Filamentous Cyanobacterium Anabaena Sp Research Articles

Use of segment-based microarray in the analysis of global gene expression in response to various environmental stresses in the cyanobacterium Anabaena sp. PCC 7120.

We prepared microarrays that contain genomic sequences of a heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120. The complete genome of this cyanobacterium codes for about 5,368 protein-coding genes in the main chromosome of 6.4 Mbp. In total, 2,407 DNA segments were selected from the sequencing clones, and amplified by PCR, then spotted on glass slides in duplicate. These microarrays differ from the widely used commercial or custom-made ones for other microorganisms in that each DNA segment was 3-4 kbp long, and contained about 3-4 predicted genes on average. This feature, however, did not decrease the usefulness of the microarrays, since we were able to detect a number of potentially novel genes that are induced in response to nitrogen deprivation, low temperature and drought. In addition, we found some genomic regions in which dozens of contiguous genes are simultaneously regulated. These results suggest that these segment-based microarrays are useful especially for such large genomes as Anabaena, for which the number of genes exceeds either technical or practical limitations.

Effect of iron deficiency on heterocyst differentiation and physiology of the filamentous cyanobacteriumAnabaena sp. PCC 7120

The effect of iron deficiency on heterocyst differentiation and some physiological properties of the filamentous cyanobacteriumAnabaena sp. PCC 7120 was investigated. Under moderate iron limitation conditions, achieved by addition of iron chelator 2, 2′-Dipyridyl (<80 μmol/L) led to delayed heterocyst differentiation, no heterocyst differentiation was observed under severe iron limitation conditions, when the concentration of 2,2′-Dipyridyl in the medium was more than 100 μmol/L. it seemed that there are certain iron-regulated genes or operons whose function is to control heterocyst development. In addition, iron deficiency impaired the growth. Low iron cells had a decrease in the quantities of pigment content (chlorophyll and phycocyanin content), the whole cellin vivo absorbance spectra confirmed the decrease, the protein electrophoretic profiles revealed that iron-deficient cells had less protein bands, with the increase of 2,2′-Dipyridyl, the protein bands was more and more less. And differently, iron deficiency also caused an increase of ROS (Reactive Oxygen Species) and SOD activity, it suggests that iron deficiency led to oxidative stress, which generally occured under high-iron conditions.

Description of five mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst differentiation and identification of the transposon-tagged genes

When growing on N 2 as sole nitrogen source, the filamentous cyanobacterium Anabaena sp. PCC 7120 forms N 2 fixing heterocysts in a semi-regular pattern. To identify genes involved in heterocyst differentiation we characterised five transposon-generated mutants that were not able to form mature heterocysts. After recovering the transposon together with the flanking region of the Anabaena chromosome the affected genes were identified. Four of the genes could be involved in formation of the heterocyst-specific envelope: alr2887, encoding a probable outer membrane efflux protein, alr3698, a glycosyl transferase, all4388, a putative periplasmic polysaccharide export protein and alr5357, the formerly described gene hglB/ hetM, encoding a fatty-acid synthetase. Another gene, all0049/ mutS2, may be important in one of the genome rearrangements that occur during heterocyst differentiation. By transcriptional fusion to reporter genes luxAB differential expression of alr2887, alr3698 and alr5357 could be monitored during heterocyst differentiation.

Heterocyst-Specific Expression of patB , a Gene Required for Nitrogen Fixation in Anabaena sp. Strain PCC 7120

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.

Comprehensive Analysis of Tandem Repeat Sequences in Cyanobacteria Genome

Over the past year, two complete genome sequences of the cyanobacteria (filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 [3] and thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 [6]) became available in addition to the Synechocystis sp. PCC6803 genome [4]. Additionally, five species are in draft to finishing phase and other eight genome projects are also in progress. Among these genomes, some repeat sequences have been identified, however, their functions are not yet characterized well [5]. In this research, we have applied comprehensive search for tandem repeat sequences on each cyanobacterium genome. From this screening, we have found new tandem repeat units in addition to previously reported repeat sequences such as STRRs (short tandemly repeated repetitive) and LTRRs (long tandemly repeated repetitive) sequences [5].

Calcium transients in response to salinity and osmotic stress in the nitrogen‐fixing cyanobacterium Anabaena sp. PCC7120, expressing cytosolic apoaequorin

AbstractWe show here that both salinity and osmotic stress trigger transient increases in intracellular free Ca2+ concentration ([Ca2+]i) in cells of the nitrogen‐fixing filamentous cyanobacterium Anabaena sp. PCC7120, which constitutively expresses apoaequorin. Isoosmolar concentrations of salt (NaCl) and osmoticum (sucrose) induced calcium transients of similar magnitude and shape, suggesting that cells sense, via Ca2+ signalling, mostly osmotic stress. The Ca2+ transients induced by NaCl and sucrose were completely blocked by the calcium chelator ethylene glycol‐bis(b‐aminoethylether)N,N,N¢,N¢‐tetraacetic acid (EGTA) and were partially inhibited by the calcium channel blocker verapamil. Increased external Ca2+ and the Ca2+ ionophore calcimycin (compound A23187) enhanced Ca2+ influx further, suggesting the involvement of extracellular Ca2+ in the observed response to salinity and osmotic stress. However, the plant hormone abscisic acid (ABA) did not provoke any effect on the Ca2+ transients induced by both stresses, indicating that it may not be acting upstream of Ca2+ in the signalling of salinity and/or osmotic stress in Anabaena sp. PCC7120.

PatS and products of nitrogen fixation control heterocyst pattern.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. The patS gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of patS expression and heterocyst pattern formation. A patS-gfp reporter strain revealed clusters of patS-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription of patS during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5' end of a transcript for patS was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5' end at -39 bases replaced the vegetative cell transcript. A patS mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. The patS mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern.

HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2‐FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)

The process of N2 fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin‐containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.

Developmental regulation of the cell division protein FtsZ in Anabaena sp. strain PCC 7120, a cyanobacterium capable of terminal differentiation.

Heterocysts are terminally differentiated cells devoted to nitrogen fixation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. We show here that the cell division protein FtsZ is present in vegetative cells but undetectable in heterocysts. These results provide a first rational explanation for the inability of mature heterocysts to undergo cell division.

Metal-induced expressing of mammal Metallothionein-1 gene in cyanobacteria to promote cadmium-binding preferences

The metal(zinc)-inducible smtA gene promoter (smt O-P) from cyanobacteria was applied for the expression of mouse MT-1 cDNA in the filamentous cyanobacterium Anabaena sp. PCC 7120 to enhance its metal-binding capability and to change its main binding specificity from zinc to cadmium. Shuttle expression vector pKT-MRE transformed the cyanobacterial cells by triparent conjugal transfer. Positive clones were screened and identified by streptomycin, DNA dot blot, SDS-PAGE and Western blot analysis. Photosynthetic oxygen evolution and metal atom absorption indicated that under the cadmium stress the metal-induced expression of foreign mMT-1 doubled their cadmium resistance and developed cells showing a much higher preference to absorb cadmium other than zinc in medium. The cadmium content in cell extract rose from 11% to 36%, and the cadmium cleared from media by transgenic cells rose from 18% to 62%. There was only a slight enhancement for zinc binding in the wild or transgenic type.

Genome rearrangements during Anabaena heterocyst differentiation

The filamentous cyanobacterium Anabaena sp. PCC 7120 produces specialized, terminally differentiated cells called heterocysts that are the sites of nitrogen fixation. The genome of Anabaena undergoes at least two specific developmentally regulated DNA rearrangements during heterocyst differentiation. One rearrangement involves the nitrogen-fixation genes nifH, nifD, and nifK. During heterocyst differentiation, an 11-kilobase (kb) DNA element is excised from the 3′ coding region of the nifD gene by site-specific recombination within 11 base pair, directly repeated sequences present at the ends of the element. The excision results in formation of a complete nifD coding sequence and allows transcription of all three genes from a single promoter upstream of nifH. The gene xisA, located at one end of the 11-kb element, is believed to encode the site-specific recombinase responsible for excision of the element from the nifD gene. A second DNA rearrangement occurs close to the 5′ end of the nifS gene and results in placing the genes rbcL and rbcS approximately 10 kb from the nif gene cluster. This rearrangement is also a deletion from the chromosome but involves a significantly larger segment of the genome, 55 kb. The genomic breakpoints of the nifS rearrangement have been cloned and sequenced. The recombination sites show no homology to those involved in the nifD rearrangement. This suggests that each rearrangement is catalyzed by a different site-specific recombination system and that the two arrangements may be independently regulated.

Genes encoding ferredoxins from Anabaena sp. PCC 7937 and Synechococcus sp. PCC 7942: structure and regulation

The gene encoding ferredoxin I (petF1) from the filamentous cyanobacterium Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413) was cloned by low stringency hybridization with the ferredoxin cDNA from the higher plant Silene pratensis. The petF1 gene from the unicellular cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) was cloned by low stringency hybridization with the petF1 gene from Anabaena sp. PCC 7937. One copy of the petF genes was detected in both organisms, and a single transcript of about 630 b was found for Synechococcus sp. PCC 7942. Both the Synechococcus sp. PCC 7942 and the Anabaena sp. PCC 7937 petF1 genes contain a 297 bp open reading frame coding for a small acidic protein, consisting of 98 amino-acid residues, with a molecular mass of about 10.5 kDa.The ferredoxin content of Synechococcus sp. PCC 7942 is strongly reduced under ironlimited growth conditions. The slight decrease in the amount of ferredoxin transcript found under iron limitation does not account for the more severe reduction in ferredoxin protein observed. The main regulation of the ferredoxin content probably is effected at the level of translation and/or degradation. Although ferredoxin expression can be strongly reduced by iron stress, the ferredoxin function seems to be indispensable, as Synechococcus sp. PCC 7942 appeared refractory to yield mutants lacking the petF1 gene.