Secondary metabolites of cyanobacteriaNostoc sp.

Cyanobacteria attracted much attention recently because of their secondary metabolites with potent biological activities and unusual structures. This paper reviews some recent studies on the isolation, structural, elucidation and biological activities of the bioactive compounds from cyanobacteriaNostoc species.

     

Construction of a new gene integration platform system for theSynechococcus sp. PCC7942

According to the known sequence of iron stress-induced gene (isiAB operon), we cloned its 1.5 kb fragment by PCR, and used this fragment as integration homologous fragment. After several steps of subcloning donor DNA into theisiAB fragment, a donor plasmid pZL which could be integrated into the chromosomal DNA ofSynechococcus sp. PCC7942 was constructed. In order to express the heterologous gene at a high level through the integration platform system, we constructed the donor DNA by the following steps. We cloned the strong promoter (240 bp) of heat shock genegroESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS),rbcS polyA into the downstream of thegroESL promoter. The kanamycin resistance gene, as the marker gene, was also subcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment and several expression elements, such asgroESL promoter, MCS,rbcS polyA terminator and kanamycin resistance gene, were all included.

After naturally transformed and introduced the donor plasmid pZL intoSynechococcus sp. PCC7942, as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homologous to theisiAB fragment ofSynechococcus sp. PCC7942, the homologous DNA can recombine with the chromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologous DNA were selected. The efficiency of transformation is about 1×10⁻⁶. By southern blot analysis, it was confirmed that the donor DNA had been integrated into the chromosomal DNA ofSynechococcus sp. PCC7942, located on the site of theisiAB gene, and can be replicated with the chromosomal DNA.

     

Cloning and sequencing of the ferredoxin gene of blue-green algaAnabaena siamensis

The structure gene for ferredoxin,petFI, fromAnabaena siamensis has been amplified by polymerase chain reaction(PCR) and cloned into cloning vector pGEM-3zf(+). The nucleotide sequence ofpetFI has been determined with silver staining sequencing method. There is 96.8% homology between coding region ofpetFI fromA. siamensis and that ofpetFI fromA. sp. 7120. Amino acid sequences of seven strains of blue-green algae are compared.

     

Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council     

Extracellular release of β-lactamase by osmotic shock inSynechococcus transformant

A unicellular cyanobacteriumSynechococcus sp. strain PCC 7002 was transformed with plasmid pQL1, on which β-lactamase gene (bla) and β-galactosidase gene (lacZ) were encoded. The transformant cells released β-lactamase into medium by an abrupt drop of osmotic pressure. This result indicates that this cyanobacterium recognizes and processes the signal sequence of β-lactamase, and accumulates the enzyme in periplasm. Repeated release of β-lactamase was possible by repeated osmotic shocks wihout, impairing cell viability. On the other hand, most of the β-galactosidase remained in cytoplasm under the osmotic shock.

     

Optimization of conditions for tetraspore release and assessment of photosynthetic activities for different generation branches of Gracilaria lemaneiformis Bory

Gracilaria lemaneiformis Bory is an economically important alga that is primarily used for agar production. Although tetraspores are ideal seeds for the cultivation of G. lemaneiformis, the most popular culture method is currently based on vegetative fragments, which is labor-intensive and time-consuming. In this study, we optimized the conditions for tetraspore release and evaluated the photosynthetic activities of different colonies formed from the branches of G. lemaneiformis using a PAM (pulse-amplitude-modulated) measuring system. The results showed that variations in temperature and salinityhad significant effects on tetraspore yield. However, variations in the photon flux density (from 15 μmol m⁻² s⁻¹ to 480 μmol m⁻² s⁻¹) had no apparent effect on tetraspore yield. Moreover, the PAM-parameters Y(I), Y(II), ETR(I), ETR(II) and F _v/F _m of colonies formed from different branches showed the same trend: parameter values of first generation branches>second generation branches>third generation branches. These results suggest that the photosynthetic activities of different colonies of branches changed with the same trend. Furthermore, photosynthesis in G. lemaneiformis was found to be involved in vegetative reproduction and tetraspore formation. Finally, the first generation branches grew slowly, but accumulated organic compounds to form large numbers of tetraspores. Taken together, these results showed that the first generation branches are ideal materials for the release of tetraspores.     

The genetic diversity,individual relatedness and possible mating system of an isolated population of the Cyprinid species Megalobrama pellgrini in upper reaches of the Changjiang (Yangtze) River,China

Megalobrama pellegrini is a cyprinid fish endemic to upper reaches of the Changjiang(Yangtze) River, China, which is also an important economic species in the local area. In recent years,resources of this species have decreased sharply and its conservation has drawn great attention. In the present study, we collected 120 individuals from the Longxi River, a tributary isolated from the main channel of the Changjiang River, where M. pellegrini is still relatively abundant. Using two different molecular markers, mitochondrial cytochrome b(cyt b) gene, and nuclear microsatellite(simple sequence repeat,SSR), we analyzed the genetic diversity of this isolated population. The results show that sequence genetic diversity was low(H_d:0.290 and P_i: 0.000 77 for cyt b gene),while the SSR genetic diversity was high(H_o:0.824 4±0.147 2,H_e: 0.823 510.145 1). Analysis indicated that this population had experienced a bottleneck,with inbreeding and small effective population size(around 50). Based on SSR data, relatedness analyzing revealed that the 120 samples were grouped into 10 completely independent clusters. It was inferred that the mating system of M. pellegrini was polygamy. We suggested that the low genetic diversity could be induced by the overfishing and inbreeding depression. Therefore, we suggested that the urgent conservation measures should be taken to control the overfishing and give better conditions for the fish to grow and spawn, then to restore population size.     

16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.     

Insertal orientation has no influence on the expression ofgfp gene and the growth of the hostSynechococcus sp. PCC7942

In transgenic process, a foreign gene can be integrated in the host genome in two directions, which may influence its expression. In order to study the effects of insertal orientation, the gfp reporter gene was inserted in the isiAB locus of Synechococcus sp.PCC7942 in different directions, and the GFP expression levels and the growth of the transgenic algae were compared. It was showed that the gfp gene could express in each direction, and no significant difference was detected on algal growth and GFP expression levels between the two recombinant algae.     

Genetic transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) using particle bombardment and glass-bead agitation

Platymonas (Tetraselmis) subcordiformis is a unicellular marine green alga. It was found to be very sensitive to the herbicide Basta through a sensitivity test indicating it could be employed as a selective agent. The bar gene is a practicable and selectable marker gene. The vector containing the expression cassette of the bar gene was transferred to P. subcordiformis by both particle bombardment and glass-bead agitation and transformants were then selected using Basta. Finally, Southern blotting analysis indicated that the bar gene had been successfully integrated into the nuclear genome of P. subcordiformis using both of the transgenic techniques, with the transformation efficiency of the glass-bead method being slightly higher than that of particle bombardment. This is the first report on stable transformation of P. subcordiformis, and will improve fundamental research and enlarge application of this alga.     

Physiological aspects of a high-co2 requiring mutant and the high-co2 growing cells of the cyanobacteriumSynechococcus pcc7942

A new chemically mutagenic mutant ofSynechococcus PCC7942 named high-CO₂ requiring mutant, which could grow at 4% CO₂ but could not grow at air levels of CO₂, was isolated. Comparative studies on some physiological aspects of the mutant and high-CO₂ growing cells (growing at 4% CO₂) were conducted. The result showed that the mutant had lower growing rate, about 1/40th photosynthetic affinity to inorganic carbon, 25% lower carbonic anhydrase (CA) activity, lower quenching rate of chlorophyll fluorescence, and about 1/2 alkalinization rate of the medium. The CA activity responses of the two types of cells to different concentration of CO₂ were determined. Upon the addition, of inorganic carbon (Ci), the rate of active Ci uptake described by the rate of chlorophyll fluorescence quenching of the mutant was obviously lower compared with that of the high-CO₂ growing cells; the size of the internal inorganic carbon pool size detemined by the extent of fluorescence quenching of the mutant was also smaller.

     

The loss of genetic diversity during captive breeding of the endangered sculpin, Trachidermus fasciatus, based on ISSR markers: implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of cultured and wild populations of Trachidermus fasciatus, an endangered catadromous fish species in China. Six selected primers were used to amplify DNA samples from 85 individuals, and 353 loci were detected. Relatively low genetic diversity was detected in the cultured population (the percentage of polymorphic loci PPL = 73.80%, Nei’s gene diversity h = 0.178 2, Shannon information index ...     

Preliminary Study on the Responses of Three Marine Algae,Ulva pertusa （ Chlorophyta ） , Gelidium amansii （ Rhodophyta ）and Sargassum enerve （ Phaeophyta）, to Nitrogen Source and Its Availability

1　Introduction　Inrecentyears ,eutrophicationhasbecomeaworld wideenvironmentalprobleminthecoastalarea.Aqua culturebecomesaseriouspollutionsourcelikesewagedischarge .Wasteproductsfromfishfarmsconsistma inlyofnitrogen ,phosphorusandcarbondioxide .Inpen bas…     

Fractal dimensions of flocs between clay particles and HAB organisms

The impact of harmful algal blooms (HABs) on public health and related economics have been increasing in many coastal regions of the world. Sedimentation of algal cells through flocculation with clay particles is a promising strategy for controlling HABs. Previous studies found that removal efficiency (RE) was influenced by many factors, including clay type and concentration, algal growth stage, and physiological aspects of HAB cells. To estimate the effect of morphological characteristics of the aggregates on HAB cell removal, fractal dimensions were measured and the RE of three species of HAB organism, Heterosigma akashiwo, Alexandrium tamarense, and Skeletonema costatum, by original clay and modified clay, was determined. For all HAB species, the modified clay had a higher RE than original clay. For the original clay, the two-dimensional fractal dimension (D2) was 1.92 and three-dimensional fractal dimension (D3) 2.81, while for the modified clay, D2 was 1.84 and D3 was 2.50. The addition of polyaluminum chloride (PACl) lead to a decrease of the repulsive barrier between clay particles, and resulted in lower D2 and D3. Due to the decrease of D3, and the increase of the effective sticking coefficient, the flocculation rate between modified clay particles and HAB organisms increased, and thus resulted in a high RE. The fractal dimensions of flocs differed in HAB species with different cell morphologies. For example, Alexandrium tamarense cells are ellipsoidal, and the D3 and D2 of flocs were the highest, while for Skeletonema costatum, which has filamentous cells, the D3 and D2 of flocs were the lowest.     

Rotifer community structure and assessment of water quality in Yangcheng Lake

Rotifer community structure was investigated in Yangcheng Lake in 2008. Dominant species and species diversity indices were determined and QB/T was used to assess water quality. The annual average density and biomass of the rotifers in Yangcheng Lake was 2 894 ± 1 006 ind./L and 12.47 ± 10.28 mg/L, respectively. The highest densities were observed in the western portion of the lake in March, but the highest biomass occurred in inflowing creeks in September. Within a year of monitoring in Yangcheng Lake, 93 species were identified and the dominant species were found to be Polyarthra trigla, Brachionus angularis, Keratella cochlearis, Keratella valga, Brachionus calyciflorus, and Filinia major. Of the species recorded, 75 were pollution indicator species. Density and biomass exhibited significant positive correlations with water temperature (R = 0.209, P = 0.003; R = 0.446, P = 0.000), but the peak density showed two lags in response to chl a. According to the Jaccard similarity index (SJ), the greatest similarity among dominant species occurred between creeks and the eastern part of the lake. The annual average Shannon-Wiener diversity index H’, Margalef richness index D and Pielou evenness index J were 1.96 ± 0.34, 1.61 ± 0.50 and 0.77 ± 0.10, respectively. In all four areas of Yangcheng Lake, β- mesosaprobic species comprised the largest share of pollution indicator species. These data suggest that Yangcheng Lake is mesosaprobic.     

Sequence comparison of partial cytochrome b genes of two Coilia species

Sequence variation of partial cytochrome b genes between two Coilia species, C. ectenes and C. mystus, was investigated. Of the 402 nucleotides, twenty-seven (6.72%) are polymorphic and all are synonymous substitutions. At the third positions of genetic condon of cytochrome b gene, the two species show an extreme anti-G bias (<4%) and a pronounced bias towards A and C (>68%). There is no amino acid sequence divergence between the partial cytochrome b genes of the two species, indicating a close genetic relationship between them. The k-2p genetic distance of partial cytochrome b segment of the two species is 0.072, suggesting that the species were separated 3.6 Ma ago, in the middle Pliocene. Our result reveals that the cytochrome b gene is an appropriate marker for studies of population genetic structures and phylogeographic patterns of the two species.     

Microalgae cultivation in a tubular bioreactor and utilization of their cells

In this study on the possiblities of microalgae technology as an option for CO₂ mitigation, many microalgae were isolated from seawater. Some species of the isolates,Chlamydomonas sp. strain YA-SH-1, which accumulates starch in cells under light and ferment ethanol in dark and anaerobic condition, was grown outdoors by using 50-L tubular bioreactors in batch cultivation and harvested. Using these cells, the performance of ethanol production was examined quantitatively in a 0.5-L scale fermentor. Another species,Tetraselmis sp. strain Tt-1, was cultivated in a semi-batch manner by a similar type of tubular bioreactor indoors and examined for its utilization. Tests showed these cells could be used as partial substitute for wood and kenaf pulp for processing into paper. With the idea of making microalgae produce cellulose by genetic engineering in their minds, the authors studied the structure of bacterial cellulose synthase genes and the low temperature-induced, reversible flocculation in a thermophilic blue green alga (Cyanobacterium),Synechocystis vulcanus in order to examine the feasibility of using these genes as gene source and the cynanobacterium as host.

     

Production of γ-linolenic acid and stearidonic acid by <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.     

Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.