Microbial diversity, genomics, and phage-host interactions of cyanobacterial harmful algal blooms.

The occurrence of cyanobacterial harmful algal blooms (cyanoHABs) is related to their physical and chemical environment. However, less is known about their associated microbial interactions and processes. In this study, cyanoHABs were analyzed as a microbial ecosystem, using 1 year of 16S rRNA sequencing and 70 metagenomes collected during the bloom season from Lake Okeechobee (Florida, USA). Biogeographical patterns observed in microbial community composition and function reflected ecological zones distinct in their physical and chemical parameters that resulted in bloom "hotspots" near major lake inflows. Changes in relative abundances of taxa within multiple phyla followed increasing bloom severity. Functional pathways that correlated with increasing bloom severity encoded organic nitrogen and phosphorus utilization, storage of nutrients, exchange of genetic material, phage defense, and protection against oxidative stress, suggesting that microbial interactions may promote cyanoHAB resilience. Cyanobacterial communities were highly diverse, with picocyanobacteria ubiquitous and oftentimes most abundant, especially in the absence of blooms. The identification of novel bloom-forming cyanobacteria and genomic comparisons indicated a functionally diverse cyanobacterial community with differences in its capability to store nitrogen using cyanophycin and to defend against phage using CRISPR and restriction-modification systems. Considering blooms in the context of a microbial ecosystem and their interactions in nature, physiologies and interactions supporting the proliferation and stability of cyanoHABs are proposed, including a role for phage infection of picocyanobacteria. This study displayed the power of "-omics" to reveal important biological processes that could support the effective management and prediction of cyanoHABs. IMPORTANCE: Cyanobacterial harmful algal blooms pose a significant threat to aquatic ecosystems and human health. Although physical and chemical conditions in aquatic systems that facilitate bloom development are well studied, there are fundamental gaps in the biological understanding of the microbial ecosystem that makes a cyanobacterial bloom. High-throughput sequencing was used to determine the drivers of cyanobacteria blooms in nature. Multiple functions and interactions important to consider in cyanobacterial bloom ecology were identified. The microbial biodiversity of blooms revealed microbial functions, genomic characteristics, and interactions between cyanobacterial populations that could be involved in bloom stability and more coherently define cyanobacteria blooms. Our results highlight the importance of considering cyanobacterial blooms as a microbial ecosystem to predict, prevent, and mitigate them.

Change and stasis of distinct sediment microbiomes across Port Everglades Inlet (PEI) and the adjacent coral reefs.

Deep water ports are human built coastal structures that by definition welcome ship traffic and disturbance. Evidence is accumulating that enhanced port activities such as dredging or deepening have negatively affected nearby natural habitats. Port Everglades Inlet (PEI) is a large active South Florida cargo port for over two million people and lies adjacent to coral reefs, dwindling mangroves, and recreational beaches. In this study, the microbial communities of PEI and adjacent reef sediments were characterized to serve as indicators for change due to dredging and assess anthropogenic influence on these sensitive ecosystems by sequencing the V4 region of 16S rRNA ahead of a large-scale port deepening event. For the first time, this study established baseline bacterial community characterizations and their patterns of diversity prior to and after a maintenance dredging event. PEI samples were collected for two consecutive years 2020 (Phase I, before maintenance dredging) and 2021 (Phase II, after maintenance dredging) from PEI sediments and adjacent coral reef sediments. In spite of their proximity and tidal connections through the PEI, reef and PEI sediment microbial communities were distinct. Changes in microbial diversity within the intracoastal waterway (ICW), a route for community exchange or transfers, were the greatest after maintenance dredging occurred. Microbial diversity in reef sediments also changed after dredging, indicating potential influence from resuspended sediments due to an associated increase in trace metals and decrease in cyanobacterial diversity. Sediments were identified as a possible source of human and coral pathogens, although dredging did not affect the relative abundances of these indicator microorganisms. This study highlighted the utility and relative ease of applying current molecular ecology methods to address macroscale questions with environmental management ramifications.

Three complete mitochondrial genomes of shortfin mako sharks, Isurus oxyrinchus, from the Atlantic and Pacific Oceans.

We present complete mitogenome sequences of three shortfin mako sharks (Isurus oxyrinchus) sampled from the western Pacific, and eastern and western Atlantic oceans. Mitogenome sequence lengths ranged between 16,699 bp and 16,702 bp, and all three mitogenomes contained one non-coding control region, two rRNA genes, 22 tRNA genes, and 13 protein-coding genes. Comparative assessment of five mitogenomes from globally distributed shortfin makos (the current three and two previously published mitogenomes) yielded 98.4% identity, with the protein-coding genes ATP8, ATP6, and ND5 as the most variable regions (sequence identities of 96.4%, 96.5%, and 97.6%, respectively). These mitogenome sequences contribute resources for assessing the genetic population dynamics of this endangered oceanic apex predator.

Periodically Disturbing the Spatial Structure of Biofilms Can Affect the Production of an Essential Virulence Factor in Pseudomonas aeruginosa.

Understanding the environmental factors that affect the production of virulence factors has major implications in evolution and medicine. While spatial structure is important in virulence factor production, observations of this relationship have occurred in undisturbed or continuously disturbed environments. However, natural environments are subject to periodic fluctuations, including changes in physical forces, which could alter the spatial structure of bacterial populations and impact virulence factor production. Using Pseudomonas aeruginosa PA14, we periodically applied a physical force to biofilms and examined production of pyoverdine. Intermediate frequencies of disturbance reduced the amount of pyoverdine produced compared to undisturbed or frequently disturbed conditions. To explore the generality of this finding, we examined how an intermediate disturbance frequency affected pyoverdine production in 21 different strains of P. aeruginosa. Periodic disturbance increased, decreased, or did not change the amount of pyoverdine produced relative to undisturbed populations. Mathematical modeling predicts that interactions between pyoverdine synthesis rate and biofilm density determine the amount of pyoverdine synthesized. When the pyoverdine synthesis rates are high, depletion of the biofilm due to disturbance reduces the accumulation of pyoverdine. At intermediate synthesis rates, production of pyoverdine increases during disturbance as bacteria dispersed into the planktonic state enjoy increased growth and pyoverdine production rates. At low synthesis rates, disturbance does not alter the amount of pyoverdine produced since disturbance-driven access to nutrients does not augment pyoverdine synthesis. Our results suggest that environmental conditions shape robustness in the production of virulence factors and may lead to novel approaches to treat infections. IMPORTANCE Virulence factors are required to cause infections. Previous work has shown that the spatial organization of a population, such as a biofilm, can increase the production of some virulence factors, including pyoverdine, which is produced by Pseudomonas aeruginosa. Pyoverdine is essential for the infection process, and reducing its production can limit infections. We have discovered that periodically changing the spatial structure of a biofilm of P. aeruginosa strain PA14 using a physical force can reduce the production of pyoverdine. A mathematical model suggests that this is due to the disruption of spatial organization. Using additional strains of P. aeruginosa isolated from patients and the environment, we use experiments and modeling to show that this reduction in pyoverdine is due to interactions between biofilm density and the synthesis rate of pyoverdine. Our results identify conditions where pyoverdine production is reduced and may lead to novel ways to treat infections.

Elevated pH Conditions Associated With Microcystis spp. Blooms Decrease Viability of the Cultured Diatom Fragilaria crotonensis and Natural Diatoms in Lake Erie.

Cyanobacterial Harmful Algal Blooms (CyanoHABs) commonly increase water column pH to alkaline levels ≥9.2, and to as high as 11. This elevated pH has been suggested to confer a competitive advantage to cyanobacteria such as Microcystis aeruginosa. Yet, there is limited information regarding the restrictive effects bloom-induced pH levels may impose on this cyanobacterium's competitors. Due to the pH-dependency of biosilicification processes, diatoms (which seasonally both precede and proceed Microcystis blooms in many fresh waters) may be unable to synthesize frustules at these pH levels. We assessed the effects of pH on the ecologically relevant diatom Fragilaria crotonensis in vitro, and on a Lake Erie diatom community in situ. In vitro assays revealed F. crotonensis monocultures exhibited lower growth rates and abundances when cultivated at a starting pH of 9.2 in comparison to pH 7.7. The suppressed growth trends in F. crotonensis were exacerbated when co-cultured with M. aeruginosa at pH conditions and cell densities that simulated a cyanobacteria bloom. Estimates demonstrated a significant decrease in silica (Si) deposition at alkaline pH in both in vitro F. crotonensis cultures and in situ Lake Erie diatom assemblages, after as little as 48 h of alkaline pH-exposure. These observations indicate elevated pH negatively affected growth rate and diatom silica deposition; in total providing a competitive disadvantage for diatoms. Our observations demonstrate pH likely plays a significant role in bloom succession, creating a potential to prolong summer Microcystis blooms and constrain diatom fall resurgence.

Complete Genome Sequence of Microcystis aeruginosa FD4, Isolated from a Subtropical River in Southwest Florida.

We report the first complete genome of Microcystis aeruginosa from North America. A harmful bloom that occurred in the Caloosahatchee River in 2018 led to a state of emergency declaration in Florida. Although strain FD4 was isolated from this toxic bloom, the genome did not have a microcystin biosynthetic gene cluster.

Nitrogen flux into metabolites and microcystins changes in response to different nitrogen sources in Microcystis aeruginosa NIES-843.

The over-enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non-N2 -fixing Microcystis spp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable-labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins in Microcystis aeruginosa NIES-843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing 15 N-labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N-rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However, 15 N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N-speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.

The "Neglected Viruses" of Taihu: Abundant Transcripts for Viruses Infecting Eukaryotes and Their Potential Role in Phytoplankton Succession.

Drivers of algal bloom dynamics remain poorly understood, but viruses have been implicated as important players. Research addressing bloom dynamics has generally been restricted to the virus-infection of the numerically dominant (i.e. bloom forming) taxa. Yet this approach neglects a broad diversity of viral groups, limiting our knowledge of viral interactions and constraints within these systems. We examined hallmark virus marker genes in metatranscriptomic libraries from a seasonal and spatial survey of a Microcystis aeruginosa bloom in Lake Tai (Taihu) China to identify active infections by nucleocytoplasmic large DNA viruses (NCLDVs), RNA viruses, ssDNA viruses, bacteriophage, and virophage. Phylogenetic analyses revealed a diverse virus population with seasonal and spatial variability. We observed disproportionately high expression of markers associated with NCLDVs and ssRNA viruses (consistent with viruses that infect photosynthetic protists) relative to bacteriophage infecting heterotrophic bacteria or cyanobacteria during the height of the Microcystis bloom event. Under a modified kill-the-winner scheme, we hypothesize viruses infecting protists help suppress the photosynthetic eukaryotic community and allow for the proliferation of cyanobacteria such as Microcystis. Our observations provide a foundation for a little considered factor promoting algal blooms.

Metatranscriptomic Analyses of Diel Metabolic Functions During a Microcystis Bloom in Western Lake Erie (United States).

This study examined diel shifts in metabolic functions of Microcystis spp. during a 48-h Lagrangian survey of a toxin-producing cyanobacterial bloom in western Lake Erie in the aftermath of the 2014 Toledo Water Crisis. Transcripts mapped to the genomes of recently sequenced lower Great Lakes Microcystis isolates showed distinct patterns of gene expression between samples collected across day (10:00 h, 16:00 h) and night (22:00 h, 04:00 h). Daytime transcripts were enriched in functions related to Photosystem II (e.g., psbA), nitrogen and phosphate acquisition, cell division (ftsHZ), heat shock response (dnaK, groEL), and uptake of inorganic carbon (rbc, bicA). Genes transcribed during nighttime included those involved in phycobilisome protein synthesis and Photosystem I core subunits. Hierarchical clustering and principal component analysis (PCA) showed a tightly clustered group of nighttime expressed genes, whereas daytime transcripts were separated from each other over the 48-h duration. Lack of uniform clustering within the daytime transcripts suggested that the partitioning of gene expression in Microcystis is dependent on both circadian regulation and physicochemical changes within the environment.

Urea Is Both a Carbon and Nitrogen Source for Microcystis aeruginosa: Tracking 13C Incorporation at Bloom pH Conditions.

The use of urea as a nitrogenous fertilizer has increased over the past two decades, with urea itself being readily detected at high concentrations in many lakes. Urea has been linked to cyanobacterial blooms as it is a readily assimilated nitrogen (N) - source for cyanobacteria that possess the enzyme urease. We tested the hypothesis that urea may also act as a carbon (C) source to supplemental growth requirements during the alkaline conditions created by dense cyanobacterial blooms, when concentrations of dissolved CO2 are vanishingly low. High rates of photosynthesis markedly reduce dissolved CO2 concentrations and drive up pH. This was observed in Lake Erie during the largest bloom on record (2015) over long periods (months) and short periods (days) of time, suggesting blooms experience periods of CO2-limitation on a seasonal and daily basis. We used 13C-urea to demonstrate that axenic cultures of the model toxic cyanobacterium, Microcystis aeruginosa NIES843, assimilated C at varying environmentally relevant pH conditions directly into a spectrum of metabolic pools during urea hydrolysis. Primarily, 13C from urea was assimilated into central C metabolism and amino acid biosynthesis pathways, including those important for the production of the hepatotoxin, microcystin, and incorporation into these pathways was at a higher percentage during growth at higher pH. This corresponded to increased growth rates on urea as the sole N source with increasing pH. We propose this ability to incorporate C from urea represents yet another competitive advantage for this cyanobacterium during dense algal blooms.

Insight Into the Molecular Mechanisms for Microcystin Biodegradation in Lake Erie and Lake Taihu.

Microcystins are potent hepatotoxins that are frequently detected in fresh water lakes plagued by toxic cyanobacteria. Microbial biodegradation has been referred to as the most important avenue for removal of microcystin from aquatic environments. The biochemical pathway most commonly associated with the degradation of microcystin is encoded by the mlrABCD (mlr) cassette. The ecological significance of this pathway remains unclear as no studies have examined the expression of these genes in natural environments. Six metatranscriptomes were generated from microcystin-producing Microcystis blooms and analyzed to assess the activity of this pathway in environmental samples. Seventy-eight samples were collected from Lake Erie, United States/Canada and Lake Tai (Taihu), China, and screened for the presence of mlr gene transcripts. Read mapping to the mlr cassette indicated transcripts for these genes were absent, with only 77 of the collective 3.7 billion reads mapping to any part of the mlr cassette. Analysis of the assembled metatranscriptomes supported this, with only distantly related sequences identified as mlrABC-like. These observations were made despite the presence of microcystin and over 500,000 reads mapping to the mcy cassette for microcystin production. Glutathione S-transferases and alkaline proteases have been previously hypothesized to be alternative pathways for microcystin biodegradation, and expression of these genes was detected across space and time in both lakes. While the activity of these alternative pathways needs to be experimentally confirmed, they may be individually or collectively more important than mlr genes in the natural environment. Importantly, the lack of mlr expression could indicate microcystin biodegradation was not occurring in the analyzed samples. This study raises interesting questions about the ubiquity, specificity and locality of microcystin biodegradation, and highlights the need for the characterization of relevant mechanisms in natural communities to understand the fate of microcystin in the environment and risk to public health.

Seasonal Gene Expression and the Ecophysiological Implications of Toxic Microcystis aeruginosa Blooms in Lake Taihu.

Harmful cyanobacterial blooms represent an increasing threat to freshwater resources globally. Despite increased research, the physiological basis of how the dominant bloom-forming cyanobacteria, Microcystis spp., proliferate and then maintain high population densities through changing environmental conditions is poorly understood. In this study, we examined the transcriptional profiles of the microbial community in Lake Taihu, China at 9 stations sampled monthly from June to October in 2014. To target Microcystis populations, we collected metatranscriptomic data and mapped reads to the M. aeruginosa NIES 843 genome. Our results revealed significant temporal gene expression patterns, with many genes separating into either early or late bloom clusters. About one-third of genes observed from M. aeruginosa were differentially expressed between these two clusters. Conductivity and nutrient availability appeared to be the environmental factors most strongly associated with these temporal gene expression shifts. Compared with the early bloom season (June and July), genes involved in N and P transport, energy metabolism, translation, and amino acid biosynthesis were down-regulated during the later season (August to October). In parallel, genes involved in regulatory functions as well as transposases and the production of microcystin and extracellular polysaccharides were up-regulated in the later season. Our observation indicates an eco-physiological shift occurs within the Microcystis spp. transcriptome as cells move from the rapid growth of early summer to bloom maintenance in late summer and autumn.

Molecular prediction of lytic vs lysogenic states for Microcystis phage: Metatranscriptomic evidence of lysogeny during large bloom events.

Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium capable of producing the potent hepatotoxin, microcystin. Despite increased interest in this organism, little is known about the viruses that infect it and drive nutrient mobilization and transfer of genetic material between organisms. The genomic complement of sequenced phage suggests these viruses are capable of integrating into the host genome, though this activity has not been observed in the laboratory. While analyzing RNA-sequence data obtained from Microcystis blooms in Lake Tai (Taihu, China), we observed that a series of lysogeny-associated genes were highly expressed when genes involved in lytic infection were down-regulated. This pattern was consistent, though not always statistically significant, across multiple spatial and temporally distinct samples. For example, samples from Lake Tai (2014) showed a predominance of lytic virus activity from late July through October, while genes associated with lysogeny were strongly expressed in the early months (June-July) and toward the end of bloom season (October). Analyses of whole phage genome expression shows that transcription patterns are shared across sampling locations and that genes consistently clustered by co-expression into lytic and lysogenic groups. Expression of lytic-cycle associated genes was positively correlated to total dissolved nitrogen, ammonium concentration, and salinity. Lysogeny-associated gene expression was positively correlated with pH and total dissolved phosphorous. Our results suggest that lysogeny may be prevalent in Microcystis blooms and support the hypothesis that environmental conditions drive switching between temperate and lytic life cycles during bloom proliferation.

Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OH.

Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for >2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of reoccurring Lake Erie blooms and thus may reoccur in the future.

Spatial and temporal variability in the nitrogen cyclers of hypereutrophic Lake Taihu.

Harmful cyanobacterial blooms (cyanoHABs) are a major threat to freshwater ecosystems worldwide. Evidence suggests that both nitrogen and phosphorus are important nutrients in the development and proliferation of blooms, yet much less is known about nitrogen cycling dynamics in these systems. To assess the potential nitrogen cycling function of the cyanoHAB community, surface water samples were collected in Lake Tai (Taihu), China over a 5-month bloom event in 2014. The expression of six nitrogen cycling genes (nifH, hzsA, nxrB, nrfA, amoA, nosZ) was surveyed using a targeted microarray with probes designed to provide phylogenetic information. N-Cycling gene expression varied spatially across Taihu, most notably near the mouth of the Dapu River. Expression of nifH was observed across the lake and attributable to both Proteobacteria and Cyanobacteria: Proteobacteria were major contributors to nifH signal near shore. Other N transformations such as anaerobic ammonia oxidation and denitrification were evident in the surface waters as well. Observations in this study highlight the potential importance of heterotrophic bacteria in N-cycling associated with cyanoHABs.