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Considerable studies have been conducted to quantify and examine free cyanophage particles across a wide range of aquatic systems, but few studies have analyzed the dynamics of host cell fraction cyanophages, and even less is known about tropical freshwater cyanophages. Here, we employed qPCR to examine the abundances of four ecologically relevant novel cyanophages (PA-SR01, S-SRP01, S-SRP02 and S-SRM01) in a tropical urban lake in Singapore from July to November 2019. Besides quantifying the extracellular free cyanophage particles, host cell fraction cyanophage were also examined to represent the abundance of intracellular and attached phages. Our study revealed differing fate and dynamics of each cyanophage target. Host cell fraction phage abundance was found to be more abundant than free phage abundance, indicating high level of infection and rapid dispersal of phage particles upon lysis. By further investigating the correlations between free phage particle abundance and numerous biotic (total cyanobacteria, Microcystis abundances, etc) as well as abiotic (nutrients and physiochemical water quality) variables, multiple correlations across different sampling sites were obtained, suggesting pH and rainfall to be potential key factors driving shifts in both cyanophage and cyanobacteria population. This study demonstrated the ecological importance and key roles of the cyanophage PA-SR01, S-SRP01, S-SRP02 and S-SRM01 in tropical freshwater lakes not only as regulators of the cyanobacterial community, but also as promoters of certain cyanotoxin production.

Rapid proliferation of cyanobacteria in both benthic and suspended (planktonic) habitats is a major threat to environmental safety, as they produce nuisance compounds such as cytotoxins and off-flavors, which degrade the safety and quality of water supplies. Temperature and light irradiance are two of the key factors in regulating the occurrence of algal blooms and production of major off-flavors. However, the role of these factors in regulating the growth and metabolism is poorly explored for both benthic and planktonic cyanobacteria. To fill this gap, we studied the effects of light and temperature on the growth and metabolic profiling of both benthic (Hapalosiphon sp. MRB220) and planktonic (Planktothricoides sp. SR001) environmental species collected from a freshwater reservoir in Singapore. Moreover, this study is the first report on the metabolic profiling of cyanobacteria belonging to two different habitats in response to altered environmental conditions. The highest growth rate of both species was observed at the highest light intensity (100 μmol photons/m²/s) and at a temperature of 33 °C. Systematic metabolite profiling analysis suggested that temperature had a more profound effect on metabolome of the Hapalosiphon, whereas light had a greater effect in the case of Planktothricoides. Interestingly, Planktothricoides sp. SR001 showed a specialized adaptation mechanism via biosynthesis of arginine, and metabolism of cysteine and methionine to survive and withstand higher temperatures of 38 °C and higher. Hence, the mode of strategies for coping with different light and temperature conditions was correlated with the growth and alteration in metabolic activities for physiological and ecological adaptations in both species. In addition, we putatively identified a number of unique metabolites with a broad range of antimicrobial activities in both species in response to both light and temperature. These metabolites could play a role in the dominant behavior of these species in suppressing competition during bloom formation. Overall, this study elucidated novel insights into the effects of environmental factors on the growth, metabolism, and adaptation strategies of cyanobacteria from two different habitats, and could be useful in controlling their harmful effects on human health and environmental concerns.

Microcystin is an algal toxin that is commonly found in eutrophic freshwaters throughout the world. Many studies have been conducted to elucidate the factors affecting its production, but few studies have attempted mechanistic models of its production to aid water managers in predicting its occurrence. Here, a mechanistic model was developed based on microcystin production by Microcystis spp. under laboratory culture and ambient field conditions. The model was built on STELLA, a dynamic modelling software, and is based on constitutive cell quota that varies with nitrogen, phosphorus, and temperature. In addition to these factors, varying the decay rate of microcystin according to its proportion in the intracellular and extracellular phase was important for the model’s performance. With all these effects, the model predicted most of the observations with a model efficiency that was >0.72 and >0.45 for the lab and field conditions respectively. However, some large discrepancies were observed. These may have arisen from the non-constitutive microcystin production that appear to have a precondition of nitrogen abundance. Another reason for the large root mean square error is that cell quota is affected by factors differently between strains.

Abstract Planktothricoides is a free-living filamentous cyanobacterium belonging to the order Oscillatoriales and the family Phormidiaceae, capable of forming bloom in fresh and brackish waters. A unicyanobacterial non-axenic culture dominated by Planktothricoides sp. SR001 was obtained from a freshwater reservoir in Singapore. The draft genome presented here is the first tropical freshwater Planktothricoides sp. ever sequenced. The genome of 7.0Mbp contains 5,776 genes predicted using the JGI IMG pipeline. The whole genome sequence allows identification of genes encoding for nitrogen-fixation, accessory photosynthetic pigments and biosynthesis of an off-flavor compound, 2-methylisoborneol, which has been experimentally verified here based on metabolite detection. In addition, strain SR001 genome contains an operon putatively involved in the production of a linear tripeptide cyanobactin related to viridisamide A and aeruginosamide, with the later known to possess anti-microbial or cytotoxic effect.

In this study, Illumina MiSeq sequencing technique was employed to explore the characteristics and dynamics of cyanobacteria–heterotrophic bacteria between two estuarine reservoirs in sub-tropical (reservoir A in Shanghai) and tropical (reservoir B in Singapore) regions. The results indicated that significant differences in bacterial community composition were found between two estuarine reservoirs, which influenced by varied environmental variables. The environmental heterogeneity in reservoir A was much higher, which indicated that the composition of bacterial community in reservoir A was more complex. In contrast, reservoir B provided a suitable and temperate water environment conditions for bacterial growth, which resulted in higher community diversity and less co-exclusion correlations. The molecular ecological network indicated that the presence of dominant bacterial community in each of the reservoir were significant different. These differences mainly reflected the responses of bacterial community to the variations of environmental variables. Although Synechococcus was the dominant cyanobacterial species in both reservoirs, it exhibited co-occurrence patterns with different heterotrophic bacteria between reservoirs. In addition, the cyanobacteria–heterotrophic bacteria interaction exhibited highly dynamic variations, which was affected by nutrition and survive space. Also, the co-occurrence of Microcystis and Pseudanabaena found in reservoir B implied that the non-N-fixing Microcystis accompanied with N-fixing Pseudanabeana occurrence in freshwater lakes, so as to better meet the demand for nitrogen source.

In this study, we focused on the dynamics of bacterial community composition in a large reservoir in the Yangtze estuary during spring and summer seasons, especially the variations of functional mechanisms of microbial community during the seasonal alternation between spring and summer. Both 16S rRNA gene sequencing and shotgun metagenomic sequencing technology were used for these purposes. The results indicated that obvious variations of bacterial community structures were found at different sites. Particle-associated bacterial taxa exhibited higher abundance at the inlet site, which was closer to the Yangtze River with a high level of turbidity. In other sites, Synechococcus, as the most dominant cyanobacterial species, revealed high abundance driven by increased temperature. Moreover, some heterotrophic bacterial taxa revealed high abundance following the increased Synechococcus in summer, which indicated potential correlations about carbon source utilization between these microorganisms. In addition, the shotgun metagenomic data indicated during the period of seasonal alternation between spring and summer, the carbohydrate transport and metabolism, energy production and conversion, translation/ribosomal biogenesis, and cell wall/membrane/envelope biogenesis were significantly enhanced at the exit site. However, the course of cell cycle control/division was more active at the internal site.

The increased antibiotic pollutants in aquatic environments pose severe threats on microbial ecology due to their extensive distribution and antibacterial properties. A total of 16 antibiotics including fluoroquinolones (FQs) (ofloxacin (OFX), ciprofloxacin (CFX), norfloxacin (NFX)), Sulfonamides (SAs) (sulfamonomethoxine (SMM), sulfadiazine (SDZ), sulfaquinoxaline (SQX)), Tetracyclines (TCs) (tetracycline (TC), doxycycline (DC)), β-lactams (penicillin G (PEN G), penicillin V (PEN V), cefalexin (LEX)), Macrolides (MLs) (erythromycin-H2O (ETM), tylosin (TYL)) and other antibiotics (Polymix-B (POL), Vancomycin (VAN), Lincomycin (LIN)) were detected in the surface water of the Qingcaosha Reservoir. Multivariate statistical analysis indicated that both water quality and physicochemical indexes have less contributions on variations of these antibiotics, suggesting the concentrations of antibiotics inside the reservoir are mainly affected by upstream runoff and anthropic activity along the river. Antibiotics including TYL, PEN G and ETM showed significant correlations with variations of bacterial community composition, and closely connected with various gram-negative bacteria in co-occurrence/exclusion patterns of the network, suggesting these bacterial taxa play important roles in the course of migration and transformation of related antibiotics. In conclusion, further research is required to evaluate the potential risk of genetic transfer of resistance to related bacteria induced by long-term exposure to low levels of antibiotics in the environment.

The identification of phytoplankton species and microbial biodiversity is necessary to assess water ecosystem health and the quality of water resources. We investigated the short-term (2 days) vertical and diel variations in bacterial community structure and microbially derived secondary metabolites during a cyanobacterial bloom that emerged in a highly urbanized tropical reservoir. The waterbody was largely dominated by the cyanobacteria Planktothricoides spp., together with the Synechococcus, Pseudanabaena, Prochlorothrix, and Limnothrix. Spatial differences (i.e., water depth) rather than temporal differences (i.e., day versus night) better-explained the short-term variability in water quality parameters and bacterial community composition. Difference in bacterial structure suggested a resource-driven distribution pattern for the community. We found that the freshwater bacterial community associated with cyanobacterial blooms is largely conserved at the phylum level, with Proteobacteria (ß-proteobateria), Bacteroidetes, and Actinobacteria as the main taxa despite the cyanobacterial species present and geographical (Asia, Europe, Australia, and North America) or climatic distinctions. Through multivariate statistical analyses of the bacterial community, environmental parameters, and secondary metabolite concentrations, we observed positive relationships between the occurrences of cyanobacterial groups and off-flavor compounds (2-methyisoborneol and ß-ionone), suggesting a cyanobacterial origin. This study demonstrates the potential of 16S rRNA gene amplicon sequencing as a supporting tool in algal bloom monitoring or water-resource management. [ABSTRACT FROM AUTHOR]

A non-axenic unialgal culture containing a Subsection V (Stigonematales) cyanobacterium, Hapalosiphon strain MRB 220, was obtained from a benthic freshwater algal mat through multiple transfers following growth in sterile media. Physiological characterization demonstrated the culture was capable of nitrogen-fixation and production of the off flavor compound 2-methylisoborneol (2-MIB). Total DNA isolated from this culture was sequenced using Illumina HiSeq and de novo assembled into contigs. The genome of MRB 220 was separated from co-occurring heterotrophic bacteria using sequence homology and compositional approaches, and its purity was confirmed based on best BLAST hit classification and principle component analysis of the tetranucleotide frequencies of fragmented contigs. The genome of ~7.4 Mbp contains 6,345 protein coding genes with 4,320 of these having functional prediction including predicted pathways for biosynthesis of the secondary metabolite welwitindolinone. Analyses of 16S rRNA gene and whole genome sequence average nucleotide identity indicated close relatedness of MRB 220 to the genera Hapalosiphon and Fischerella within the order Stigonematales. Microscopic examination showed that MRB 220 formed heterocystous branched filaments, thereby supporting identification of strain MRB 220 as a morphospecies of Hapalosiphon. Availability of the draft genome of Hapalosiphon strain MRB 220 enables future work to elucidate the pathway and dynamics for biosynthesis of 2-MIB and other secondary metabolites and understand the ecology and physiology of Stigonematales cyanobacteria in tropical freshwaters. [ABSTRACT FROM AUTHOR]

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]