic affinity to the external inorganic carbon significantly increased; the number of the carboxysomes, which is one of the most important components of CCM in cyanobacteria also increased. These facts indicated that the CCM activity of Anabaena PCC 7120 was induced. When the pH in the medium increased from 6 to 9, the photosynthetic affinity to external inorganic carbon of both HCG and LCG declined, while the apparent photosynthetic affinity to external CO2 increased. In the light of these findings, this inducible CCM in cyanobacteria provided a good model for the study of the photosynthetic Ci utilization in the phototrophic microoganisms.