Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET.

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryoelectron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder, a site that may act as an elongation center. Subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a ß sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

Triclosan toxicity in a model cyanobacterium (Anabaena flos-aquae): Growth, photosynthesis and transcriptomic response.

Exposure to triclosan (TCS) has been reported to reduce photosynthetic pigments, suppress photosynthesis, and inhibit growth in both prokaryotic and eukaryotic algae including Anabaena flos-aquae (a model cyanobacterium). In particular, cyanobacteria are more sensitive to TCS toxicity compared to eukaryotic algae possibly due to the structural similarity to bacteria (target organisms); however, whether TCS exerts its toxicity to cyanobacteria by targeting signaling pathways of fatty acid biosynthesis as in bacteria remains virtually unknown, particularly at environmental exposure levels. With the complete genome sequence of A. flos-aquae presented in this study, the transcriptomic alterations and potential toxic mechanisms in A. flos-aquae under TCS stress were revealed. The growth, pigments and photosynthetic activity of A. flos-aquae were markedly suppressed following a 7-day TCS exposure at 0.5 µg/L but not 0.1 µg/L (both concentrations applied are environmentally relevant). The transcriptomic sequencing analysis showed that signaling pathways, such as biofilm formation - Pseudomonas aeruginosa, two-component system, starch and sucrose metabolism, and photosynthesis were closely related to the TCS-induced growth inhibition in the 0.5 µg/L TCS treatment. Photosynthesis systems and potentially two-component system were identified to be sensitive targets of TCS toxicity in A. flos-aquae. The present study provides novel insights on TCS toxicity at the transcriptomic level in A. flos-aquae.

The effects of aqueous extract from watermelon (Citrullus lanatus) peel on the growth and physiological characteristics of Dolichospermum flos-aquae.

Nowadays, the increasing Dolichospermum (Anabaena) blooms pose a major threat to the aquatic environment and public health worldwide. The use of naturally derived chemicals from plants to control cyanobacteria blooms has recently received a tremendous amount of attention. This study investigates the possibility of transforming watermelon peel (WMP) into a biological resource to allelopathically inhibit Dolichospermum flos-aquae blooms. The results demonstrated that the growth of D. flos-aquae was efficiently restricted by the aqueous extract of watermelon peel (WMPAE) in a concentration-dependent manner. Cell viability decreased quickly, intracellular structural damage occurred, chlorophyll a in algal cells degraded, and photosynthesis was clearly inhibited. At the same time, the levels of reactive oxygen species in viable cells increased significantly, as did malondialdehyde levels, indicating that WMPAE elucidated strong oxidative stress and corresponding damage to D. flos-aquae. Capsular polysaccharide (CPS) levels increased in all treatment groups, which represents an adaptive response indicative of the development of resistance to WMPAE stress and oxidative damage. Despite this, WMPAE had clear inhibitory effects on D. flos-aquae. These findings provide fundamental information on an allelopathic system that could be a novel and attractive approach for suppressing D. flos-aquae blooms in small aquatic environments, especially aquaculture ponds.

Morphologically-different cells and colonies cause distinctive performance of coagulative colloidal ozone microbubbles in simultaneously removing bloom-forming cyanobacteria and microcystin-LR.

Morphology, the important feature of bloom-forming cyanobacteria, was studied for its impacts on the harmful cyanobacterial bloom (HCB) treatment by coagulative colloidal ozone microbubbles (CCOMBs). The globally-appeared HCB species - Microcystis aeruginosa (spherical cells, block mass colonies), Microcystis panniformis (spherical cells, flat penniform-like colonies) and Anabaena flos-aquae (filamentous morphology) were chosen as representative species. CCOMBs were generated by modifying the bubble surface and the gas core with coagulant and ozone, respectively. The removal of spherical cells and filaments was > 99.5% and ≤ 34.6%, individually, and the latter was ascribed to chain breakage. CCOMBs collected Microcystis panniformis via complexing with the fluorescent and non-fluorescent functional groups of cell colonies but captured Anabaena flos-aquae through the fluorescent ones. More Microcystis aeruginosa got membrane-damaged than Microcystis panniformis; nevertheless, the microcystin-LR (MC-LR) removal was guaranteed through efficiently oxidizing the released MC-LR. Although the outer peptidoglycan sheet of Anabaena flos-aquae was destroyed, the inner cyte membrane remained intact, preventing intracellular MC-LR from releasing. The HCBs dominated by single species with spherical cells were more readily treated than those with co-occurred species. The toxicological tests imply that, as a robust tool for HCB treatment, the CCOMB technology could be eco-environmentally friendly to the aquatic environment.

A Glimpse at Siderophores Production by Anabaena flos-aquae UTEX 1444.

In this study, a strain of Anabaena flos-aquae UTEX 1444 was cultivated in six different concentrations of iron (III). Cultures were extracted with organic solvents and analyzed using our dereplication strategy, based on the combined use of high-resolution tandem mass spectrometry and molecular networking. The analysis showed the presence of the siderophores' family, named synechobactins, only in the zero iron (III) treatment culture. Seven unknown synechobactin variants were present in the extract, and their structures have been determined by a careful HRMS/MS analysis. This study unveils the capability of Anabaena flos-aquae UTEX 1444 to produce a large array of siderophores and may be a suitable model organism for a sustainable scale-up exploitation of such bioactive molecules, for the bioremediation of contaminated ecosystems, as well as in drug discovery.

Effect of complex iron on the phosphorus absorption by two freshwater algae.

Iron plays an important role in physiological processes of microalgae and also affects the absorption of other nutrients by algae cells. Therefore, iron is one of the important controlling factors for algae bloom formation. This study investigated the effect of four kinds of complex iron (EDTA-Fe, ferric humate, ferric oxalate and ferric ammonium citrate) on the phosphorus absorption by two freshwater algae (Scenedesmus quadricauda and Anabaena flos-aquae). The results showed that the species and concentration of complex iron had a significant effect on the phosphorus uptake rate of S. quadricauda, but had only a slight effect on that of A. flos-aquae. The former exhibits positive influences on phosphorus absorption and was in the following order: ferric oxalate and EDTA-Fe > ferric humate and ammonium ferric citrate, and these effects depended on whether the presence of complex iron constitutes an environmental pressure for the growth of algal cells.

Effect of iron and phosphorus on the microalgae growth in co-culture.

Iron and phosphorus (P) are the important micro- and macro-nutrient for microalgae growth, respectively. However, the effect of iron and P on microalgae growth in co-culture associating with the formation of dominate algae has not been investigated before. In the current study, Anabaene flos-aquae, Chlorella vulgaris and Melosira sp. were co-cultivated under the addition of different initial iron and P to reveal the effect of iron and phosphorus on the growth of microalgae. The results showed that the mean growth rate of A. flos-aquae, C. vulgaris and Melosira was 0.270, 0.261 and 0.062, respectively, indicating that the A. flos-aquae and C. vulgaris algae are liable to be the dominant algae while the growth of Melosira was restrained when co-cultured. The ratio of Fe to P has a significant impact on the growth of microalgae and could be regarded as an indicator of algae growth. Microalgae showed a much more obvious uptake of iron compared to that of P. The information obtained in the current study was useful for the forecast of water quality and the control of microalgae bloom.

Effective biosorption of uranium from aqueous solution by cyanobacterium Anabaena flos-aquae.

Anabaena flos-aquae, a typical species of cyanobacterial bloom, was employed as a useful biosorbent for uranium removal. Batch experiments were conducted to examine the effects of different parameters on the uranium uptake amount of Anabaena flos-aquae. The maximum adsorption capacity of 196.4 mg/g was obtained under the optimized experimental conditions. The calculations of kinetic and thermodynamic results proved the adsorption process was endothermic, chemisorption, and spontaneous. The adsorption of uranium onto Anabaena flos-aquae was better defined by the Langmuir model, which indicated the process was a monolayer sorption. In addition, the characterization of the biosorbent before and after uranium sorption implied that the dominant functional groups participated in the uranium adsorption process were hydroxyl, amino, and carboxyl. In conclusion, the environmentally friendly and biocompatible characteristics of Anabaena flos-aquae suggest that it can be a promising biosorbent for uranium removal.

Inactivation of harmful Anabaena flos-aquae by ultrasound irradiation: Cell disruption mechanism and enhanced coagulation.

Harmful algal blooms pose a potential threat to the safety of drinking water sources. Ultrasound is an effective method for algae removal. However, this method can lead to the release of algal organic matter and the effects and toxic mechanisms of ultrasound on Anabaena are still poorly understood. The destruction mechanism of Anabaena flos-aquae cells under different ultrasonic conditions, the safety of intracellular organic matter (IOM) release to water and the enhanced coagulation efficiency of ultrasound were studied. Results showed that high-frequency ultrasound was effective in breaking down algae cells. After 10 min ultrasonication at 20 kHz, 5 min at 740 kHz and 1 min at 1120 kHz, the algae cells were inactivated and algae growth was halted. Ultrasound radiation can lead to the release of IOM, primarily chlorophyll a and phycocyanin, followed by some tryptophan and humic substances, polysaccharides, and proteins. The sonicated ribosomes were considerably reduced, and the antioxidant system of cells was also damaged to some extent. The coagulation effect of algae cells was substantially improved after ultrasonication. Thus, the safety of algae cell removal could be improved by controlling the changes in physiological structure and IOM release of algae cells by adjusting the ultrasound parameters.

Effect of four kinds of complexing iron on the process of iron uptake by Anabaena flos-aquae.

AbstractIron (Fe), which is a necessary micronutrient for algal growth, plays an important role in the physiological metabolism and enzymatic reactions of algae. This study aimed to investigate the absorption process of four kinds of complexing iron absorbed by Anabaena flos-aquae. Results showed that the absorptive capacity of A. flos-aquae to complex iron was inversely proportional to the stability of the complex bond of complex iron. Complex iron with weak binding ability can be quickly adsorbed by A. flos-aquae. The absorptive rate was as follows: ferric humate, ferric oxalate >ammonium ferric citrate >EDTA Fe. For EDTA-Fe with a strong binding ability, a moderate iron concentration (e.g. 0.6 mg l-1) is favourable for iron uptake by A. flos-aquae. Our experiments also revealed that the process of separating iron from complex iron before entering algal cells was probably as follows: iron complexed with organic ligands were firstly adsorbed on the surface of algae cells; afterwards, iron ions were captured by organic matter on the surface of algae cells, accompanied by the rupture of the bond between Fe3+ and ligand; finally, the Fe3+ entered into the cell of algae while the organic ligands returned to the medium.

Occurrence, distribution, and source track of antibiotics and antibiotic resistance genes in the main rivers of Chongqing city, Southwest China.

In this study, the occurrence of 14 antibiotics, four corresponding antibiotic resistance genes (ARGs) and two microbial source tracker (MST) indicators were analyzed in two rivers of Chongqing city, southwest China. The results showed that 13 antibiotics were detected in all 12 sites and their detection frequencies were much higher in September, but concentrations were lower than that in March. Of them, erythromycin (ETM) and ofloxacin (OFL) were the predominant antibiotics in both seasons. The remarkably higher concentration of antibiotics in sediments of these rivers than those in other rivers were found. Environmental risk assessment found that four antibiotics posed high risk toward some sensitive algae. For ARGs, their relative abundances were higher in waters than those in sediments, higher in March than in September. Correlation analysis showed that antibiotics were not the exclusive selective pressure of ARGs; many environmental factors like dry matter contents on a mass basis, organic matter, total organic carbon, dissolved organic carbon, temperature, oxidation reduction potential and nitrite could affect the occurrence of ARGs. MST indicators analysis demonstrated that this river basin was largely polluted by human and pig feces, and human feces might be one main source of the four ARGs and five antibiotics.

Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation.

Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 µmol/mg/min) and A. flos-aquae (30.5 µmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30°C, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.

Ultrasound imaging of gene expression in mammalian cells.

The study of cellular processes occurring inside intact organisms requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling noninvasive imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce mammalian acoustic reporter genes. Starting with a gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of intracellular air-filled protein nanostructures called gas vesicles, which produce ultrasound contrast. Mammalian acoustic reporter genes allow cells to be visualized at volumetric densities below 0.5% and permit high-resolution imaging of gene expression in living animals.

Effect of different kinds of complex iron on the growth of Anabaena flos-aquae.

As a necessary micronutrient for algal growth, iron (Fe) has important effects on the physiological metabolism and enzymatic reactions of algae. In this study, a series of experiments were designed to compare the effects of different kinds of iron on the growth of Anabaena flos-aquae. Results showed that the promotion of iron species on algae growth was in order of ferric ammonium citrate > EDTA-Fe > iron ions > iron oxalate. When the concentration of iron is in the range of 0.1-0.8 mg/L, iron species have more significant influences on Anabaena bloom compared with iron concentration. Our experiments also revealed that A. flos-aquae has the strongest adsorption capacity for iron ion among the four iron sources and further induced. This leads to the toxicity of the iron ion group to algal cells at low concentration.

Mutual Dependence of Nitrogen and Phosphorus as Key Nutrient Elements: One Facilitates Dolichospermum flos-aquae to Overcome the Limitations of the Other.

Dolichospermum flos-aquae (formerly Anabaena flos-aquae) is a diazotrophic cyanobacterium causing harmful blooms worldwide, which is partly attributed to its capacity to compete for nitrogen (N) and phosphorus (P). Preventing the blooms by reducing P alone or both N and P has caused debate. To test the effects alone and together on the growth of cyanobacteria, we performed culture experiments in different eutrophication scenarios. N2 fixation in terms of heterocyst density, nitrogenase activity and nifH expression increased significantly in P-replete cultures, suggesting that P enrichment facilitates N2 fixation. Correspondingly, the expression of genes involved in P uptake, e.g., those involved in P-transport ( pstS) and the hydrolysis of phosphomonoesters ( phoD), was upregulated in P-deficient cultures. Interestingly, N addition enhanced not only the expression of these genes but also polyphosphate formation and alkaline phosphatase activity in P-deficient cultures relative to the P-replete cultures, as evidenced by qualitative (enzyme-labeled fluorescence) and quantitative (fluorogenic spectrophotometry) measurements. Furthermore, after N addition, cell activity and growth increased in the P-deficient cultures, underscoring the risk of N enrichment in P-limited systems. The eco-physiological responses shown here help further our understanding of the mechanism of N and P colimitation and underscore the importance of dual N and P reduction in controlling cyanobacterial blooms.

Biological activities and nitrogen and phosphorus removal during the anabaena flos-aquae biofilm growth using different nutrient form.

This work investigated the biological activities and nitrogen and phosphorus removal during the anabaena flos-aquae biofilm growth on the polyvinyl chloride (PVC) carriers, in different nutrient form mediums. The study showed that the production of dehydrogenase activity (DHA) and extracellular polymeric substances (EPS) can reach 40.4 g/(h·m2) and 115 × 10-2 g/m2 in an 11-day period, respectively, indicating that the anabaena flos-aquae biofilm had high biological activities. The results showed that the nitrogen and phosphorus removal reached 94.9 and 96.8%, respectively, in the ammonium form nitrogen group; while 97.7% of phosphorus were removed in the orthophosphate form phosphorous group. A comparison study was conducted and results showed that the present anabaena flos-aquae based biofilm provided a better removal of nitrogen and phosphorus than the other microalgae biofilms.

Recyclable and Intrinsically Anti-cyanobacterial Polyanionic Membranes.

Cyanobacteria blooms possess serious threats to water resources. Herein, we report the synthesis of polyanionic membranes (PA-M) by in situ photo-crosslinking of a sulfate-based anionic monomer followed by cation-exchange with metal cations, Fe3+ (PA-Fe), Cu2+ (PA-Cu), or Zn2+ (PA-Zn). The effect of cations on the anti-cyanobacterial activities against both Microcystis aeruginosa (M. aeruginosa) and Anabaena flos-aquae (A. flos-aquae) was investigated. All the prepared metal-containing membranes (PA-Fe, PA-Cu, PA-Zn) exhibit high anti-cyanobacterial activities and long-term anti-cyanobacterial stability, demonstrating that the synthesized PA-M membranes can be used as an effective and safe inhibitor to control cyanobacterial blooms.

Using stable isotope labeling to study the nitrogen metabolism in Anabaena flos-aquae growth and anatoxin biosynthesis.

Freshwater resources are under stress around the world due to rapid urbanization and excessive water consumption. Cyanobacterial blooms have occurred frequently in surface waters, which produced toxic secondary metabolites causing a potential harm to aquatic ecosystems and humans. In this study, the relationship between different types of nitrogen source and the algal growth of Anabaena flos-aquae, which was isolated from Dianchi Lake in southern China, was investigated. Experiments were accomplished by using four types of isotope tracers including 15N-ammonium chloride, 15N-sodium nitrate, 15N-urea, 15N-l-alanine in culture medium to characterize the biosynthesis of 15N-anatoxin-a (ATX-A), which is a major algal toxin from A. flos-aquae, through liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that all these four types of nitrogen can be incorporated into algal cells. The ATX-A production with urea as the nitrogen source was much higher than that with the other three types of nitrogen. The 15N labeling experiments further demonstrated that the uptake of organic nitrogen nutrients was significantly greater than that of inorganic nitrogen. These results provide new evidence and deeper insight to explore the biosynthesis of ATX-A in the specific strain of A. flos-aquae.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica

Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica.

Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.