Isolation of a novel cyanophage infectious to the filamentous cyanobacterium Planktothrix agardhii (Cyanophyceae) from Lake Donghu, China

A previously unknown cyanophage, PaV-LD (Planktothrix agardhii Virus isolated from Lake Donghu), which causes lysis of the bloom-forming filamentous cyanobacterium P. agardhii, was isolated from Lake Donghu, Wuhan, China. PaV-LD only lysed P. agardhii strains isolated from Lake Donghu and not those isolated from other lakes. The PaV-LD particle has an icosahedral, non-tailed structure, ca. 70 to 85 nm (mean +/- SD = 76 +/- 6 nm) in diameter. PaV-LD was stable at freezing temperature, but lost its infectivity at temperatures >50 degrees C. Lysis of host cells was delayed about 3 d after the PaV-LD treatment with chloroform, and the virus was inactivated by exposure to low pH (<= 4). The latent period and burst size of the PaV-LD were estimated to be 48 to 72 h and about 340 infectious units per cell, respectively. The regrowth cultures of surviving host filaments were not lysed by the PaV-LD suspension. To our knowledge, this is the first isolation and cultivation of a virus infectious to the filamentous bloom-forming cyanobacterium Planktothrix from a freshwater lake.

Cytochemical changes in the developmental process of Nostoc sphaeroides (cyanobacterium)

There are several apparent developmental stages in the life cycle of Nostoc sphaeroides Kutzing, an edible cyanobacterium found mainly in paddy fields in central China. The cytochemical changes in developmental stages such as hormogonia, aseriate stage, filamentous stage and colony in N. sphaeroides were examined using fluorescent staining and colorimetric methods. The staining of acidic and sulfated polysaccharides increased with development when hormogonia were used as the starting point. Acidic polysaccharides (AP) were most abundant at the aseriate stage and then decreased, while the sulfated polysaccharides (SP) were highest at the colony stage. Quantitatively, along the developmental process from hormogonia to colony, total carbohydrates first increased, then became stable, and then reached their highest level at the colony stage, while reducing sugars were highest at the hormogonia stage and then decreased sharply once development began. SP were not detectable in the hot water soluble polysaccharides (HWSP), and hormogonia had the lowest content of AP, while old colonies had the highest. The AP content of the aseriate stage, filamentous stage and young colony stage were very similar. The evolutionary relationships reflected in the developmental stages of N. sphaeroides are discussed.

Microcystin-RR induces physiological stress and cell death in the cyanobacterium Aphanizomenon sp DC01 isolated from Lake Dianchi, China

The phytoplankton community in Lake Dianchi (Yunnan Province, Southwestern China) is dominated in April by a bloom of Aphanizomenon, that disappears Suddenly and is displaced by a Microcystis bloom in May. The reasons for the rapid bloom disappearance phenomenon and the temporal variability in the composition of phytoplankton assemblages are poorly understood. Cell growth, ultrastructure and physiological changes were examined in cultures of Aphanizomenon sp. DC01 isolated from Lake Dianchi exposed to different closes of rnicrocystin-RR (MC-RR) produced by the Microcystis bloom. MC-RR concentrations above 100 mu g L-1 markedly inhibited the pigment (chlorophyll-a, phycocyanin) synthesis and caused an increase of soluble carbohydrate and protein contents and nitrate reductase activity of toxin-treated blue-green algae. A drastic. reduction in photochemical efficiency of PSII (Fv/Fm) was also found. Morphological examinationn showed that the Aphanizomenon filaments disintegrated and file cells lysed gradually after 48 h Of toxin exposure. Transmission electron microscopy revealed that cellular inclusions of stressed cells almost leaked out completely and the cell membranes were grossly damaged. These findings demonstrate the allelopathic activity of Microcystis aeruginosa inducing physiological stress and cell death of Aphanizomenon sp. DC01 Although the active concentrations of microcystin were rather high, we propose that microcystin may function as allelopathic Substance due to inhomogeneous toxin concentrations close to Microcystis cells. Hence, it may play a role in species Succession of Aphanizomenon and Microcystis in Lake Dianchi.

Different tolerances and responses to low temperature and darkness between waterbloom forming cyanobacterium Microcystis and a green alga Scenedesmus

The dynamics of planktonic cyanobacteria in eutrophicated freshwaters play an important role in formation of annual summer blooms, yet overwintering mechanisms of these water bloom forming cyanobacteria remain unknown. The responses to darkness and low temperature of three strains (unicellular Microcystis aeruginosa FACHB-905, colonial M. aeruginosa FACHB-938, and a green alga Scenedesmus quadricauda FACHB-45) were investigated in the present study. After a 30-day incubation under darkness and low temperature, cell morphology, cell numbers, chlorophyll a, photosynthetic activity (ETRmax and I-k), and malodialdehyde (MDA) content exhibited significant changes in Scenedesmus. In contrast, Microcystis aeruginosa cells did not change markedly in morphology, chlorophyll a, photosynthetic activity, and MDA content. The stress caused by low temperature and darkness resulted in an increase of the antioxidative enzyme-catalase (CAT) in all three strains. When the three strains re-grew under routine cultivated condition subjected to darkness and low temperature, specific growth rate of Scenedesmus was lower than that of Microcystis. Flow cytometry (FCM) examination indicated that two distinct types of metabolic response to darkness and low temperature existed in the three strains. The results from the present study reveal that the cyanobacterium Microcystis, especially colonial Microcystis, has greater endurance and adaptation ability to the stress of darkness and low temperature than the green alga Scenedesmus.

Solar UV radiation drives CO2 fixation in marine phytoplankton: A double-edged sword

Photosynthesis by phytoplankton cells in aquatic environments contributes to more than 40% of the global primary production (Behrenfeld et al., 2006). Within the euphotic zone (down to 1% of surface photosynthetically active radiation [PAR]), cells are exposed not only to PAR (400-700 nm) but also to UV radiation (UVR; 280-400 nm) that can penetrate to considerable depths (Hargreaves, 2003). In contrast to PAR, which is energizing to photosynthesis, UVR is usually regarded as a stressor (Hader, 2003) and suggested to affect CO2-concentrating mechanisms in phytoplankton (Beardall et al., 2002). Solar UVR is known to reduce photosynthetic rates (Steemann Nielsen, 1964; Helbling et al., 2003), and damage cellular components such as D1 proteins (Sass et al., 1997) and DNA molecules (Buma et al., 2003). It can also decrease the growth (Villafane et al., 2003) and alter the rate of nutrient uptake (Fauchot et al., 2000) and the fatty acid composition (Goes et al., 1994) of phytoplankton. Recently, it has been found that natural levels of UVR can alter the morphology of the cyanobacterium Arthrospira (Spirulina) platensis (Wu et al., 2005b). On the other hand, positive effects of UVR, especially of UV- A (315-400 nm), have also been reported. UV- A enhances carbon fixation of phytoplankton under reduced (Nilawati et al., 1997; Barbieri et al., 2002) or fast-fluctuating (Helbling et al., 2003) solar irradiance and allows photorepair of UV- B-induced DNA damage (Buma et al., 2003). Furthermore, the presence of UV-A resulted in higher biomass production of A. platensis as compared to that under PAR alone (Wu et al., 2005a). Energy of UVR absorbed by the diatom Pseudo-nitzschia multiseries was found to cause fluorescence (Orellana et al., 2004). In addition, fluorescent pigments in corals and their algal symbiont are known to absorb UVR and play positive roles for the symbiotic photosynthesis and photoprotection (Schlichter et al., 1986; Salih et al., 2000). However, despite the positive effects that solar UVR may have on aquatic photosynthetic organisms, there is no direct evidence to what extent and howUVR per se is utilized by phytoplankton. In addition, estimations of aquatic biological production have been carried out in incubations considering only PAR (i. e. using UV-opaque vials made of glass or polycarbonate; Donk et al., 2001) without UVR being considered (Hein and Sand-Jensen, 1997; Schippers and Lurling, 2004). Here, we have found that UVR can act as an additional source of energy for photosynthesis in tropical marine phytoplankton, though it occasionally causes photoinhibition at high PAR levels. While UVR is usually thought of as damaging, our results indicate that UVR can enhance primary production of phytoplankton. Therefore, oceanic carbon fixation estimates may be underestimated by a large percentage if UVR is not taken into account.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 mug(-1) microcystin-RR. The results showed that the growth of Synechococcus elongatus ( expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Signi. cant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Photosynthetic physiology and growth as a function of colony size in the cyanobacterium Nostoc sphaeroides

Algal size can affect the rate of metabolism and of growth. Different sized colonies of Nostoc sphaeroides were used with the aim of determining the effects of colony size on photosynthetic physiology and growth. Small colonies showed higher maximum photosynthetic rates per unit chlorophyll, higher light saturation point, and higher photosynthetic efficiency (a) than large colonies. Furthermore, small colonies had a higher affinity for DIC and higher DIC-saturated photosynthetic rates. In addition, small colonies showed higher photosynthetic rates from 5-45degreesC than large colonies. There was a greater decrease in Fv/Fm after exposure to high irradiance and less recovery in darkness for large colonies than for small colonies. Relative growth rate decreased with increasing colony size. Small colonies had less chl a and mass per unit surface area. The results indicate that small colonies can harvest light and acquire DIC more efficiently and have higher maximum photosynthetic rates and growth rates than large colonies.

Culture of the terrestrial cyanobacterium, Nostoc flagelliforme (Cyanophyceae), under aquatic conditions

Both colonies and free-living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free-living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30degreesC at either 20 or 60 mumol photons.m(-2).s(-1); colonies did not develop at 180 mumol photons.m(-2).s(-1), though this light level resulted in the most rapid growth of the cells. Conditions of 60 mumol photons.m(-2).s(-1) and 25degrees C appeared to result in the best colonial development and faster growth of the sheath-held colonies of N. flagelliforme when cultured indoor under aquatic conditions.

Effects of CO2 enrichment on the bloom-forming cyanobacterium Microcystis aeruginosa (Cyanophyceae): Physiological responses and relationships with the availability of dissolved inorganic carbon

Microcystis aeruginosa Kutz. 7820 was cultured at 350 and 700 muL.L-1 CO2 to assess the impacts of doubled atmospheric CO2 concentration on this bloom-forming cyanobacterium. Doubling Of CO2 concentration in the airflow enhanced its growth by 52%-77%, with pH values decreased and dissolved inorganic carbon (DIC) increased in the medium. Photosynthetic efficiencies and dark respiratory rates expressed per unit chl a tended to increase with the doubling of CO2. However, saturating irradiances for photosynthesis and light-saturated photosynthetic rates normalized to cell number tended to decrease with the increase of DIC in the medium. Doubling of CO2 concentration in the airflow had less effect on DIC-saturated photosynthetic rates and apparent photosynthetic affinities for DIC. In the exponential phase, CO2 and HCO3- levels in the medium were higher than those required to saturate photosynthesis. Cultures with surface aeration were DIC limited in the stationary phase. The rate of CO2 dissolution into the liquid increased proportionally when CO2 in air was raised from 350 to 700 muL.L-1, thus increasing the availability of DIC in the medium and enhancing the rate of photosynthesis. Doubled CO2 could enhance CO2 dissolution, lower pH values, and influence the ionization fractions of various DIC species even when the photosynthesis was not DIC limited. Consequently, HCO3- concentrations in cultures were significantly higher than in controls, and the photosynthetic energy cost for the operation of CO2 concentrating mechanism might decrease.

Photosynthetic bicarbonate utilization by a terrestrial cyanobacterium, Nostoc flagelliforme (Cyanophyceae)

The photosynthetic characteristics of the terrestrial cyanobacterium, Nostoc flagelliforme, after complete recovery by rewetting, was investigated to see whether it could use bicarbonate as the external inorganic carbon source when submerged. The photosynthesis-pH relationship and high pH compensation point suggested that the terrestrial alga could use bicarbonate to photosynthesize when submerged. The photosynthetic oxygen evolution rates were significantly inhibited in Na+-free and Na+ + Li+ media but were not affected by the absence of Cl-, implying that the bicarbonate uptake was associated with Na+/HCO3- symport rather than Cl-/HCO3- exchange system.

Genomic cloning, expression and recombinant protein purification of alpha and beta subunits of the allophycocyanin gene (apc) from the cyanobacterium Anacystis nidulans UTEX 625

A genomic fragment encoding alpha(APC) and beta(APC) (i.e., alpha and beta units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP ( maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. alpha(APC) and beta(APC) were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.

Intergeneric conjugation in holomycin-producing marine Streptomyces sp strain M095

Marine Streptomyces are potential candidates for novel natural products and industrial catalysts. In order to set up biosynthesis approach for a holomycin-producing strain M095 isotated from Jiaozhou Bay, China, a genetic transformation system was established using intergeneric conjugation. The plasmid pIJ8600 consists of an origin of replication for Escherichia coli, a phage integrase directing efficient site-specific integration in bacterial chromosome, thiostrepton-induced promoter and an attP sequence. Using E. coli ET12567 (pUZ8002) carrying pIJ8600 as a conjugal donor, while it was mated with strain M095, pIJ8600 was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. The frequency of exconjugants was 1.9 +/- 0.13 x 10(-4) per recipient cell. Analysis of eight exconjugants showed pIJ8600 was stable integrated at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of growth and antimicrobial activity analysis indicated that the integration of pIJ8600 did not seem to affect the biosynthesis of antibiotics or other essential amino acids. To demonstrate the feasibility of above gene transfer system, the allophycocyanin gene (apc) from cyanobacterium Anacystis nidulans UTEX625 was expressed in strain M095, and the results indicated heterologous allophycocyanin could be expressed and folded effectively. (c) 2006 Elsevier GmbH. All rights reserved.

Post-cyclic Strength Degradation of Undisturbed and Remolded Marine Silty Clay

A series of static and cyclic-static tri-axial compression tests under consolidated-undrained conditions are carried out to study the characteristics of post-cyclic strength of the undisturbed and the remolded samples of marine silty clay. It is found that the post-cyclic monotonic strength decreases if the cyclic strain or pore pressure is over a certain value. The maximum degradation is 10% for undisturbed samples while 70% for remolded ones. The relationship between normalized undrained shear strength and apparent overconsolidation ratio, which is determined by the excess pore pressure induced by cyclic loading, is also established. Static consolidated-undrained tests on overconsolidated remolded samples are also performed. It is proposed that the static consolidated-undrained tests may be substituted for the cyclic-static consolidated-undrained tests if the post-cyclic strength degradation of remolded silty clay is needed to be evaluated simply.