Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H₂ producers.     

The structural nif genes of four symbiotic Anabaena azollae show a highly conserved physical arrangement

Cloned DNA from Anabaena sp. PCC 7120 was used to determine the distribution of restriction sites around NifHDK genes of endosymbionts extracted from Azolla species. Although many of the restriction sites of the symbiotic Anabaena nifHDK genes differed from those of the free-living Anabaeba sp. PCC 7120, three apparently identical restriction sites were found in and around the nifD region. The arrangement of A. azollae and Anabaena sp. PCC 7120 nif H,D,K genes appears similar, with nifH and nifD linked and nifK some distance away from nifD. The A. azollae nifHDK genes appear strongly conserved among the Azolla species examined, regardless of the geographical origin of the ferns.     

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium,Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacteriumAnabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDGTNF. The expression of the rhTNF gene inEscherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced intoAnabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-³²P labeled hTNF cDNA probes, while the expression of the hTNF gene inAnabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic c~anobacteriumAnabaena sp. PCC 7120.     

Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131

Summary Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from aXhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cureA. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain ofAnabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication inAnabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged form after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation toA. variabilis, where they appeared to recombine with pRDS1.     

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more ¹³NO₃⁻ into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.     

Nutrient enrichment of a heterotrophic stream alters leaf litter nutritional quality and shredder physiological condition via the microbial pathway

We studied if biomass, cell dimensions and microcystin concentration of the cyanobacterium Anabaena sp. (strain BIR 257; recently also referred to as Dolichospermum) change in the presence of turbidity caused by clay and a eukaryotic green algal species. Anabaena sp. was incubated in experimental lab bioassays, with added clay and the chlorophyte Brachiomonas submarina. A control was established containing a single species of Anabaena sp. Biomass of Anabaena, given as carbon concentration increased in response to clay addition, suggesting that Anabaena was stimulated by lower light conditions whereas B. submarina was not. Cell lengths increased most likely as a response to lower light conditions. Dissolved toxin concentration decreased in treatments containing clay. Increased clay turbidity may favour the cyanobacterium Anabaena sp. over its eukaryotic competitors in the future Baltic Sea, especially in low-salinity estuaries.     

The response of the TonB-dependent transport network in Anabaena sp. PCC 7120 to cell density and metal availability

TonB dependent transporters (TBDT) are an essential protein family in bacteria involved in the uptake of a broad variety of molecules such as siderophore-chelated iron, which was the first described substrate. Meanwhile it is known that TBDTs are involved in the uptake of many metals, sugars and polypeptides. The action of TBDTs is regulated and energized by the plasma membrane anchored TonB, which is charged by a proton pump. The number of the genes coding for TBDTs varies in different species, which might reflect environmental adaptations or evolutionary variations of the system. For example, in the cyanobacterium Anabaena sp. PCC 7120 the large number of 22 genes coding for TBDTs has been identified and the expression of these genes has been explored in the absence of iron or copper as well as under nitrogen starvation. We describe the analysis of the expression of the TBDT genes and the according cytoplasmic-membrane localized components; the latter appear to have a lower degree of complexity in Anabaena sp. PCC 7120. This analysis unravels that the response is not sole dependent on the metal supply, but also on cell culture densities. In addition, we present a large group of FhuA-like genes which is expressed highest under standard conditions suggesting a function distinct from iron or copper transport. The genes are clustered according to the expression profile and the consequences for our understanding of the transport systems in Anabaena sp. PCC 7120 are discussed.     

Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。     

Effect of cyanobacterial blooms on thermal stratification

Enclosure experiments were performed at Akanoi Bay, Lake Biwa, in 1995 to determine whether the blooms of cyanobacterial algae changed thermal stratification in the lake. We used four rectangular enclosures, each 10 m × 10 m, with a volume of 200 m³, which were open to the sediments. Two enclosures, A and B, were mixed artificially by aquatic pumps from 1000 to 1400 every day, and the other two enclosures, C and D, were controls with no mixing. The experiment was conducted during late summer from August 3 to September 27. Chlorophyll a concentrations were highest in enclosure D, followed by enclosure C, both of which were controls without mixing. Enclosure A had lower concentrations than enclosures C and D, and enclosure B had the lowest concentrations. No large cyanobacterial algae blooms of Anabaena sp. and Microcystis sp. were seen in the mixed enclosures A and B. In enclosures C and D, blooms of Anabaena sp. occurred in the middle of August, and Microcystis sp. later became dominant in enclosure D at the end of August. In enclosure D, the water temperature changed over the diel cycle before August 17, with thermal stratification during the day and complete mixing at night. After August 17, as Anabaena sp. and Microcystis sp. became dominant, the temperature at the bottom of the enclosure did not change clearly over the 24-h cycle. The APE (available potential energy) density (a measure of water column stability) in the enclosures increased by almost 100% when the biovolume of Anabaena sp. + Microcystis sp. exceeded 20 mm³ l⁻¹. These results indicate that blooms of Anabaena sp. and Microcystis sp. can increase the available potential energy in the water column and create more stable stratification for their growth. Received: September 25, 1999 / Accepted: January 6, 2000     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.     

in-silico analysis suggests alterations in the function of XisA protein as a possible mechanism of butachlor toxicity in the nitrogen fixing cyanobacterium Anabaena sp. PCC 7120

Butachlor, a commonly used herbicide adversely affects the nitrogen fixing capability of Anabaena, an acclaimed nitrogen fixer in the Indian paddy fields. The nitrogen fixation in Anabaena is triggered by the excision of nifD element by xisA gene leading to rearrangement of nifD forming nifHDK operon in the heterocyst of Anabaena sp. PCC7120. Functional elucidation adjudged through in-silico analysis revealed that xisA belongs to integrase family of tyrosine recombinase. The predicted functional partners with XisA protein that have shown cooccurence with this protein in a network are mainly hypothetical proteins with unknown functions except psaK1 whose exact function in photosystem I is not yet known. The focus of this study was to find out the relation between XisA and butachlor using in-silico approaches. The XisA protein was modeled and its active sites were identified. Docking studies revealed that butachlor binds at the active site of XisA protein hampering its excision ability vis-à-vis nif genes in Anabaena sp. PCC7120. This study reveals that butachlor is not directly involved in hampering the nitrogen fixing ability of Anabaena sp. PCC7120 but by arresting the excision ability of XisA protein necessary for the functioning of nif gene and nitrogen fixation.     

Characterization of two naturally truncated, Ssb-like proteins from the nitrogen-fixing cyanobacterium, Anabaena sp. PCC7120

Single-stranded (ss) DNA-binding (Ssb) proteins are vital for all DNA metabolic processes and are characterized by an N-terminal OB-fold followed by P/G-rich spacer region and a C-terminal tail. In the genome of the heterocystous, nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC 7120, two genes alr0088 and alr7579 are annotated as ssb, but the corresponding proteins have only the N-terminal OB-fold and no P/G-rich region or acidic tail, thereby rendering them unable to interact with genome maintenance proteins. Both the proteins were expressed under normal growth conditions in Anabaena PCC7120 and regulated differentially under abiotic stresses which induce DNA damage, indicating that these are functional genes. Constitutive overexpression of Alr0088 in Anabaena enhanced the tolerance to DNA-damaging stresses which caused formation of DNA adducts such as UV and MitomycinC, but significantly decreased the tolerance to γ-irradiation, which causes single- and double-stranded DNA breaks. On the other hand, overexpression of Alr7579 had no significant effect on normal growth or stress tolerance of Anabaena. Thus, of the two truncated Ssb-like proteins, Alr0088 may be involved in protection of ssDNA from damage, but due to the absence of acidic tail, it may not aid in repair of damaged DNA. These two proteins are present across cyanobacterial genera and unique to them. These initial studies pave the way to the understanding of DNA repair in cyanobacteria, which is not very well documented.