Sara E. Roggensack

Single-cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus.

Cyanobacterial Diversity What does it mean to be a global species? The marine cyanobacterium Prochlorococcus is ubiquitous and, arguably, the most abundant and productive of all living organisms. Although to our eyes the seas look uniform, to a bacterium the oceans bulk is a plethora of microhabitats, and by large-scale single-cell genomic analysis of uncultured cells, Kashtan et al. (p. 416; see the Perspective by Bowler and Scanlan) reveal that Prochlorococcus has diversified to match. This “species” constitutes a mass of subpopulations—each with million-year ancestry—that vary seasonally in abundance. The subpopulations in turn have clades nested within that show covariation between sets of core alleles and variable gene content, indicating flexibility of responses to rapid environmental changes. Large sets of coexisting populations could be a general feature of other free-living bacterial species living in highly mixed habitats. Covariation between the core alleles and flexible gene content of a marine cyanobacterium underpins vast diversity. [Also see Perspective by Bowler and Scanlan] Extensive genomic diversity within coexisting members of a microbial species has been revealed through selected cultured isolates and metagenomic assemblies. Yet, the cell-by-cell genomic composition of wild uncultured populations of co-occurring cells is largely unknown. In this work, we applied large-scale single-cell genomics to study populations of the globally abundant marine cyanobacterium Prochlorococcus. We show that they are composed of hundreds of subpopulations with distinct “genomic backbones,” each backbone consisting of a different set of core gene alleles linked to a small distinctive set of flexible genes. These subpopulations are estimated to have diverged at least a few million years ago, suggesting ancient, stable niche partitioning. Such a large set of coexisting subpopulations may be a general feature of free-living bacterial species with huge populations in highly mixed habitats.

Bacterial Vesicles in Marine Ecosystems

Carbon Budding in the Ocean Bacterial vesicles are gaining increasing attention for their roles in pathogenesis, but the abundance of these structures and their ecological roles in nonpathogenic contexts have received little notice. Biller et al. (p. 183; see the Perspective by Scanlan) provide evidence that membrane vesicles ∼100 nm in diameter are released by marine cyanobacteria and are a major feature of marine ecosystems. Studies of cultures of Prochlorococcus—the most abundant photoautotroph in the oligotrophic oceans—show that vesicles are continually released by this cyanobacterium and are abundant in the marine environment. These vesicles have properties that change the way we think about genetic and biogeochemical exchange among plankton and the dissolved organic carbon pool in marine ecosystems. The abundant marine cyanobacterium Prochlorococcus sheds membrane vesicles that may contribute to carbon budgets. [Also see Perspective by Scanlan] Many heterotrophic bacteria are known to release extracellular vesicles, facilitating interactions between cells and their environment from a distance. Vesicle production has not been described in photoautotrophs, however, and the prevalence and characteristics of vesicles in natural ecosystems is unknown. Here, we report that cultures of Prochlorococcus, a numerically dominant marine cyanobacterium, continuously release lipid vesicles containing proteins, DNA, and RNA. We also show that vesicles carrying DNA from diverse bacteria are abundant in coastal and open-ocean seawater samples. Prochlorococcus vesicles can support the growth of heterotrophic bacterial cultures, which implicates these structures in marine carbon flux. The ability of vesicles to deliver diverse compounds in discrete packages adds another layer of complexity to the flow of information, energy, and biomolecules in marine microbial communities.

Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis

Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world’s oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome—that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome.

Genomes of marine cyanopodoviruses reveal multiple origins of diversity

The marine cyanobacteria Prochlorococcus and Synechococcus are highly abundant in the global oceans, as are the cyanophage with which they co-evolve. While genomic analyses have been relatively extensive for cyanomyoviruses, only three cyanopodoviruses isolated on marine cyanobacteria have been sequenced. Here we present nine new cyanopodovirus genomes, and analyse them in the context of the broader group. The genomes range from 42.2 to 47.7 kb, with G+C contents consistent with those of their hosts. They share 12 core genes, and the pan-genome is not close to being fully sampled. The genomes contain three variable island regions, with the most hypervariable genes concentrated at one end of the genome. Concatenated core-gene phylogeny clusters all but one of the phage into three distinct groups (MPP-A and two discrete clades within MPP-B). The outlier, P-RSP2, has the smallest genome and lacks RNA polymerase, a hallmark of the Autographivirinae subfamily. The phage in group MPP-B contain photosynthesis and carbon metabolism associated genes, while group MPP-A and the outlier P-RSP2 do not, suggesting different constraints on their lytic cycles. Four of the phage encode integrases and three have a host integration signature. Metagenomic analyses reveal that cyanopodoviruses may be more abundant in the oceans than previously thought.

Physiology and evolution of nitrate acquisition in Prochlorococcus.

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ∼17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.

Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus

The marine cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism in the oligotrophic oceans, and a model system in marine microbial ecology. Here we report 27 new whole genome sequences (2 complete and closed; 25 of draft quality) of cultured isolates, representing five major phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from diverse regions of the oceans, facilitating studies of the drivers of microbial diversity—both in the lab and in the field. To improve the utility of these genomes for comparative genomics, we also define pre-computed clusters of orthologous groups of proteins (COGs), indicating how genes are distributed among these and other publicly available Prochlorococcus genomes. These data represent a significant expansion of Prochlorococcus reference genomes that are useful for numerous applications in microbial ecology, evolution and oceanography.

Fundamental differences in diversity and genomic population structure between Atlantic and Pacific Prochlorococcus

The Atlantic and Pacific Oceans represent different biogeochemical regimes in which the abundant marine cyanobacterium Prochlorococcus thrives. We have shown that Prochlorococcus populations in the Atlantic are composed of hundreds of genomically, and likely ecologically, distinct coexisting subpopulations with distinct genomic backbones. Here we ask if differences in the ecology and selection pressures between the Atlantic and Pacific are reflected in the diversity and genomic composition of their indigenous Prochlorococcus populations. We applied large-scale single-cell genomics and compared the cell-by-cell genomic composition of wild populations of co-occurring cells from samples from Station ALOHA off Hawaii, and from Bermuda Atlantic Time Series Station off Bermuda. We reveal fundamental differences in diversity and genomic structure of populations between the sites. The Pacific populations are more diverse than those in the Atlantic, composed of significantly more coexisting subpopulations and lacking dominant subpopulations. Prochlorococcus from the two sites seem to be composed of mostly non-overlapping distinct sets of subpopulations with different genomic backbones—likely reflecting different sets of ocean-specific micro-niches. Furthermore, phylogenetically closely related strains carry ocean-associated nutrient acquisition genes likely reflecting differences in major selection pressures between the oceans. This differential selection, along with geographic separation, clearly has a significant role in shaping these populations.

Heterotroph Interactions Alter Prochlorococcus Transcriptome Dynamics during Extended Periods of Darkness

Prochlorococcus is the most abundant photosynthetic organism on the planet. These cells play a central role in the physiology of surrounding heterotrophs by supplying them with fixed organic carbon. It is becoming increasingly clear, however, that interactions with heterotrophs can affect autotrophs as well. Here we show that such interactions have a marked impact on the response of Prochlorococcus to the stress of extended periods of darkness, as reflected in transcriptional dynamics. These data suggest that diel transcriptional rhythms within Prochlorococcus, which are generally considered to be strictly under the control of light quantity, quality, and timing, can also be influenced by biotic interactions. Together, these findings provide new insights into the importance of microbial interactions on Prochlorococcus physiology and reveal conditions where heterotroph-derived compounds may support autotrophs—contrary to the canonical autotroph-to-heterotroph trophic paradigm. ABSTRACT Microbes evolve within complex ecological communities where biotic interactions impact both individual cells and the environment as a whole. Here we examine how cellular regulation in the marine cyanobacterium Prochlorococcus is influenced by a heterotrophic bacterium, Alteromonas macleodii, under different light conditions. We monitored the transcriptome of Prochlorococcus, grown either alone or in coculture, across a diel light:dark cycle and under the stress of extended darkness—a condition that cells would experience when mixed below the ocean’s euphotic zone. More Prochlorococcus transcripts exhibited 24-h periodic oscillations in coculture than in pure culture, both over the normal diel cycle and after the shift to extended darkness. This demonstrates that biotic interactions, and not just light, can affect timing mechanisms in Prochlorococcus, which lacks a self-sustaining circadian oscillator. The transcriptomes of replicate pure cultures of Prochlorococcus lost their synchrony within 5 h of extended darkness and reflected changes in stress responses and metabolic functions consistent with growth cessation. In contrast, when grown with Alteromonas, replicate Prochlorococcus transcriptomes tracked each other for at least 13 h in the dark and showed signs of continued biosynthetic and metabolic activity. The transcriptome patterns suggest that the heterotroph may be providing energy or essential biosynthetic substrates to Prochlorococcus in the form of organic compounds, sustaining this autotroph when it is deprived of solar energy. Our findings reveal conditions where mixotrophic metabolism may benefit marine cyanobacteria and highlight new impacts of community interactions on basic Prochlorococcus cellular processes. IMPORTANCE Prochlorococcus is the most abundant photosynthetic organism on the planet. These cells play a central role in the physiology of surrounding heterotrophs by supplying them with fixed organic carbon. It is becoming increasingly clear, however, that interactions with heterotrophs can affect autotrophs as well. Here we show that such interactions have a marked impact on the response of Prochlorococcus to the stress of extended periods of darkness, as reflected in transcriptional dynamics. These data suggest that diel transcriptional rhythms within Prochlorococcus, which are generally considered to be strictly under the control of light quantity, quality, and timing, can also be influenced by biotic interactions. Together, these findings provide new insights into the importance of microbial interactions on Prochlorococcus physiology and reveal conditions where heterotroph-derived compounds may support autotrophs—contrary to the canonical autotroph-to-heterotroph trophic paradigm.