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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.

Prochlorococcus is a diverse picocyanobacterial genus and the most abundant phototroph on Earth. Its photosynthetic diversity divides it into high-light (HL)- or low-light (LL)-adapted groups representing broad phylogenetic grades-each composed of several monophyletic clades. Here, we physiologically characterize four new Prochlorococcus strains isolated from below the deep chlorophyll maximum in the North Pacific Ocean. We combine these physiological properties with genomic analyses to explore the evolution of photosynthetic antennae and discuss potential macroevolutionary implications. The isolates belong to deeply branching low-light-adapted clades that have no other cultivated representatives and display some unusual characteristics. For example, despite its otherwise low-light-adapted physiological characteristics, strain MIT1223 has low chl b 2 content similar to high-light-adapted strains. Isolate genomes revealed that each strain contains a unique arsenal of pigment biosynthesis and binding alleles that have been horizontally acquired, contributing to the observed physiological diversity. Comparative genomic analysis of all picocyanobacteria reveals that Pcb, the major pigment carrying protein in Prochlorococcus , greatly increased in copy number and diversity per genome along a branch that coincides with the loss of facultative particle attachment. Collectively, these observations support a recently developed macroevolutionary model, in which niche-constructing radiations allowed ancestral lineages of picocyanobacteria to transition from a particle-attached to planktonic lifestyle and broadly colonize the euphotic zone. IMPORTANCE The marine cyanobacterium, Prochlorococcus , is among the Earth's most abundant organisms, and much of its genetic and physiological diversity remains uncharacterized. Although field studies help reveal the scope of diversity, cultured isolates allow us to link genomic potential to physiological processes, illuminate eco-evolutionary feedbacks, and test theories arising from comparative genomics of wild cells. Here, we report the isolation and characterization of novel low-light (LL)-adapted Prochlorococcus strains that fill in multiple evolutionary gaps. These new strains are the first cultivated representatives of the LLVII and LLVIII paraphyletic grades of Prochlorococcus , which are broadly distributed in the lower regions of the ocean euphotic zone. Each of these grades is a unique, highly diverse section of the Prochlorococcus tree that separates distinct ecological groups: the LLVII grade branches between monophyletic clades that have facultatively particle-associated and constitutively planktonic lifestyles, whereas the LLVIII grade lies along the branch that leads to all high-light (HL)-adapted clades. Characterizing strains and genomes from these grades yields insights into the large-scale evolution of Prochlorococcus . The new LLVII and LLVIII strains are adapted to growth at very low irradiance levels and possess unique light-harvesting gene signatures and pigmentation. The LLVII strains represent the most basal Prochlorococcus group with a major expansion in photosynthetic antenna genes. Furthermore, a strain from the LLVIII grade challenges the paradigm that all LL-adapted Prochlorococcus exhibit high ratios of chl b:a 2 . These findings provide insights into the photophysiological evolution of Prochlorococcus and redefine what it means to be a low- vs high-light-adapted Prochlorococcus cell.

Microbial associations and interactions drive and regulate nutrient fluxes in the ocean. However, physical contact between cells of marine cyanobacteria has not been studied thus far. Here, we show a mechanism of direct interaction between the marine cyanobacteria Prochlorococcus and Synechococcus , the intercellular membrane nanotubes. We present evidence of inter- and intra-genus exchange of cytoplasmic material between neighboring and distant cells of cyanobacteria mediated by nanotubes. We visualized and measured these structures in xenic and axenic cultures and in natural samples. We show that nanotubes are produced between living cells, suggesting that this is a relevant system of exchange material in vivo. The discovery of nanotubes acting as exchange bridges in the most abundant photosynthetic organisms in the ocean may have important implications for their interactions with other organisms and their population dynamics.

Chitin is an abundant, carbon-rich polymer in the marine environment. Chitinase activity has been detected in spent media of Synechococcus WH7803 cultures-yet it was unclear which specific enzymes were involved. Here we delivered a CRISPR tool into the cells via electroporation to generate loss-of-function mutants of putative candidates and identified ChiA as the enzyme required for the activity detected in the wild type., (© 2023. The Author(s).)

Importance: Approximately half of the annual carbon fixation on Earth occurs in the surface ocean through the photosynthetic activities of phytoplankton such as the ubiquitous picocyanobacterium Prochlorococcus . Ecologically distinct subpopulations (or ecotypes) of Prochlorococcus are central conduits of organic substrates into the ocean microbiome, thus playing important roles in surface ocean production. We measured the chemical profile of three cultured ecotype strains, observing striking differences among them that have implications for the likely chemical impact of Prochlorococcus subpopulations on their surroundings in the wild. Subpopulations differ in abundance along gradients of temperature, light, and nutrient concentrations, suggesting that these chemical differences could affect carbon cycling in different ocean strata and should be considered in models of Prochlorococcus physiology and marine carbon dynamics., Competing Interests: The authors declare no conflict of interest.

Prochlorococcus is an abundant photosynthetic bacterium in the open ocean, where nitrogen (N) often limits phytoplankton growth. In the low-light-adapted LLI clade of Prochlorococcus , nearly all cells can assimilate nitrite (NO 2 - ), with a subset capable of assimilating nitrate (NO 3 - ). LLI cells are maximally abundant near the primary NO 2 - maximum layer, an oceanographic feature that may, in part, be due to incomplete assimilatory NO 3 - reduction and subsequent NO 2 - release by phytoplankton. We hypothesized that some Prochlorococcus exhibit incomplete assimilatory NO 3 - reduction and examined NO 2 - accumulation in cultures of three Prochlorococcus strains (MIT0915, MIT0917, and SB) and two Synechococcus strains (WH8102 and WH7803). Only MIT0917 and SB accumulated external NO 2 - during growth on NO 3 - . Approximately 20-30% of the NO 3 - transported into the cell by MIT0917 was released as NO 2 - , with the rest assimilated into biomass. We further observed that co-cultures using NO 3 - as the sole N source could be established for MIT0917 and Prochlorococcus strain MIT1214 that can assimilate NO 2 - but not NO 3 - . In these co-cultures, the NO 2 - released by MIT0917 is efficiently consumed by its partner strain, MIT1214. Our findings highlight the potential for emergent metabolic partnerships that are mediated by the production and consumption of N cycle intermediates within Prochlorococcus populations. IMPORTANCE Earth's biogeochemical cycles are substantially driven by microorganisms and their interactions. Given that N often limits marine photosynthesis, we investigated the potential for N cross-feeding within populations of Prochlorococcus , the numerically dominant photosynthetic cell in the subtropical open ocean. In laboratory cultures, some Prochlorococcus cells release extracellular NO 2 - during growth on NO 3 - . In the wild, Prochlorococcus populations are composed of multiple functional types, including those that cannot use NO 3 - but can still assimilate NO 2 - . We show that metabolic dependencies arise when Prochlorococcus strains with complementary NO 2 - production and consumption phenotypes are grown together on NO 3 - . These findings demonstrate the potential for emergent metabolic partnerships, possibly modulating ocean nutrient gradients, that are mediated by cross-feeding of N cycle intermediates., Competing Interests: The authors declare no conflict of interest.

Extracellular vesicles are small (approximately 50 to 250 nm in diameter), membrane-bound structures that are released by cells into their surrounding environment. Heterogeneous populations of vesicles are abundant in the global oceans, and they likely play a number of ecological roles in these microbially dominated ecosystems. Here, we examine how vesicle production and size vary among different strains of cultivated marine microbes as well as explore the degree to which this is influenced by key environmental variables. We show that both vesicle production rates and vesicle sizes significantly differ among cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Further, these properties vary within individual strains as a function of differences in environmental conditions, such as nutrients, temperature, and light irradiance. Thus, both community composition and the local abiotic environment are expected to modulate the production and standing stock of vesicles in the oceans. Examining samples from the oligotrophic North Pacific Gyre, we show depth-dependent changes in the abundance of vesicle-like particles in the upper water column in a manner that is broadly consistent with culture observations: the highest vesicle abundances are found near the surface, where the light irradiances and the temperatures are the greatest, and they then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans, which is essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems. IMPORTANCE Bacteria release extracellular vesicles that contain a wide variety of cellular compounds, including lipids, proteins, nucleic acids, and small molecules, into their surrounding environment. These structures are found in diverse microbial habitats, including the oceans, where their distributions vary throughout the water column and likely affect their functional impacts within microbial ecosystems. Using a quantitative analysis of marine microbial cultures, we show that bacterial vesicle production in the oceans is shaped by a combination of biotic and abiotic factors. Different marine taxa release vesicles at rates that vary across an order of magnitude, and vesicle production changes dynamically as a function of environmental conditions. These findings represent a step forward in our understanding of bacterial extracellular vesicle production dynamics and provide a basis for the quantitative exploration of the factors that shape vesicle dynamics in natural ecosystems., Competing Interests: The authors declare no conflict of interest.

Marine picocyanobacteria Prochlorococcus and Synechococcus , the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus . Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Objectives: The marine cyanobacterium Prochlorococcus is a critical part of warm ocean ecosystems and a model for studying microbial evolution and ecology. To expand the representation of this organism's vast wild diversity in sequence collections, we performed a set of isolation efforts targeting low light-adapted Prochlorococcus. Three genomes resulting from this larger body of work are described here., Data Description: We present draft-quality Prochlorococcus genomes from enrichment cultures P1344, P1361, and P1363, sampled in the North Pacific. The genomes were built from Illumina paired reads assembled de novo. Supporting datasets of raw reads, assessments, and sequences from co-enriched heterotrophic marine bacteria are also provided. These three genomes represent members of the low light-adapted LLIV Prochlorococcus clade that are closely related, with 99.9% average nucleotide identity between pairs, yet vary in gene content. Expanding the powerful toolkit of Prochlorococcus genomes, these sequences provide an opportunity to study fine-scale variation and microevolutionary processes., (© 2023. The Author(s).)

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans., Competing Interests: Declaration of interests J.B. is an employee of Oxford Nanopore Technologies and is a shareholder and/or share option holder., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Prochlorococcus and Synechococcus are the most abundant photosynthesizing organisms in the oceans. Gene content variation among picocyanobacterial populations in separate ocean basins often mirrors the selective pressures imposed by the region's distinct biogeochemistry. By pairing genomic datasets with trace metal concentrations from across the global ocean, we show that the genomic capacity for siderophore-mediated iron uptake is widespread in Synechococcus and low-light adapted Prochlorococcus populations from deep chlorophyll maximum layers of iron-depleted regions of the oligotrophic Pacific and S. Atlantic oceans: Prochlorococcus siderophore consumers were absent in the N. Atlantic ocean (higher new iron flux) but constituted up to half of all Prochlorococcus genomes from metagenomes in the N. Pacific (lower new iron flux). Picocyanobacterial siderophore consumers, like many other bacteria with this trait, also lack siderophore biosynthesis genes indicating that they scavenge exogenous siderophores from seawater. Statistical modeling suggests that the capacity for siderophore uptake is endemic to remote ocean regions where atmospheric iron fluxes are the smallest, especially at deep chlorophyll maximum and primary nitrite maximum layers. We argue that abundant siderophore consumers at these two common oceanographic features could be a symptom of wider community iron stress, consistent with prior hypotheses. Our results provide a clear example of iron as a selective force driving the evolution of marine picocyanobacteria., (© 2022. The Author(s).)

SignificancePhosphonates are a class of phosphorus metabolites characterized by a highly stable C-P bond. Phosphonates accumulate to high concentrations in seawater, fuel a large fraction of marine methane production, and serve as a source of phosphorus to microbes inhabiting nutrient-limited regions of the oligotrophic ocean. Here, we show that 15% of all bacterioplankton in the surface ocean have genes phosphonate synthesis and that most belong to the abundant groups Prochlorococcus and SAR11. Genomic and chemical evidence suggests that phosphonates are incorporated into cell-surface phosphonoglycoproteins that may act to mitigate cell mortality by grazing and viral lysis. These results underscore the large global biogeochemical impact of relatively rare but highly expressed traits in numerically abundant groups of marine bacteria.

Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus , are often accompanied by diverse heterotrophic "contaminating" bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as direct streak-plating, are often unsuccessful with this particular group of microorganisms. While they will grow in pour plates, colonies often remain contaminated with heterotrophic bacteria that can migrate through the soft agar. Additionally, axenic clones of picocyanobacteria can be recovered via dilution-to-extinction in liquid medium, but the efficiency of recovery is low, often requiring large numbers of 96-well plates. Here, we detail a simple and effective protocol for rendering cultures of Synechococcus and Prochlorococcus strains free of bacterial contaminants while at the same time yielding clonal isolates. We build on the fact that co-culture with specific heterotrophs-"helper heterotrophs"-is often necessary to grow colonies of picocyanobacteria from single cells in agar. Suspecting that direct physical contact between the helper and the picocyanobacterial cells was not necessary for the "helper effect," we developed a protocol in which the helper cells are embedded in soft agar pour plates, a filter overlaid on the surface, and a picocyanobacterial culture is diluted and then spotted on top of the filter. With this approach, motile contaminants cannot swim to the colonies, and it is possible to obtain the expected number of colonies from a given input (i.e., a Poisson distribution of colonies with an expected value equal to the input number of cells), thus ensuring clonal colonies. Using this protocol, we rendered three strains of Synechococcus , two strains of Prochlorococcus , and 19 new strains of Synechococcus from coastal seawater clonal and free of heterotrophic bacteria. The simplicity of this approach should expand the repertoire of axenic picocyanobacterial strains available for controlled physiological experiments. It will also enable the study of microdiversity in populations of picocyanobacteria by facilitating large-scale isolation of picocyanobacterial clones from a single source, including direct isolation from natural seawater., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kearney, Coe, Castro and Chisholm.)

Marine cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, spanning vast regions of the oceans and contributing significantly to global primary production. Their viruses (cyanophages) greatly influence cyanobacterial ecology and evolution. Although many cyanophage genomes have been sequenced, insight into the functional role of cyanophage genes is limited by the lack of a cyanophage genetic engineering system. Here, we describe a simple, generalizable method for genetic engineering of cyanophages from multiple families, that we named REEP for REcombination, Enrichment and PCR screening. This method enables direct investigation of key cyanophage genes, and its simplicity makes it adaptable to other ecologically relevant host-virus systems. T7-like cyanophages often carry integrase genes and attachment sites, yet exhibit lytic infection dynamics. Here, using REEP, we investigated their ability to integrate and maintain a lysogenic life cycle. We found that these cyanophages integrate into the host genome and that the integrase and attachment site are required for integration. However, stable lysogens did not form. The frequency of integration was found to be low in both lab cultures and the oceans. These findings suggest that T7-like cyanophage integration is transient and is not part of a classical lysogenic cycle., (© 2021. The Author(s).)

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the ocean's euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this region, depriving them of light for extended periods of time. Here, we demonstrate that members of these genera can adapt to tolerate repeated periods of light energy deprivation. Strains kept in the dark for 3 d and then returned to the light initially required 18-26 d to resume growth, but after multiple rounds of dark exposure they began to regrow after only 1-2 d. This dark-tolerant phenotype was stable and heritable; some cultures retained the trait for over 132 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is likely responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation-one potential mechanism mediating epigenetic inheritance in bacteria-occurs in Prochlorococcus . LC-MS/MS analysis showed that while DNA methylations, including 6 mA and 5 mC, are found in some other Prochlorococcus strains, there were no methylations detected in either the parental or dark-tolerant NATL2A strains. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation, and highlights phenotypic heterogeneity as an additional dimension of Prochlorococcus diversity., Competing Interests: None declared., (© 2021 The Authors. Limnology and Oceanography published by Wiley Periodicals LLC on behalf of Association for the Sciences of Limnology and Oceanography.)

Background: The cyanobacteria Prochlorococcus and Synechococcus are responsible for around 10% of global net primary productivity, serving as part of the foundation of marine food webs. Heterotrophic bacteria are often co-isolated with these picocyanobacteria in seawater enrichment cultures that contain no added organic carbon; heterotrophs grow on organic carbon supplied by the photolithoautotrophs. For examining the selective pressures shaping autotroph/heterotroph interactions, we have made use of unialgal enrichment cultures of Prochlorococcus and Synechococcus maintained for hundreds to thousands of generations in the lab. We examine the diversity of heterotrophs in 74 enrichment cultures of these picocyanobacteria obtained from diverse areas of the global oceans., Results: Heterotroph community composition differed between clades and ecotypes of the autotrophic 'hosts' but there was significant overlap in heterotroph community composition across these cultures. Collectively, the cultures were comprised of many shared taxa, even at the genus level. Yet, observed differences in community composition were associated with time since isolation, location, depth, and methods of isolation. The majority of heterotrophs in the cultures are rare in the global ocean, but enrichment conditions favor the opportunistic outgrowth of these rare bacteria. However, we found a few examples, such as bacteria in the family Rhodobacteraceae, of heterotrophs that were ubiquitous and abundant in cultures and in the global oceans. We found their abundance in the wild is also positively correlated with that of picocyanobacteria., Conclusions: Particular conditions surrounding isolation have a persistent effect on long-term culture composition, likely from bottlenecking and selection that happen during the early stages of enrichment for the picocyanobacteria. We highlight the potential for examining ecologically relevant relationships by identifying patterns of distribution of culture-enriched organisms in the global oceans.

Prochlorococcus cells are the numerically dominant phototrophs in the open ocean. Cyanophages that infect them are a notable fraction of the total viral population in the euphotic zone, and, as vehicles of horizontal gene transfer, appear to drive their evolution. Here we examine the propensity of three cyanophages-a podovirus, a siphovirus, and a myovirus-to mispackage host DNA in their capsids while infecting Prochlorococcus, the first step in phage-mediated horizontal gene transfer. We find the mispackaging frequencies are distinctly different among the three phages. Myoviruses mispackage host DNA at low and seemingly fixed frequencies, while podo- and siphoviruses vary in their mispackaging frequencies by orders of magnitude depending on growth light intensity. We link this difference to the concentration of intracellular reactive oxygen species and protein synthesis rates, both parameters increasing in response to higher light intensity. Based on our findings, we propose a model of mispackaging frequency determined by the imbalance between the production of capsids and the number of phage genome copies during infection: when protein synthesis rate increase to levels that the phage cannot regulate, they lead to an accumulation of empty capsids, in turn triggering more frequent host DNA mispackaging errors.

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms., Competing Interests: The authors declare that there are no conflicts of interest., (© 2020 The Authors.)

Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prochlorococcus and SAR11 are among the smallest and most abundant organisms on Earth. With a combined global population of about 2.7 × 10 28 cells, they numerically dominate bacterioplankton communities in oligotrophic ocean gyres and yet they have never been grown together in vitro. Here we describe co-cultures of Prochlorococcus and SAR11 isolates representing both high- and low-light adapted clades. We examined: (1) the influence of Prochlorococcus on the growth of SAR11 and vice-versa, (2) whether Prochlorococcus can meet specific nutrient requirements of SAR11, and (3) how co-culture dynamics vary when Prochlorococcus is grown with SAR11 compared with sympatric copiotrophic bacteria. SAR11 grew 15-70% faster in co-culture with Prochlorococcus, while the growth of the latter was unaffected. When Prochlorococcus populations entered stationary phase, this commensal relationship rapidly became amensal, as SAR11 abundances decreased dramatically. In parallel experiments with copiotrophic bacteria; however, the heterotrophic partner increased in abundance as Prochlorococcus densities leveled off. The presence of Prochlorococcus was able to meet SAR11's central requirement for organic carbon, but not reduced sulfur. Prochlorococcus strain MIT9313, but not MED4, could meet the unique glycine requirement of SAR11, which could be due to the production and release of glycine betaine by MIT9313, as supported by comparative genomic evidence. Our findings also suggest, but do not confirm, that Prochlorococcus MIT9313 may compete with SAR11 for the uptake of 3-dimethylsulfoniopropionate (DMSP). To give our results an ecological context, we assessed the relative contribution of Prochlorococcus and SAR11 genome equivalents to those of identifiable bacteria and archaea in over 800 marine metagenomes. At many locations, more than half of the identifiable genome equivalents in the euphotic zone belonged to Prochlorococcus and SAR11 - highlighting the biogeochemical potential of these two groups.

Sea spray is the largest aerosol source on Earth. Bubble bursting mechanisms at the ocean surface create smaller film burst and larger jet drop particles. This study quantified the effects of particle chemistry on the depositional ice nucleation efficiency of laboratory-generated sea spray aerosols under the cirrus-relevant conditions. Cultures of Prochlorococcus, the most abundant phytoplankton species in the global ocean, were used as a model source of organic sea spray aerosols. We showed that smaller particles generated from lysed Prochlorococcus cultures are organically enriched and nucleate more effectively than larger particles generated from the same cultures. We then quantified the ice nucleation efficiency of single component organic molecules that mimic Prochlorococcus proteins, lipids, and saccharides. Amylopectin, agarose, and aspartic acid exhibited similar critical ice saturations, fractional activations, and ice nucleation active site number densities to particles generated from Prochlorococcus cultures. These findings indicate that saccharides and proteins with numerous and well-ordered hydrophilic functional groups may determine the ice nucleation abilities of organic sea spray aerosols.

Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing intraspecific allocation of ecologically important functions. Nitrate assimilation genes are absent in basal lineages but occur at an intermediate frequency that is randomly distributed within recently emerged clades. The distribution of nitrate assimilation genes within clades appears largely governed by vertical inheritance, gene loss, and homologous recombination. By mapping this process onto a model of Prochlorococcus' macroevolution, we propose that niche-constructing adaptive radiations and subsequent niche partitioning set the stage for loss of nitrate assimilation genes from basal lineages as they specialized to lower light levels. Retention of these genes in recently emerged lineages has likely been facilitated by selection as they sequentially partitioned into niches where nitrate assimilation conferred a fitness benefit., Competing Interests: PB, AR, RB, RS, SC No competing interests declared, (© 2019, Berube et al.)

Prochlorococcus and Synechococcus are the dominant primary producers in marine ecosystems and perform a significant fraction of ocean carbon fixation. These cyanobacteria interact with a diverse microbial community that coexists with them. Comparative genomics of cultivated isolates has helped address questions regarding patterns of evolution and diversity among microbes, but the fraction that can be cultivated is miniscule compared to the diversity in the wild. To further probe the diversity of these groups and extend the utility of reference sequence databases, we report a data set of single cell genomes for 489 Prochlorococcus, 50 Synechococcus, 9 extracellular virus particles, and 190 additional microorganisms from a diverse range of bacterial, archaeal, and viral groups. Many of these uncultivated single cell genomes are derived from samples obtained on GEOTRACES cruises and at well-studied oceanographic stations, each with extensive suites of physical, chemical, and biological measurements. The genomic data reported here greatly increases the number of available Prochlorococcus genomes and will facilitate studies on evolutionary biology, microbial ecology, and biological oceanography.

Recent advances in understanding the ecology of marine systems have been greatly facilitated by the growing availability of metagenomic data, which provide information on the identity, diversity and functional potential of the microbial community in a particular place and time. Here we present a dataset comprising over 5 terabases of metagenomic data from 610 samples spanning diverse regions of the Atlantic and Pacific Oceans. One set of metagenomes, collected on GEOTRACES cruises, captures large geographic transects at multiple depths per station. The second set represents two years of time-series data, collected at roughly monthly intervals from 3 depths at two long-term ocean sampling sites, Station ALOHA and BATS. These metagenomes contain genomic information from a diverse range of bacteria, archaea, eukaryotes and viruses. The data's utility is strengthened by the availability of extensive physical, chemical, and biological measurements associated with each sample. We expect that these metagenomes will facilitate a wide range of comparative studies that seek to illuminate new aspects of marine microbial ecosystems.

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.

Prochlorococcus is a globally abundant marine cyanobacterium with many adaptations that reduce cellular nutrient requirements, facilitating growth in its nutrient-poor environment. One such genomic adaptation is the preferential utilization of amino acids containing fewer N-atoms, which minimizes cellular nitrogen requirements. We predicted that transcriptional regulation might further reduce cellular N budgets during transient N limitation. To explore this, we compared transcription start sites (TSSs) in Prochlorococcus MED4 under N-deprived and N-replete conditions. Of 64 genes with primary and internal TSSs in both conditions, N-deprived cells initiated transcription downstream of primary TSSs more frequently than N-replete cells. Additionally, 117 genes with only an internal TSS demonstrated increased internal transcription under N-deprivation. These shortened transcripts encode predicted proteins with an average of 21% less N content compared to full-length transcripts. We hypothesized that low translation rates, which afford greater control over protein abundances, would be beneficial to relatively slow-growing organisms like Prochlorococcus. Consistent with this idea, we found that Prochlorococcus exhibits greater usage of glycine-glycine motifs, which causes translational pausing, when compared to faster growing microbes. Our findings indicate that structural changes occur within the Prochlorococcus MED4 transcriptome during N-deprivation, potentially altering the size and structure of proteins expressed under nutrient limitation.

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.

Lanthipeptides are ribosomally derived peptide secondary metabolites that undergo extensive posttranslational modification. Prochlorosins are a group of lanthipeptides produced by certain strains of the ubiquitous marine picocyanobacteria Prochlorococcus and Synechococcus Unlike other lanthipeptide-producing bacteria, picocyanobacteria use an unprecedented mechanism of substrate promiscuity for the production of numerous and diverse lanthipeptides using a single lanthionine synthetase. Through a cross-scale analysis of prochlorosin biosynthesis genes-from genomes to oceanic populations-we show that marine picocyanobacteria have the collective capacity to encode thousands of different cyclic peptides, few of which would display similar ring topologies. To understand how this extensive structural diversity arises, we used deep sequencing of wild populations to reveal genetic variation patterns in prochlorosin genes. We present evidence that structural variability among prochlorosins is the result of a diversifying selection process that favors large, rather than small, sequence changes in the precursor peptide genes. This mode of molecular evolution disregards any conservation of the ancestral structure and enables the emergence of extensively different cyclic peptides through short mutational paths based on indels. Contrary to its fast-evolving peptide substrates, the prochlorosin lanthionine synthetase evolves under a strong purifying selection, indicating that the diversification of prochlorosins is not constrained by commensurate changes in the biosynthetic enzyme. This evolutionary interplay between the prochlorosin peptide substrates and the lanthionine synthetase suggests that structure diversification, rather than structure refinement, is the driving force behind the creation of new prochlorosin structures and represents an intriguing mechanism by which natural product diversity arises., Competing Interests: The authors declare no conflict of interest.

Move over plants-make way for tiny Prochlorococcus, the smallest and most abundant photosynthetic cell on earth! Penny Chisholm tells us all about this powerhouse marine bacterium., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Metabolism mediates the flow of matter and energy through the biosphere. We examined how metabolic evolution shapes ecosystems by reconstructing it in the globally abundant oceanic phytoplankter Prochlorococcus To understand what drove observed evolutionary patterns, we interpreted them in the context of its population dynamics, growth rate, and light adaptation, and the size and macromolecular and elemental composition of cells. This multilevel view suggests that, over the course of evolution, there was a steady increase in Prochlorococcus ' metabolic rate and excretion of organic carbon. We derived a mathematical framework that suggests these adaptations lower the minimal subsistence nutrient concentration of cells, which results in a drawdown of nutrients in oceanic surface waters. This, in turn, increases total ecosystem biomass and promotes the coevolution of all cells in the ecosystem. Additional reconstructions suggest that Prochlorococcus and the dominant cooccurring heterotrophic bacterium SAR11 form a coevolved mutualism that maximizes their collective metabolic rate by recycling organic carbon through complementary excretion and uptake pathways. Moreover, the metabolic codependencies of Prochlorococcus and SAR11 are highly similar to those of chloroplasts and mitochondria within plant cells. These observations lead us to propose a general theory relating metabolic evolution to the self-amplification and self-organization of the biosphere. We discuss the implications of this framework for the evolution of Earth's biogeochemical cycles and the rise of atmospheric oxygen.

Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events. Subtomogram classification and averaging revealed three main conformations characterized by different angles between the phage tail and the cell surface. Namely, phage tails were (i) parallel to, (ii) ~45 degrees to, or (iii) perpendicular to the cell surface. Furthermore, different conformations of phage tail fibers correlated with the aforementioned orientations of the tails. We also observed density beyond the tail tip in vertically-oriented phages that had penetrated the cell wall, capturing the final stage of adsorption. Together, our data provide a quantitative characterization of the orientation of phages as they adsorb onto cells, and suggest that cyanophages that abut their cellular targets are only transiently in the "perpendicular" orientation required for successful infection.

Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell's biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean.

Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (<0.01-1%) of vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time.

Microbial interactions, whether direct or indirect, profoundly affect the physiology of individual cells and ultimately have the potential to shape the biogeochemistry of the Earth. For example, the growth of Prochlorococcus, the numerically dominant cyanobacterium in the oceans, can be improved by the activity of co-occurring heterotrophs. This effect has been largely attributed to the role of heterotrophs in detoxifying reactive oxygen species that Prochlorococcus, which lacks catalase, cannot. Here, we explore this phenomenon further by examining how the entire transcriptome of Prochlorococcus NATL2A changes in the presence of a naturally co-occurring heterotroph, Alteromonas macleodii MIT1002, with which it was co-cultured for years, separated and then reunited. Significant changes in the Prochlorococcus transcriptome were evident within 6 h of initiating co-culture, with groups of transcripts changing in different temporal waves. Many transcriptional changes persisted throughout the 48 h experiment, suggesting that the presence of the heterotroph affected a stable shift in Prochlorococcus physiology. These initial transcriptome changes largely corresponded to reduced stress conditions for Prochlorococcus, as inferred from the depletion of transcripts encoding DNA repair enzymes and many members of the 'high light inducible' family of stress-response proteins. Later, notable changes were seen in transcripts encoding components of the photosynthetic apparatus (particularly, an increase in PSI subunits and chlorophyll synthesis enzymes), ribosomal proteins and biosynthetic enzymes, suggesting that the introduction of the heterotroph may have induced increased production of reduced carbon compounds for export. Changes in secretion-related proteins and transporters also highlight the potential for metabolic exchange between the two strains.

The assimilation of organic nutrients by autotrophs, a form of mixotrophy, has been demonstrated in the globally abundant marine picocyanobacterial genera Prochlorococcus and Synechococcus. However, the range of compounds used and the distribution of organic compound uptake genes within picocyanobacteria are unknown. Here we analyze genomic and metagenomic data from around the world to determine the extent and distribution of mixotrophy in these phototrophs. Analysis of 49 Prochlorococcus and 18 Synechococcus isolate genomes reveals that all have the transporters necessary to take up amino acids, peptides and sugars. However, the number and type of transporters and associated catabolic genes differ between different phylogenetic groups, with low-light IV Prochlorococcus, and 5.1B, 5.2 and 5.3 Synechococcus strains having the largest number. Metagenomic data from 68 stations from the Tara Oceans expedition indicate that the genetic potential for mixotrophy in picocyanobacteria is globally distributed and differs between clades. Phylogenetic analyses indicate gradual organic nutrient transporter gene loss from the low-light IV to the high-light II Prochlorococcus. The phylogenetic differences in genetic capacity for mixotrophy, combined with the ubiquity of picocyanobacterial organic compound uptake genes suggests that mixotrophy has a more central role in picocyanobacterial ecology than was previously thought.

Cyanophage infecting the marine cyanobacteria Prochlorococcus and Synechococcus require light and host photosystem activity for optimal reproduction. Many cyanophages encode multiple photosynthetic electron transport (PET) proteins, which are presumed to maintain electron flow and produce ATP and NADPH for nucleotide biosynthesis and phage genome replication. However, evidence suggests phage augment NADPH production via the pentose phosphate pathway (PPP), thus calling into question the need for NADPH production by PET. Genes implicated in cyclic PET have since been identified in cyanophage genomes. It remains an open question which mode of PET, cyclic or linear, predominates in infected cyanobacteria, and thus whether the balance is towards producing ATP or NADPH. We sequenced transcriptomes of a cyanophage (P-HM2) and its host (Prochlorococcus MED4) throughout infection in the light or in the dark, and analyzed these data in the context of phage replication and metabolite measurements. Infection was robust in the light, but phage were not produced in the dark. Host gene transcripts encoding high-light inducible proteins and two terminal oxidases (plastoquinol terminal oxidase and cytochrome c oxidase)-implicated in protecting the photosynthetic membrane from light stress-were the most enriched in light but not dark infection. Among the most diminished transcripts in both light and dark infection was ferredoxin-NADP+ reductase (FNR), which uses the electron acceptor NADP+ to generate NADPH in linear photosynthesis. The phage gene for CP12, which putatively inhibits the Calvin cycle enzyme that receives NADPH from FNR, was highly expressed in light infection. Therefore, both PET production of NADPH and its consumption by carbon fixation are putatively repressed during phage infection in light. Transcriptomic evidence is thus consistent with cyclic photophosphorylation using oxygen as the terminal electron acceptor as the dominant mode of PET under infection, with ATP from PET and NADPH from the PPP producing the energy and reducing equivalents for phage nucleotide biosynthesis and replication., Competing Interests: The authors have declared that no competing interests exist.

Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2-540 pg alkanes per mL per day, which translates into a global ocean yield of ∼ 308-771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities.

Carbon fixation has a central role in determining cellular redox poise, increasingly understood to be a key parameter in cyanobacterial physiology. In the cyanobacterium Prochlorococcus-the most abundant phototroph in the oligotrophic oceans-the carbon-concentrating mechanism is reduced to the bare essentials. Given the ability of Prochlorococcus populations to grow under a wide range of oxygen concentrations in the ocean, we wondered how carbon and oxygen physiology intersect in this minimal phototroph. Thus, we examined how CO2:O2 gas balance influenced growth and chlorophyll fluorescence in Prochlorococcus strain MED4. Under O2 limitation, per-cell chlorophyll fluorescence fell at all CO2 levels, but still permitted substantial growth at moderate and high CO2. Under CO2 limitation, we observed little growth at any O2 level, although per-cell chlorophyll fluorescence fell less sharply when O2 was available. We explored this pattern further by monitoring genome-wide transcription in cells shocked with acute limitation of CO2, O2 or both. O2 limitation produced much smaller transcriptional changes than the broad suppression seen under CO2 limitation and CO2/O2 co-limitation. Strikingly, both CO2 limitation conditions initially evoked a transcriptional response that resembled the pattern previously seen in high-light stress, but at later timepoints we observed O2-dependent recovery of photosynthesis-related transcripts. These results suggest that oxygen has a protective role in Prochlorococcus when carbon fixation is not a sufficient sink for light energy.

Alteromonas spp. are heterotrophic gammaproteobacteria commonly found in marine environments. We present here the draft genome sequence of Alteromonas macleodii MIT1002, which was isolated from an enrichment culture of the marine cyanobacterium Prochlorococcus NATL2A. This genome contains a mixture of features previously seen only within either the "surface" or "deep" Alteromonas ecotype., (Copyright © 2015 Biller et al.)

The marine cyanobacterium Prochlorococcus is the smallest and most abundant photosynthetic organism on Earth. In this Review, we summarize our understanding of the diversity of this remarkable phototroph and describe its role in ocean ecosystems. We discuss the importance of interactions of Prochlorococcus with the physical environment, with phages and with heterotrophs in shaping the ecology and evolution of this group. In light of recent studies, we have come to view Prochlorococcus as a 'federation' of diverse cells that sustains its broad distribution, stability and abundance in the oceans via extensive genomic and phenotypic diversity. Thus, it is proving to be a useful model system for elucidating the forces that shape microbial populations and ecosystems.

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.

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.

Production of dissolved organic matter (DOM) by marine phytoplankton supplies the majority of organic substrate consumed by heterotrophic bacterioplankton in the sea. This production and subsequent consumption converts a vast quantity of carbon, nitrogen, and phosphorus between organic and inorganic forms, directly impacting global cycles of these biologically important elements. Details regarding the chemical composition of DOM produced by marine phytoplankton are sparse, and while often assumed, it is not currently known if phylogenetically distinct groups of marine phytoplankton release characteristic suites of DOM. To investigate the relationship between specific phytoplankton groups and the DOM they release, hydrophobic phytoplankton-derived dissolved organic matter (DOMP) from eight axenic strains was analyzed using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Identification of DOM features derived from Prochlorococcus, Synechococcus, Thalassiosira, and Phaeodactylum revealed DOMP to be complex and highly strain dependent. Connections between DOMP features and the phylogenetic relatedness of these strains were identified on multiple levels of phylogenetic distance, suggesting that marine phytoplankton produce DOM that in part reflects its phylogenetic origin. Chemical information regarding the size and polarity ranges of features from defined biological sources was also obtained. Our findings reveal DOMP composition to be partially conserved among related phytoplankton species, and implicate marine DOM as a potential factor influencing microbial diversity in the sea by acting as a link between autotrophic and heterotrophic microbial community structures.