Selection and characterization of cyanophage resistance in marine Synechococcus strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Lake Superior supports novel clusters of cyanobacterial picoplankton

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the "picocyanobacterial clade" consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10(5) cells ml-1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.     

Development of phoH as a novel signature gene for assessing marine phage diversity

Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment.     

Prevalence of highly host-specific cyanophages in the estuarine environment

Cyanophages that infect coastal and oceanic Synechococcus have been studied extensively. However, no cyanophages infecting estuarine Synechococcus have been reported. In this study, seven cyanophages (three podoviruses, three siphoviruses and one myovirus) isolated from four estuarine Synechococcus strains were characterized in terms of their morphology, host range, growth and genetic features. All the podoviruses and siphoviruses were highly host specific. For the first time, the photosynthesis gene ( psbA ) was found in two podoviruses infecting estuarine Synechococcus . However, the psbA gene was not detected in the three siphoviruses. The psbA sequences from the two Synechococcus podoviruses clustered with some environmental psbA sequences, forming a unique cluster distantly related to previous known psbA clusters. Our results suggest that the psbA among Synechococcus podoviruses may evolve independently from the psbA of Synechococcus myoviruses. All three estuarine Synechococcus podoviruses contained the DNA polymerase ( pol ) gene, and clustered with other podoviruses that infect oceanic Synechococcus and Prochlorococcus , suggesting that the DNA pol is conserved among marine picocyanobacterial podoviruses. Prevalence of host-specific cyanophages in the estuary suggests that Synechococcus and their phages in the estuarine ecosystem may develop a host–phage relationship different from what have been found in the open ocean.     

The capsid of the T4 phage superfamily: the evolution, diversity, and structure of some of the most prevalent proteins in the biosphere

The Escherichia coli bacteriophage T4 has served as a classic system in phage biology for more than 60 years. Only recently have phylogenetic analyses and genomic comparisons demonstrated the existence of a large, diverse, and widespread superfamily of T4-like phages in the environment. We report here on the T4-like major capsid protein (MCP) sequences that were obtained by targeted polymerase chain reaction (PCR) of marine environmental samples. This analysis was then expanded to include 1,000 s of new sequences of T4-like capsid genes from the metagenomic data obtained during the Sorcerer II Global Ocean Sampling (GOS) expedition. This data compilation reveals that the diversity of the major and minor capsid proteins from the GOS metagenome follows the same general patterns as the sequences from cultured phage genomes. Interestingly, the new MCP sequences obtained by PCR targeted to MCP sequences in environmental samples are more divergent (deeper branching) than the vast majority of the MCP sequences coming from the other sources. The marine T4-like phage population appears to be largely dominated by the T4-like cyanophages. Using approximately 1,400 T4-like MCP sequences from various sources, we mapped the degree of sequence conservation on a structural model of the T4-like MCP. The results indicate that within the T4 superfamily there are some clear phylogenetic groups with regard to the more conserved and more variable domains of the MCP. Such differences can be correlated with variations in capsid morphology, the arrangement of the MCP lattice, and the presence of different capsid accessory proteins between the subgroups of the T4 superfamily.     

Multi-locus sequence analysis, taxonomic resolution and biogeography of marine Synechococcus

Conserved markers such as the 16S rRNA gene do not provide sufficient molecular resolution to identify spatially structured populations of marine Synechococcus, or 'ecotypes' adapted to distinct ecological niches. Multi-locus sequence analysis targeting seven 'core' genes was employed to taxonomically resolve Synechococcus isolates and correlate previous phylogenetic analyses encompassing a range of markers. Despite the recognized importance of lateral gene transfer in shaping the genomes of marine cyanobacteria, multi-locus sequence analysis of more than 120 isolates reflects a clonal population structure of major lineages and subgroups. A single core genome locus, petB, encoding the cytochrome b(6) subunit of the cytochrome b(6) f complex, was selected to expand our understanding of the diversity and ecology of marine Synechococcus populations. Environmental petB sequences cloned from contrasting sites highlight numerous genetically and ecologically distinct clusters, some of which represent novel, environmentally abundant clades without cultured representatives. With a view to scaling ecological analyses, the short sequence, taxonomic resolution and accurate automated alignment of petB is ideally suited to high-throughput and high-resolution sequencing projects to explore links between the ecology, evolution and biology of marine Synechococcus.     

Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria.

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.     

Phages of the marine cyanobacterial picophytoplankton

Cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the picophytoplankton in the oceans. It is still less than 10 years since the discovery of phages that infect marine Synechococcus and the beginning of the characterisation of these phages and assessment of their ecological impact. Estimations of the contribution of phages to Synechococcus mortality are highly variable, but there is clear evidence that phages exert a significant selection pressure on Synechococcus community structure. In turn, there are strong selection pressures on the phage community, in terms of both abundance and composition. This review focuses on the factors affecting the diversity of cyanophages in the marine environment, cyanophage interactions with their hosts, and the selective pressures in the marine environment that affect cyanophage evolutionary biology.     

A freshwater cyanophage whose genome indicates close relationships to photosynthetic marine cyanomyophages

Bacteriophage S-CRM01 has been isolated from a freshwater strain of Synechococcus and shown to be present in the upper Klamath River valley in northern California and Oregon. The genome of this lytic T4-like phage has a 178,563 bp circular genetic map with 297 predicted protein-coding genes and 33 tRNA genes that represent all 20-amino-acid specificities. Analyses based on gene sequence and gene content indicate a close phylogenetic relationship to the 'photosynthetic' marine cyanomyophages infecting Synechococcus and Prochlorococcus. Such relatedness suggests that freshwater and marine phages can draw on a common gene pool. The genome can be considered as being comprised of three regions. Region 1 is populated predominantly with structural genes, recognized as such by homology to other T4-like phages and by identification in a proteomic analysis of purified virions. Region 2 contains most of the genes with roles in replication, recombination, nucleotide metabolism and regulation of gene expression, as well as 5 of the 6 signature genes of the photosynthetic cyanomyophages (hli03, hsp20, mazG, phoH and psbA; cobS is present in Region 3). Much of Regions 1 and 2 are syntenic with marine cyanomyophage genomes, except that a segment encompassing Region 2 is inverted. Region 3 contains a high proportion (85%) of genes that are unique to S-CRM01, as well as most of the tRNA genes. Regions 1 and 2 contain many predicted late promoters, with a combination of CTAAATA and ATAAATA core sequences. Two predicted genes that are unusual in phage genomes are homologues of cellular spoT and nusG.     

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Lysogeny in the oceans: Lessons from cultivated model systems and a reanalysis of its prevalence

In the oceans, viruses that infect bacteria (phages) influence a variety of microbially mediated processes that drive global biogeochemical cycles. The nature of their influence is dependent upon infection mode, be it lytic or lysogenic. Temperate phages are predicted to be prevalent in marine systems where they are expected to execute both types of infection modes. Understanding the range and outcomes of temperate phage–host interactions is fundamental for evaluating their ecological impact. Here, we (i) review phage-mediated rewiring of host metabolism, with a focus on marine systems, (ii) consider the range and nature of temperate phage–host interactions, and (iii) draw on studies of cultivated model systems to examine the consequences of lysogeny among several dominant marine bacterial lineages. We also readdress the prevalence of lysogeny among marine bacteria by probing a collection of 1239 publicly available bacterial genomes, representing cultured and uncultivated strains, for evidence of complete prophages. Our conservative analysis, anticipated to underestimate true prevalence, predicts 18% of the genomes examined contain at least one prophage, the majority (97%) were found within genomes of cultured isolates. These results highlight the need for cultivation of additional model systems to better capture the diversity of temperate phage–host interactions in the oceans.     

Global distribution of nearly identical phage-encoded DNA sequences

Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7-like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan-associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7-like phage-encoded DNA polymerase genes from this clade were >99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7-like phages may be smaller than previously hypothesized.     

Characterisation of Host Growth after Infection with a Broad-Range Freshwater Cyanopodophage

Freshwater cyanophages are poorly characterised in comparison to their marine counterparts, however, the level of genetic diversity that exists in freshwater cyanophage communities is likely to exceed that found in marine environments, due to the habitat heterogeneity within freshwater systems. Many cyanophages are specialists, infecting a single host species or strain; however, some are less fastidious and infect a number of different host genotypes within the same species or even hosts from different genera. Few instances of host growth characterisation after infection by broad host-range phages have been described. Here we provide an initial characterisation of interactions between a cyanophage isolated from a freshwater fishing lake in the south of England and its hosts. Designated ΦMHI42, the phage is able to infect isolates from two genera of freshwater cyanobacteria, Planktothrix and Microcystis. Transmission Electron Microscopy and Atomic Force Microscopy indicate that ΦMHI42 is a member of the Podoviridae, albeit with a larger than expected capsid. The kinetics of host growth after infection with ΦMHI42 differed across host genera, species and strains in a way that was not related to the growth rate of the uninfected host. To our knowledge, this is the first characterisation of the growth of cyanobacteria in the presence of a broad host-range freshwater cyanophage.     

Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory.

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.     

Ecology of prokaryotic viruses

The finding that total viral abundance is higher than total prokaryotic abundance and that a significant fraction of the prokaryotic community is infected with phages in aquatic systems has stimulated research on the ecology of prokaryotic viruses and their role in ecosystems. This review treats the ecology of prokaryotic viruses ('phages') in marine, freshwater and soil systems from a 'virus point of view'. The abundance of viruses varies strongly in different environments and is related to bacterial abundance or activity suggesting that the majority of the viruses found in the environment are typically phages. Data on phage diversity are sparse but indicate that phages are extremely diverse in natural systems. Lytic phages are predators of prokaryotes, whereas lysogenic and chronic infections represent a parasitic interaction. Some forms of lysogeny might be described best as mutualism. The little existing ecological data on phage populations indicate a large variety of environmental niches and survival strategies. The host cell is the main resource for phages and the resource quality, i.e., the metabolic state of the host cell, is a critical factor in all steps of the phage life cycle. Virus-induced mortality of prokaryotes varies strongly on a temporal and spatial scale and shows that phages can be important predators of bacterioplankton. This mortality and the release of cell lysis products into the environment can strongly influence microbial food web processes and biogeochemical cycles. Phages can also affect host diversity, e.g., by 'killing the winner' and keeping in check competitively dominant species or populations. Moreover, they mediate gene transfer between prokaryotes, but this remains largely unknown in the environment. Genomics or proteomics are providing us now with powerful tools in phage ecology, but final testing will have to be performed in the environment.     

Genome sequences of siphoviruses infecting marine Synechococcus unveil a diverse cyanophage group and extensive phage-host genetic exchanges

Investigating the interactions between marine cyanobacteria and their viruses (phages) is important towards understanding the dynamic of ocean's primary productivity. Genome sequencing of marine cyanophages has greatly advanced our understanding about their ecology and evolution. Among 24 reported genomes of cyanophages that infect marine picocyanobacteria, 17 are from cyanomyoviruses and six from cyanopodoviruses, and only one from cyanosiphovirus (Prochlorococcus phage P-SS2). Here we present four complete genome sequences of siphoviruses (S-CBS1, S-CBS2, S-CBS3 and S-CBS4) that infect four different marine Synechococcus strains. Three distinct subtypes were recognized among the five known marine siphoviruses (including P-SS2) in terms of morphology, genome architecture, gene content and sequence similarity. Our study revealed that cyanosiphoviruses are genetically diverse with polyphyletic origin. No core genes were found across these five cyanosiphovirus genomes, and this is in contrast to the fact that many core genes have been found in cyanomyovirus or cyanopodovirus genomes. Interestingly, genes encoding three structural proteins and a lysozyme of S-CBS1 and S-CBS3 showed homology to a prophage-like genetic element in two freshwater Synechococcus elongatus genomes. Re-annotation of the prophage-like genomic region suggests that S.?elongatus may contain an intact prophage. Cyanosiphovirus genes involved in DNA metabolism and replication share high sequence homology with those in cyanobacteria, and further phylogenetic analysis based on these genes suggests that ancient and selective genetic exchanges occurred, possibly due to past prophage integration. Metagenomic analysis based on the Global Ocean Sampling database showed that cyanosiphoviruses are present in relatively low abundance in the ocean surface water compared to cyanomyoviruses and cyanopodoviruses.     

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10⁵ cells ml⁻¹ and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.