Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. Based on the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.
saxitoxin; copper transporter; FET3; trace metal uptake; Saccharomyces cerevisiae
Picocyanobacteria represented by Prochlorococcus and Synechococcus have an important role in oceanic carbon fixation and nutrient cycling. In this study, we compared the community composition of picocyanobacteria from diverse marine ecosystems ranging from estuary to open oceans, tropical to polar oceans and surface to deep water, based on the sequences of 16S-23S rRNA internal transcribed spacer (ITS). A total of 1339 ITS sequences recovered from 20 samples unveiled diverse and several previously unknown clades of Prochlorococcus and Synechococcus. Six high-light (HL)-adapted Prochlorococcus clades were identified, among which clade HLVI had not been described previously. Prochlorococcus clades HLIII, HLIV and HLV, detected in the Equatorial Pacific samples, could be related to the HNLC clades recently found in the high-nutrient, low-chlorophyll (HNLC), iron-depleted tropical oceans. At least four novel Synechococcus clades (out of six clades in total) in subcluster 5.3 were found in subtropical open oceans and the South China Sea. A niche partitioning with depth was observed in the Synechococcus subcluster 5.3. Members of Synechococcus subcluster 5.2 were dominant in the high-latitude waters (northern Bering Sea and Chukchi Sea), suggesting a possible cold-adaptation of some marine Synechococcus in this subcluster. A distinct shift of the picocyanobacterial community was observed from the Bering Sea to the Chukchi Sea, which reflected the change of water temperature. Our study demonstrates that oceanic systems contain a large pool of diverse picocyanobacteria, and further suggest that new genotypes or ecotypes of picocyanobacteria will continue to emerge, as microbial consortia are explored with advanced sequencing technology.
cyanobacteria; Prochlorococcus; Synechococcus; diversity; global ocean; 16S-23S rRNA ITS
Development and use of primer sets to amplify nucleic acid sequences of interest is fundamental to studies spanning many life science disciplines. As such, the validation of primer sets is essential. Several computer programs have been created to aid in the initial selection of primer sequences that may or may not require multiple nucleotide combinations (i.e., degeneracies). Conversely, validation of primer specificity has remained largely unchanged for several decades, and there are currently few available programs that allows for an evaluation of primers containing degenerate nucleotide bases. To alleviate this gap, we developed the program De-MetaST that performs an in silico amplification using user defined nucleotide sequence dataset(s) and primer sequences that may contain degenerate bases. The program returns an output file that contains the in silico amplicons. When De-MetaST is paired with NCBI’s BLAST (De-MetaST-BLAST), the program also returns the top 10 nr NCBI database hits for each recovered in silico amplicon. While the original motivation for development of this search tool was degenerate primer validation using the wealth of nucleotide sequences available in environmental metagenome and metatranscriptome databases, this search tool has potential utility in many data mining applications.
Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.
Viruses are the most abundant life forms on Earth, with an estimated 1031 total viruses globally. The majority of these viruses infect microbes, whether bacteria, archaea or microeukaryotes. Given the importance of microbes in driving global biogeochemical cycles, it would seem, based on numerical abundances alone, that viruses also play an important role in the global cycling of carbon and nutrients. However, the importance of viruses in controlling host populations and ecosystem functions, such as the regeneration, storage and export of carbon and other nutrients, remains unresolved. Here, we report on advances in the study of ecological effects of viruses of microbes. In doing so, we focus on an area of increasing importance: the role that ocean viruses play in shaping microbial population sizes as well as in regenerating carbon and other nutrients.
Toxic cyanobacterial blooms have persisted in freshwater systems around the world for centuries and appear to be globally increasing in frequency and severity. Toxins produced by bloom-associated cyanobacteria can have drastic impacts on the ecosystem and surrounding communities, and bloom biomass can disrupt aquatic food webs and act as a driver for hypoxia. Little is currently known regarding the genomic content of the Microcystis strains that form blooms or the companion heterotrophic community associated with bloom events. To address these issues, we examined the bloom-associated microbial communities in single samples from Lake Erie (North America), Lake Tai (Taihu, China), and Grand Lakes St. Marys (OH, USA) using comparative metagenomics. Together the Cyanobacteria and Proteobacteria comprised >90% of each bloom bacterial community sample, although the dominant phylum varied between systems. Relative to the existing Microcystis aeruginosa NIES 843 genome, sequences from Lake Erie and Taihu revealed a number of metagenomic islands that were absent in the environmental samples. Moreover, despite variation in the phylogenetic assignments of bloom-associated organisms, the functional potential of bloom members remained relatively constant between systems. This pattern was particularly noticeable in the genomic contribution of nitrogen assimilation genes. In Taihu, the genetic elements associated with the assimilation and metabolism of nitrogen were predominantly associated with Proteobacteria, while these functions in the North American lakes were primarily contributed to by the Cyanobacteria. Our observations build on an emerging body of metagenomic surveys describing the functional potential of microbial communities as more highly conserved than that of their phylogenetic makeup within natural systems.
Considerable research has shown that cyanobacteria and the viruses that infect them (cyanophage) are pervasive and diverse in global lake populations. Few studies have seasonally analyzed freshwater systems, and little is known about the bacterial and viral communities that coexist during the harsh winters of the Laurentian Great Lakes. Here, we employed quantitative PCR to estimate the abundance of cyanomyoviruses in this system, using the portal vertex g20 gene as a proxy for cyanophage abundance and to determine the potential ecological relevance of these viruses. Cyanomyoviruses were abundant in both the summer and the winter observations, with up to 3.1 × 106 copies of g20 genes ml−1 found at several stations and depths in both seasons, representing up to 4.6% of the total virus community. Lake Erie was productive during both our observations, with high chlorophyll a concentrations in the summer (up to 10.3 μg liter−1) and winter (up to 5.2 μg liter−1). Both bacterial and viral abundances were significantly higher during the summer than during the winter (P < 0.05). Summer bacterial abundances ranged from 3.3 × 106 to 1.6 × 107 ml−1 while winter abundances ranged between ∼3.4 × 105 and 1.2 × 106 ml−1. Total virus abundances were high during both months, with summer abundances significantly higher at most stations, ranging from 6.5 × 107 to 8.8 × 107 ml−1, and with winter abundances ranging from 3.4 × 107 to 6.6 × 107 ml−1. This work confirms that putative cyanomyoviruses are ubiquitous in both summer and winter months in this large freshwater lake system and that they are an abundant component of the virioplankton group.
Blooms of the potentially toxic cyanobacterium Microcystis are common events globally, and as a result significant resources continue to be dedicated to monitoring and controlling these events. Recent studies have shown that a significant proportion of total cell-associated phosphorus (P) in marine phytoplankton can be surface adsorbed; as a result studies completed to date do not accurately report the P demands of these organisms. In this study we measure the total cell-associated and intracellular P as well as growth rates of two toxic strains of Microcystis aeruginosa Kütz grown under a range of P concentrations. The results show that the intracellular P pool in Microcystis represents a percentage of total cell-associated P (50–90%) similar to what has been reported for actively growing algae in marine systems. Intracellular P concentrations (39–147 fg cell−1) generally increased with increasing P concentrations in the growth medium, but growth rate and the ratio of total cell-associated to intracellular P remained generally stable. Intracellular P quotas and growth rates in cells grown under the different P treatments illustrate the ability of this organism to successfully respond to changes in ambient P loads, and thus have implications for ecosystem scale productivity models employing P concentrations to predict algal bloom events.
cyanobacteria; phosphorus; Microcystis
Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported.
Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex® filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples.
The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.
While multiple phylogenetic markers have been used in the culture-independent study of microcystin-producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.
The diversity of microcystin-producing cyanobacteria in the western basin of Lake Erie was studied using sequence analysis of mcyA gene fragments. Distinct populations of potentially toxic Microcystis and Planktothrix were found in spatially isolated locations. This study highlights previously undocumented diversity of potentially toxic cyanobacteria.
While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolate Synechococcus sp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems.
In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.