Grazing impact of different heterotrophic nanoflagellates on eukaryotic (Ostreococcus tauri) and prokaryotic picoautotrophs (Prochlorococcus and Synechococcus)

Autotrophic picoplankton (<3 microm) composed of both prokaryotes and eukaryotes are the most abundant primary producers on Earth. In this study we examined the ingestion of the picoeukaryote Ostreococcus tauri by different marine heterotrophic nanoflagellates (HNF) with various morphologies, swimming and feeding behaviours. Cultures of specific bacterivorous nanoflagellates (Rhynchomonas nasuta, Jakoba libera, and a culture of Cafeteria sp./Monosiga sp.) and natural nanoflagellate populations were used as grazers. For comparison with Ostreococcus, we used similar-sized prokaryotes as prey, Prochlorococcus and Synechococcus. We observed large species-specific differences in terms of: use of picoautotrophs among nanoflagellates, time lag between prey addition and prey consumption (0-196 h), grazing rate (0-0.12 h(-1)), growth rate (0-0.3 h(-1)) and maximum abundance of HNF reached in experimental bottles (e.g. from 10(4) to 10(5) cells ml(-1), for a natural coastal population and a Cafeteria sp./Monosiga sp. culture feeding Ostreococcus respectively). Overall, this study shows that the nanoflagellate community composition is conclusive for picoautotrophic community structure and, vice versa, the picoautotrophic community structure favours or inhibits the growth of some nanoflagellate groups.     

Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.     

Differential grazing of two heterotrophic nanoflagellates on marine Synechococcus strains

Grazing of heterotrophic nanoflagellates on marine picophytoplankton presents a major mortality factor for this important group of primary producers. However, little is known of the selectivity of the grazing process, often merely being thought of as a general feature of cell size and motility. In this study, we tested grazing of two heterotrophic nanoflagellates, Paraphysomonas imperforata and Pteridomonas danica , on strains of marine Synechococcus . Both nanoflagellates proved to be selective in their grazing, with Paraphysomonas being able to grow on 5, and Pteridomonas on 11, of 37 Synechococcus strains tested. Additionally, a number of strains (11 for Paraphysomonas , 9 for Pteridomonas ) were shown to be ingested, but not digested (and thus did not support growth of the grazer). Both the range of prey strains that supported growth as well as those that were ingested but not digested was very similar for the two grazers, suggesting a common property of these prey strains that lent them susceptible to grazing. Subsequent experiments on selected Synechococcus strains showed a pronounced difference in grazing susceptibility between wild-type Synechococcus sp. WH7803 and a spontaneous phage-resistant mutant derivative, WH7803PHR, suggesting that cell surface properties of the Synechococcus prey are an important attribute influencing grazing vulnerability.     

Novel lineages of Prochlorococcus and Synechococcus in the global oceans

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.     

Potential photosynthesis gene recombination between Prochlorococcus and Synechococcus via viral intermediates

Genes (psbA and psbD) encoding for photosynthetically important proteins were recently found in a number of cultured cyanophage genomes. This phenomenon may be a beneficial trait to the viruses or their photosynthetic cyanobacterial hosts, or may represent an untapped pool of genes involved in the formation of the photosynthetic apparatus that are prone to lateral gene transfer. Here we show analyses of psbA genes from uncultured environmental viruses and prophage populations. We observe a statistically significant separation between viral genes and their potential Synechococcus hosts' genes, and statistical analyses under models of codon evolution indicate that the psbA genes of viruses are evolving under levels of purifying selection that are virtually indistinguishable from their hosts. Furthermore, our data also indicate the possible exchange and reshuffling of psbA genes between Synechococcus and Prochlorococcus via phage intermediates. Overall, these observations raise the possibility that marine viruses serve as a potential genetic pool in shaping the evolution of cyanobacterial photosynthesis.     

Co-culture with Synechococcus facilitates growth of Prochlorococcus under ocean acidification conditions

Anthropogenic CO₂ emissions are projected to lower the pH of the ocean 0.3 units by 2100. Previous studies suggested that Prochlorococcus and Synechococcus, the numerically dominant phytoplankton in the oceans, have different responses to elevated CO₂ that may result in a dramatic shift in their relative abundances in future oceans. Here we showed that the exponential growth rates of these two genera respond to future CO₂ conditions in a manner similar to other cyanobacteria, but Prochlorococcus strains had significantly lower realized growth rates under elevated CO₂ regimes due to poor survival after exposure to fresh culture media. Despite this, a Synechococcus strain was unable to outcompete a Prochlorococcus strain in co-culture at elevated CO₂. Under these conditions, Prochlorococcus' poor response to elevated CO₂ disappeared, and Prochlorococcus' relative fitness showed negative frequency dependence, with both competitors having significant fitness advantages when initially rare. These experiments suggested that the two strains should be able to coexist indefinitely in co-culture despite sharing nearly identical nutritional requirements. We speculate that negative frequency dependence exists due to reductive Black Queen evolution that has resulted in a passively mutualistic relationship analogous to that connecting Prochlorococcus with the ‘helper’ heterotrophic microbes in its environment.     

Factors responsible for the differences in cultural estimates and direct microscopical counts of populations of bacterivorous nanoflagellates

Bacterivorous nanoflagellates (microflagellates) have been routinely enumerated in marine and freshwater samples using either a Most Probable Number (MPN) culture method or by a direct microscopical counting method (DC). These two techniques typically yield highly disparate estimates of the density of nanoflagellates in natural samples. We compared these methods with seawater and marine snow (macroscopic detrital aggregate) samples collected from surface waters throughout the North Atlantic and in freshwater samples collected at three stations in Lake Ontario. Densities of nanoflagellates determined by the two methods differed by as much as four orders of magnitude; the MPN estimate rarely exceeded 10% of the microscopical count, and averaged 1% of this count. The MPN estimate constituted a higher percentage of the DC value in environments with high concentrations of nanoflagellates relative to environments with low concentrations of nanoflagellates. The ratio of the culture count to the microscopical count (MPNDC) increased along an environmental gradient from oligotrophy to eutrophy, and was positively correlated with the density of bacteria in the samples. In laboratory experiments with two species of bacterivorous nanoflagellates, the MPN count constituted a much greater percentage of the DC count during the exponential growth phase of the nanoflagellate than during the stationary growth phase. Differences in the estimates of nanoflagellate density obtained with these two techniques probably can be explained by the trophic mode of these protozoa, their growth stage, and the amenability of these species to laboratory culture.     

Codon usage patterns and adaptive evolution of marine unicellular cyanobacteria Synechococcus and Prochlorococcus

Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d_N/d_S values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence.     

Modelling the vertical distribution of Prochlorococcus and Synechococcus in the North Pacific Subtropical Ocean

A simple model was developed to examine the vertical distribution of Prochlorococcus and Synechococcus ecotypes in the water column, based on their adaptation to light intensity. Model simulations were compared with a 14-year time series of Prochlorococcus and Synechococcus cell abundances at Station ALOHA in the North Pacific Subtropical Gyre. Data were analysed to examine spatial and temporal patterns in abundances and their ranges of variability in the euphotic zone, the surface mixed layer and the layer in the euphotic zone but below the base of the mixed layer. Model simulations show that the apparent occupation of the whole euphotic zone by a genus can be the result of a co-occurrence of different ecotypes that segregate vertically. The segregation of ecotypes can result simply from differences in light response. A sensitivity analysis of the model, performed on the parameter alpha (initial slope of the light-response curve) and the DIN concentration in the upper water column, demonstrates that the model successfully reproduces the observed range of vertical distributions. Results support the idea that intermittent mixing events may have important ecological and geochemical impacts on the phytoplankton community at Station ALOHA.     

Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus

Cyanobacteriophage Syn9 is a large, contractile-tailed bacteriophage infecting the widespread, numerically dominant marine cyanobacteria of the genera Prochlorococcus and Synechococcus . Its 177 300 bp genome sequence encodes 226 putative proteins and six tRNAs. Experimental and computational analyses identified genes likely involved in virion formation, nucleotide synthesis, and DNA replication and repair. Syn9 shows significant mosaicism when compared with related cyanophages S-PM2, P-SSM2 and P-SSM4, although shared genes show strong purifying selection and evidence for large population sizes relative to other phages. Related to coliphage T4 – which shares 19% of Syn9's genes – Syn9 shows evidence for different patterns of DNA replication and uses homologous proteins to assemble capsids with a different overall structure that shares topology with phage SPO1 and herpes virus. Noteworthy bacteria-related sequences in the Syn9 genome potentially encode subunits of the photosynthetic reaction centre, electron transport proteins, three pentose pathway enzymes and two tryptophan halogenases. These genes suggest that Syn9 is well adapted to the physiology of its photosynthetic hosts and may affect the evolution of these sequences within marine cyanobacteria.     

Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Diversity of Synechococcus and Prochlorococcus populations determined from DNA sequences of the N-regulatory gene ntcA

The cyanobacteria Synechococcus and Prochlorococcus are abundant primary producers in the nitrogen-poor waters of the Gulf of Aqaba, northern Red Sea. Expression of the nitrogen regulatory gene ntcA is a useful indicator for determining the N-status of cyanobacteria, and preliminary work with this gene suggests that it may also serve as a useful biodiversity marker. Here we investigated the genotypic diversity of ntcA among the full spectrum of cultured Synechococcus and Prochlorococcus lineages and assessed cyanobacterial genotypic composition in environmental samples from the Gulf of Aqaba. The high level of ntcA diversification established this gene as an excellent biodiversity marker capable of distinguishing between numerous clades within each genus with high resolution. An unexpected large diversity was observed among Synechococcus populations, including the detection of four novel clades for which culture representatives have yet to be isolated. In addition, extensive microdiversity within a number of Synechococcus clades was revealed. Temporal differences in the detection of the various Synechococcus clades suggest seasonal fluctuations in the genotypic make-up of Synechococcus populations. In contrast, virtually all Prochlorococcus sequences fell within a single high-light adapted clade that was detected year round. We suggest that the limited genotypic diversity among Prochlorococcus in combination with a limited capacity for acclimation to environmental changes resulting from its small genome size led to the dramatic rise and demise of Prochlorococcus populations over the yearly cycle in the Gulf of Aqaba.     

Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes

Marine cyanobacteria of the genera Prochlorococcus and Synechococcus are important contributors to global primary production occupying a key position at the base of marine food webs. The genetically diverse nature of each genus is likely an important reason for their successful colonization of vast tracts of the world's oceans, a feature that has led to detailed analysis of the distribution of these genetic lineages at the local and ocean basin scale. Here, we extend these analyses to the global dimension, using new data from cruises in the Pacific, Indian and Arctic Oceans in combination with data from previous studies in the Atlantic Ocean, Arabian Sea, Red Sea and a circumnavigation of the southern hemisphere to form a data set which comprises most of the world's major ocean systems. We show that the distribution patterns of Prochlorococcus and Synechococcus lineages are remarkably similar in different ocean systems with comparable environmental conditions, but producing a strikingly different 'signature' in the four major ocean domains or biomes (the Polar Domain, Coastal Boundary Domain, Trade Winds Domain and Westerly Winds Domain). This clearly reiterates the idea of spatial partitioning of individual cyanobacterial lineages, but at the global scale.     

Bayesian Analysis of Congruence of Core Genes in Prochlorococcus and Synechococcus and Implications on Horizontal Gene Transfer

It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. “Universal proteins” have been used to infer the organismal phylogeny, but have been criticized as being only the “tree of one percent.” Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees – a “treecloud” – for each, as well as for a concatenated dataset based on putative “universal proteins.” We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS). We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the “tree of one percent” is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.     

Rapid Diversification of Marine Picophytoplankton with Dissimilar Light-Harvesting Structures Inferred from Sequences of Prochlorococcus and Synechococcus (Cyanobacteria)

Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions. Received: 18 January 1997 / Accepted: 18 May 1997     

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.