Effective cultivation conditions and safety evaluation of filamentous cyanobacteria producing phycocyanins with antiglycation activities.

We investigated suitable culture conditions for the production of the blue pigment phycocyanin (PC) from the unique filamentous cyanobacteria Pseudanabaena sp. ABRG5-3 and Limnothrix sp. SK1-2-1. White, green, or red LED irradiation at 30 µmol photons/m2/s was effective for phycocyanin production when compared with Arthrospira platensis (Spirulina) sp. NIES-39, which is generally grown under high light irradiation. To investigate the safety of the cyanobacteria, ABRG5-3 cells were subjected to Ames (reverse mutation) tests and single oral-dose rat studies, which revealed non-mutagenic and non-toxic properties. When three purified phycocyanins (abPC, skPC, and spPC) were subjected to agarose gel electrophoresis, they showed different mobility, indicating that each phycocyanin has unique properties. abPC exhibited strong antiglycation activities as novel function.

Production and release of 2-MIB in Pseudanabaena: Effects of growth phases on cell characteristics and 2-MIB yield.

2-methylisoborneol (2-MIB), a secondary metabolite produced by cyanobacteria, often causes a musty odour in water, threatening the safety of drinking water supplies. This study investigated the effects of the growth phases on the production of 2-MIB by Pseudanabaena. The effects of cell characteristics on the production and release of 2-MIB were also explored. The total 2-MIB concentration increased during the exponential phase and decreased during the declining phase, which was consistent with the changes in cell density. However, the total 2-MIB yield (1.12-1.27 fg cell-1) of Pseudanabaena did not significantly differ throughout the growth cycle (p > 0.05). Meanwhile, the extracellular 2-MIB yield increased significantly from 0.31 fg cell-1 in the exponential phase to 0.76 fg cell-1 in the declining phase (p < 0.05), and the corresponding proportion of extracellular 2-MIB improved from 25.13% to 59.16% (p < 0.05). The surge in extracellular 2-MIB during the declining phase could be attributed to the breaking of the Pseudanabaena filament, as indicated by the decrease in Dmean during cell ageing. The findings of this study contribute to a more inclusive comprehension and management of musty odour issues resulting from cyanobacteria in the water supply.

Maintenance of cell integrity during hydroxyl radical rapid inactivation of Pseudanabaena sp. and simultaneous mineralization of odor compound 2-methylisoborneol.

Pseudanabaena sp. and the odor compound it produces, 2-methylisoborneol (2-MIB), has been reportedly responsible for off-flavor pollution worldwide, leading to substandard drinking water sensory indicators and serious water supply crises. In this paper, the hydroxyl radical (•OH) produced by the synergistic effect of strong ionization discharge and hydrodynamic cavitation rapidly inactivated Pseudanabaena sp. and simultaneously mineralized 2-MIB to a concentration of 2.57 ng/L, which is below the odor threshold of 10 ng/L for a total reactive oxidants (TRO) concentration of 1.2 mg/L within 12 s. Crucially, the intracellular 2-MIB level was maintained in approximately 155.26- 162.29 ng/L range, indicating that 2-MIB was not released from the cells. Based on the scanning electron microscopy (SEM) results, the integrity of Pseudanabaena sp. cells was maintained with intact membranes and no intracellular organic matters (IOM) released during •OH inactivation. In contrast, ClO2 caused severe membrane rupture and massive IOM release. Based on the gas chromatograph/mass spectrometer (GC/MS) analyses and mass spectral database, the chromatogram fitted the baseline with a TRO concentration of 4 mg/L and no peaks corresponding to intermediates were detected. Moreover, •OH could mineralize 2-MIB by opening the ring structures of 1,2,3,3-tetramethyl-4-cyclopentenone, neomenthol, and 2-methylcyclohexene-1-aldehyde to produce small-molecule compounds, finally leading to CO2 and H2O formation via three reaction pathways. Therefore, the •OH not only maintained the cell integrity of Pseudanabaena sp. during inactivation but also mineralized 2-MIB simultaneously.

Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli.

Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl diphosphate by geranyl diphosphate methyl transferase (GPPMT) and subsequent cyclization into 2-MIB by 2-MIB synthase (MIBS). Pseudanabaena foetida var. intermedia is a 2-MIB-producing cyanobacterium whose GPPMT and MIBS enzymes are encoded by adjacent mtf and mtc genes. The present study identified a 2-MIB-related gene cluster composed of cnbA, mtf, mtc, and cnbB genes in P. foetida var. intermedia. The two homologous cyclic nucleotide-binding protein genes, cnbA and cnbB, were detected adjacent to the mtf and mtc genes, respectively. The nucleotide sequence of the cnbA-mtf-mtc-cnbB gene cluster showed 99.55% identity with 2-MIB synthesis-associated gene cluster of Pseudanabaena sp. dqh15. RT-PCR results revealed that mtf and mtc genes are co-expressed, while cnbA and cnbB genes are expressed independently in P. foetida var. intermedia. To investigate whether only mtf and mtc genes are sufficient for 2-MIB synthesis, the two-gene unit (mtf-mtc) was introduced into Escherichia coli strain JM109 via overexpression vector pYS1C. Gas chromatograph-mass spectrometry results showed that the E. coli strain transformed with mtf-mtc was able to produce 2-MIB. The intracellular 2-MIB level in P. foetida var. intermedia was higher than the extracellular 2-MIB level, while the transformed E. coli strain showed an opposite trend. Growth inhibition was observed in the 2-MIB-producing transformed E. coli strain. IMPORTANCE Contamination of drinking water with odiferous microbial metabolite 2-MIB is a worldwide concern. Removal of 2-MIB from drinking water burdens the water purification process. Therefore, it is important to search for alternative methods, such as suppressing the production of 2-MIB by aquatic microorganisms. For that, it is necessary to expand the current knowledge about the mechanism of 2-MIB synthesis at the genetic level. This study revealed that mtf and mtc genes of the 2-MIB-related gene cluster are transcribed as a single unit in P. foetida var. intermedia, and the expression of both mtf and mtc genes is essential and sufficient for 2-MIB synthesis in E. coli heterologous gene expression system.

Microcystins and Cyanobacterial Contaminants in the French Small-Scale Productions of Spirulina (Limnospira sp.).

Spirulina is consumed worldwide, in the form of food or dietary supplements, for its nutritional value and health potential. However, these products may contain cyanotoxins, including hepatotoxic microcystins (MCs), produced by cyanobacterial contaminants. The French spirulina market has the particularity of being supplied half-locally by approximately 180 small-scale spirulina production farms. Data about this particular production and possible contaminations with other cyanobacteria and MCs are scarce. Thus, we collected the results of MC analyses and total cyanobacteria counts, carried out between 2013 and 2021, from 95 French spirulina producers who agreed to share their data. These data consisted of MC concentrations determined with an enzyme-linked immunosorbent assay (ELISA) using 623 dry spirulina samples and 105 samples of spirulina cultures. In addition, potentially unsafe samples of dry spirulina were further investigated through mass spectrometry, as duplicate analysis. We confirmed that the situation of the French spirulina production stayed within the safe regulatory level in terms of MC levels. On the other hand, the inventory of cyanobacterial contaminants, based on 539 count results, included 14 taxa. We present their prevalence, interannual evolution and geographical distribution. We also suggested improvements in cultivation practices to limit their propagation.

Distribution, driving forces, and risk assessment of 2-MIB and its producer in a drinking water source-oriented shallow lake.

Freshwater blooms of harmful cyanobacteria in drinking water source-oriented shallow lakes affect public health and ecosystem services worldwide. Therefore, identifying 2-methylisoborneol (2-MIB)-producing cyanobacteria and predicting the risks of 2-MIB are critical for managing 2-MIB-infected water sources. Previous studies on the potential producers and risks of 2-MIB have focused on reservoirs or have been limited by the ecosystems of phytoplankton-dominated areas. We investigated the producers, distribution, and occurrence of 2-MIB in East Taihu Lake-a drinking water source-oriented shallow lake with macrophyte- and phytoplankton-dominated areas-from August 2020 to November 2021. We observed that Pseudanabaena sp. produces 2-MIB in this lake, as determined by the maximum correlation coefficient (R = 0.71, p < 0.001), maximum detection rate, and minimum false positive/negative ratio exhibited by this genus. Extreme odor events occurred in this lake during late summer and early autumn in 2021, with the mean 2-MIB concentration increasing to 727 ± 426 ng/L and 369 ± 176 ng/L in August and September, respectively. Moreover, the macrophyte-dominated area, particularly the wetland area, exhibited a significant decrease (p < 0.01) in bloom intensity and 2-MIB production during these extreme odor events. Pseudanabaena sp. outbreak was likely owing to eutrophication, seasonal gradients, and macrophyte reduction, considering that temporal trends were consistent with high water temperature, high total phosphorus levels, and low-light conditions. Moreover, 2-MIB production was sensitive to short-term hydrometeorological processes, with high water levels and radiant intensity enhancing 2-MIB production. The risk assessment results showed that the probability of 2-MIB concentration exceeding the odor threshold (10 ng/L) is up to 90% when the cell density of Pseudanabaena sp. reaches 1.8 × 107 cell/L; this risk is reduced to 50 and 25% at densities of < 3.8 × 105 cell/L and 5.6 × 104 cell/L, respectively. Our findings support calls for shallow lake management efforts to maintain a macrophyte-dominated state and control odorous cyanobacteria growth.

Corrected and Republished from: "Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena".

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena. PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. PA-SR01 is a member of Siphoviridae with a long noncontractile tail. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles. IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

A novel cyanolytic bacterium, Pseudomonas fluorescens BG-E as a potential biological control agent for freshwater bloom-forming cyanobacteria Pseudanabaena spp.

The majority of bacterial antagonists identified to date are active against Microcystis. Therefore, this study aimed to isolate and characterize novel cyanolytic bacterial strains antagonistic against bloom-forming filamentous cyanobacteria. The bacterial strain BG-E isolated from the Bandagiriya Wewa in Sri Lanka was identified as Pseudomonas fluorescens (MZ007859) based on the 16S rRNA gene sequencing. BG-E showed 82% and 73% cyanolytic activity (CA) against Pseudanabaena sp. LW2 (MW288948) and Pseudanabaena lonchoides LW1 (MW288940), respectively, after 10 days of inoculation. The light microscopic images affirmed the complete disintegration in the filamentous structures of the tested Pseudanabaena species. The bacterial cell density of 15% v/v showed the CA with 95% and 89% cell lysis, respectively, in P. lonchoides and Pseudanabaena sp. LW2. Moreover, the results showed that >50% CA could be achieved by 0.100 and 1.00 (OD730 ) cell densities for these same species. The highest CA of the cell-free supernatant of BG-E against P. lonchoides and bacterial culture against Pseudanabaena sp. LW2 illustrated the species-specific mode of action of BG-E. Although BG-E efficiently lysed the tested cyanobacterial species, the results of the MC-biodegradation assay confirmed its inability to degrade MC-LR cyanotoxin. Further, the BG-E strain lacks the mlrABCD gene cluster which is known to be responsible for the enzymatic degradation of MCs. The overall findings highlighted the applicability of P. fluorescens BG-E as a biological controlling agent to terminate blooms of freshwater filamentous cyanobacteria genus Pseudanabaena. The incorporation of cyanotoxin-degrading heterotrophic bacteria is recommended as a means of controlling toxic Pseudanabaena blooms.

Early warning of MIB episode based on gene abundance and expression in drinking water reservoirs.

Cellular 2-methylisoborneol (MIB) yield of cyanobacteria varies under different conditions according to culture studies and field investigations, the causal mechanism remains unclear and results in ineffective MIB prediction. Through an intensive field survey during an MIB episode produced by Pseudanabaena cinerea in QCS reservoir, we demonstrated that MIB synthesis (mic) gene abundance (DNA) and expression (RNA) might be useful as parameters for early warning of MIB production. It was found that the abundance of mic DNA and RNA peaked ahead of MIB concentrations by 10 and 7 days, respectively. In addition, the RNA abundance (R2 = 0.45, p < 0.01) showed a slightly higher correlation with MIB compared to DNA abundance (R2 = 0.37, p < 0.01), suggesting that the conditions for the growth of Pseudanabaena cinerea might be slightly different from those for mic gene expression, which was verified by a culture experiment. The highest cell growth was obtained under 36 µmol photons m-2 s-1, while the highest cellular MIB yield and mic gene expression level were obtained under 85 µmol photons m-2 s-1. Our results clearly supported that light intensity was the virtual regulator governing the mic gene expression within the controlled culture experiment and the actual MIB episode in the reservoir. Besides these results, we developed an early warning model using mic gene abundance as an indicator of MIB episodes, which was verified in two other reservoirs. Our findings highlight the effect of light intensity on mic gene expression and MIB synthesis and provide an early warning tool targeting MIB episode prediction, which therefore should be of importance for source water authorities.

Biosynthesis of 2-methylisoborneol is regulated by chromatic acclimation of Pseudanabaena.

Cyanobacteria can sense different light color by adjusting the components of photosynthetic pigments including chlorophyll a (Chl a), phycoerythrin (PE), and phycocyanin (PC), etc. Filamentous cyanobacteria are the main producer of 2-methylisoborneol (MIB) and many can increase their PE levels so that they are more competitive in subsurface layer where green light is more abundant, and have caused extensive odor problems in drinking water reservoirs. Here, we identified the potential correlation between MIB biosynthesis and ambient light color induced chromatic acclimation (CA) of a MIB-producing Pseudanabaena strain. The results suggest Pseudanabaena regulates the pigment proportion through Type III CA (CA3), by increasing PE abundance and decreasing PC in green light. The biosynthesis of MIB and Chl a share the common precursor, and are positively correlated with statistical significance regardless of light color (R2=0.68; p<0.001). Besides, the PE abundance is also positively correlated with Chl a in green light (R2=0.57; p=0.019) since PE is the antenna that can only transfer the energy to PC and Chl a. In addition, significantly higher MIB production was observed in green light since more Chl a was synthesized.

Heterotrophic Cultivation of the Cyanobacterium Pseudanabaena sp. on Forest Biomass Hydrolysates toward Sustainable Biodiesel Production.

Environmental pollution, greenhouse gas emissions, depletion of fossil fuels, and a growing population have sparked a search for new and renewable energy sources such as biodiesel. The use of waste or residues as substrates for microbial growth can favor the implementation of a biorefinery concept with reduced environmental footprint. Cyanobacteria constitute microorganisms with enhanced ability to use industrial effluents, wastewaters, forest residues for growth, and concomitant production of added-value compounds. In this study, a recently isolated cyanobacterium strain of Pseudanabaena sp. was cultivated on hydrolysates from pretreated forest biomass (silver birch and Norway spruce), and the production of biodiesel-grade lipids was assessed. Optimizing carbon source concentration and the (C/N) carbon-to-nitrogen ratio resulted in 66.45% w/w lipid content when microalgae were grown on glucose, compared to 62.95% and 63.79% w/w when grown on spruce and birch hydrolysate, respectively. Importantly, the lipid profile was suitable for the production of high-quality biodiesel. The present study demonstrates how this new cyanobacterial strain could be used as a biofactory, converting residual resources into green biofuel.

Green Synthesis and Characterization of Silver Nanoparticles with High Antibacterial Activity Using Cell Extracts of Cyanobacterium Pseudanabaena/Limnothrix sp.

In this work, we demonstrated the ability of the cyanobacterium Pseudanabaena/Limnothrix sp. to produce ultra-small silver nanoparticlesin the forms of metallic silver (Ag0) and silver oxides (AgxOy) via a facile green synthetic process. The biological compounds in the cyanobacterial cellular extract acted both as reducing agents for silver ions and functional stabilizing agents for the silver nanoparticles. Furthermore, the antibacterical activity of the as-synthesized nanoparticles against Gram-negative Escherichia coli and Gram-positive Corynebacterium glutamicum bacterial cells was evaluated. The experimental results revealed a remarkable bactericidal activity of the nanoparticles that was both time-dependent and dose-dependent. In addition to their excellent bactericidal properties, the developed nanoparticles can be used as nanosupports in various environmental, biological, and medical applications.

On the trail of iron uptake in ancestral Cyanobacteria on early Earth.

Cyanobacteria oxygenated Earth's atmosphere ~2.4 billion years ago, during the Great Oxygenation Event (GOE), through oxygenic photosynthesis. Their high iron requirement was presumably met by high levels of Fe(II) in the anoxic Archean environment. We found that many deeply branching Cyanobacteria, including two Gloeobacter and four Pseudanabaena spp., cannot synthesize the Fe(II) specific transporter, FeoB. Phylogenetic and relaxed molecular clock analyses find evidence that FeoB and the Fe(III) transporters, cFTR1 and FutB, were present in Proterozoic, but not earlier Archaean lineages of Cyanobacteria. Furthermore Pseudanabaena sp. PCC7367, an early diverging marine, benthic strain grown under simulated Archean conditions, constitutively expressed cftr1, even after the addition of Fe(II). Our genetic profiling suggests that, prior to the GOE, ancestral Cyanobacteria may have utilized alternative metal iron transporters such as ZIP, NRAMP, or FicI, and possibly also scavenged exogenous siderophore bound Fe(III), as they only acquired the necessary Fe(II) and Fe(III) transporters during the Proterozoic. Given that Cyanobacteria arose 3.3-3.6 billion years ago, it is possible that limitations in iron uptake may have contributed to the delay in their expansion during the Archean, and hence the oxygenation of the early Earth.

Pseudanabaena galeata CCNP1313-Biological Activity and Peptides Production.

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549ACE2/TMPRSS2) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts.

2-Methylisoborneol (2-MIB) Excretion by Pseudanabaena yagii under Low Temperature.

Outbreaks of 2-methylisoborneol (2-MIB) contamination in drinking water sources cause inconvenient odor issues in the water distribution system. In this study, microscopy-based isolation with physiological and molecular phylogenetic characterization were performed to investigate and characterize the 2-MIB odor producers that caused an odor problem in the freshwater system of the North Han River in the autumn of 2018. A benthic cyanobacterium was isolated from 2-MIB odor-issue freshwater samples and was found to be phylogenetically affiliated with Pseudanabaena yagii (99.66% sequence similarity), which was recorded in South Korea for the first time. The 2-MIB synthesis gene sequences from the odor-issue freshwater samples showed 100% similarity with those in the P. yagii strains. Protein sequences of 2-MIB synthase observed in the genome of the isolated strain showed structural and functional characteristics similar to those observed in other Pseudanabaena species. The 2-MIB production rate increased slowly during mat formation on the vessel wall; however, it rapidly increased after the temperature dropped. The 2-MIB gene was continuously expressed regardless of the temperature changes. These results suggest that the 2-MIB odor issue in the North Han River might be caused by the release of 2-MIB from the mat-forming P. yagii species in a low-temperature freshwater environment.

Development of a method for phycocyanin recovery from filamentous cyanobacteria and evaluation of its stability and antioxidant capacity.

BACKGROUND: Most commercial phycocyanins are extracted from a filamentous cyanobacterium, Arthrospira (Spirulina) platensis. Owing to the expenses of culture and complexities of the physical and chemical methods of phycocyanin purification, a more effective and simple method is required. RESULTS: We developed a new method for efficiently recovering the blue pigment protein, phycocyanin, from unique filamentous cyanobacteria, Pseudanabaena sp. ABRG5-3 and Limnothrix sp. SK1-2-1. The cells were cultivated in economy medium BG11 and lysed by adding water in a 1:16 ratio of wet cells to water. After extraction and purification, 28-30% dry cell weight of phycocyanin was obtained and its purity was confirmed. The stabilities of the phycocyanins at different pH in the presence of high temperature and light conditions and their antioxidant abilities were assessed. Results indicated that the phycocyanins were stable and possessed antioxidant properties. Interestingly, the Pseudanabaena phycocyanin was less likely to deteriorate under acidic conditions. CONCLUSIONS: Overall, we developed a promising and novel method for producing high functional phycocyanin concentrations at a low cost. The possibilities of adapting this new phycocyanin biorefinery to unique bioreactor utilization have also been discussed.

Environmental factors associated with cyanobacterial assemblages in a mesotrophic subtropical plateau lake: A focus on bloom toxicity.

Harmful algal blooms caused by cyanobacteria have been increasing in frequency worldwide. However, the main environmental drivers of this change are often difficult to identify because of the effects of the interaction between eutrophication and climate change. Recently, filamentous N2-fixing cyanobacteria and non-diazotrophic Microcystis have been observed to be co-existing and undergoing succession in some eutrophic lakes. However, the succession patterns of dominant cyanobacteria and the factors driving this in mesotrophic lakes are not well understood. We hypothesized that the changes in cyanobacterial assemblages in mesotrophic lakes could result in a relatively high risks of toxic blooms, and that these changes are associated with the global climatic changes. We tested these hypotheses using data from the subtropical mesotrophic Lake Erhai. We found that the high spatiotemporal variability in the cyanobacterial community, and the increase in biomass were driven primarily by the growth of bloom-forming cyanobacterial taxa. Species-specific biomasses were related to a different environmental stressor; increases in dissolved organic carbon (DOC) concentrations were statistically associated with an increase of Microcystis biomass, whereas increases in surface water temperature favored higher biomass of Pseudanabaena at low transparency and high concentration of phosphorus. In addition, low nitrogen- to- phosphorus ratios were identified as potential determinants of the abundance of N2-fixing Dolichospermum. Furthermore, changes in the concentration of DOC, total nitrogen, pH and water transparency levels were found to affect the composition of Microcystis morphotypes and genotypes mostly. This study highlights that the toxic to non-toxic Microcystis ratio might increase with the water darkening and browning (which occurs in many subtropical plateau lakes). Lake management strategies, therefore, need to consider the toxicity of cyanobacterial assemblages in mesotrophic lakes over the intensity of the cyanobacterial blooms.

Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena.

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. Distinct from the majority of cyanophage isolates, PA-SR01 has a tailless morphology. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles.IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

Photosynthetic adaptation to light availability shapes the ecological success of bloom-forming cyanobacterium Pseudanabaena to iron limitation.

The poorly understood filamentous cyanobacterium Pseudanabaena is commonly epiphytic on Microcystis colonies and their abundances are often highly correlated during blooms. The response and adaptation of Microcystis to iron limitation have been extensively studied, but the strategies Pseudanabaena uses to respond to iron limitation are largely unknown. Here, physiological responses to iron limitation were compared between one Pseudanabaena and two Microcystis strains grown under different light intensities. The results showed that low-intensity light exacerbated, but high-intensity light alleviated, the negative effect of iron limitation on Pseudanabaena growth relative to two Microcystis strains. It was found that robust light-harvesting and photosynthetic efficiency allowed adaptation of Pseudanabaena to low light availability relative to two Microcystis strains only during iron sufficiency. The results also indicated that a larger investment in the photosynthetic antenna probably contributed to light/iron co-limitation of Pseudanabaena relative to two Microcystis strains under both light and iron limitation. Furthermore, the lower antenna pigments/chlorophyll a ratio and photosynthetic efficiency, and higher nonphotochemical quenching and saturation irradiance provided Pseudanabaena photoadaptation and photoprotection advantages over the two Microcystis strains under the high-light condition. The lower investment in antenna pigments of Pseudanabaena than the two Microcystis strains under high-light intensity is likely an efficient strategy for both saving iron quotas and decreasing photosensitivity. Therefore, when compared with Microcystis, the high plasticity of antenna pigments, along with the excellent photoadaptation and photoprotection ability of Pseudanabaena, probably ensures its ecological success under iron limitation when light is sufficient.

Interspecific competition between Microcystis aeruginosa and Pseudanadaena and their production of T&O compounds.

Microcystis aeruginosa and Pseudanabaena are two common cyanobacterial species/genus and they can occur coincidently in many eutrophic lakes globally. These two cyanobacteria could produce Taste & Odor (T&O) compounds, and their production of T&O compounds might be changed when they are present coincidently. The amounts of T&O compounds and their producers may influence the effectiveness of water treatment processes. Therefore, the mutual interactions between Microcystis aeruginosa (FACHB-905, M) and Pseudanabaena sp. (FACHB-1277, P) on T&O compounds in co-cultures were evaluated in this study. Different initial cell concentrations of M and P, with ratios of M:P = 1:1, M:P = 1:2 and M:P = 2:1 were applied in the co-cultures. The growth of M was enhanced under all of the cyanobacterial cell ratios. The growth of P was enhanced under the ratio of M:P = 1:1, while it was inhibited under the ratios of M:P = 1:2 and M: P = 2:1. In addition, the growth of the two cyanobacteria and their production of ß-cyclocitral and 2-methylisoborneol (2-MIB) in the filtrate of P were higher than those in the filtrate of M, which may be attributed to their associated secondary metabolites. The cell integrity and photosynthetic capacity of the two studied cyanobacteria are greatly affected by exposure to ß-cyclocitral and 2-MIB. The results showed that ß-cyclocitral and 2-MIB had the allelopathic effects on the two cyanobacteria species which might influence the composition of co-existing cyanobacteria and their production of T&O compounds.