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Marine microorganisms play a critical role in regulating atmospheric CO 2 concentration via the biological carbon pump. Deposition of continental mineral dust on the sea surface increases carbon sequestration but the interaction between minerals and marine microorganisms is not well understood. We discovered that the interaction of clay minerals with dissolved organic matter and a γ-proteobacterium in seawater increases Transparent Exopolymer Particle (TEP) concentration, leading to organoclay floc formation. To explore this observation further, we conducted a microcosm experiment using surface seawater collected from the Spring 2023 phytoplankton bloom in the Gulf of Maine. Unfiltered (natural community) and filtered (200 μm and 3 μm) seawater was sprayed with clay (20 mg L - 1 and 60 mg L - 1 ) and incubated. All clay treatments led to a tenfold increase in TEP concentration. 16S rRNA gene amplicon sequence analyses of seawater and settled organoclay flocs showed the dominance of α-proteobacteria, γ-proteobacteria, and Bacteroidota. The initial seawater phytoplankton community was dominated by dinoflagellates followed by a haptophyte (Phaeocystis sp.) and diatoms. Following clay addition, dinoflagellate cell abundance declined sharply while diatom cell abundance increased. By analyzing organoclay flocs for 18S rRNA we confirmed that dinoflagellates were removed in the flocs. The clay amendment removed as much as 50% of phytoplankton organic carbon. We then explored the fate of organoclay flocs at the next trophic level by feeding clay and phytoplankton (Rhodomonas salina) to Calanus finmarchicus. The copepod ingested R. salina and organoclay flocs and egested denser fecal pellets with 1.8- to 3.6- fold higher sinking velocity compared to controls. Fecal pellet density enhancement could facilitate carbon sequestration through zooplankton diel vertical migration. These findings provide insights into how atmospheric dust-derived clay minerals interact with marine microorganisms to enhance the biological carbon pump, facilitating the burial of organic carbon at depths where it is less likely to exchange with the atmosphere., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The fastest replicating bacterium Vibrio natriegens is a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential of V. natriegens and collectively expand upon its utility as an emerging model organism for synthetic biology., Importance: Vibrio natriegens is an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of the V. natriegens model system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing "clean" derivative cells. Finally, we show the full extent of this technique's capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to the Vibrio community and specifically to the advancement of V. natriegens as a model system., Competing Interests: The authors declare no conflict of interest.

The marine cyanobacterium Prochlorococcus numerically dominates the phytoplankton community of the nutrient-limited open ocean, establishing itself as the most abundant photosynthetic organism on Earth. This ecological success has been attributed to lower cell quotas for limiting nutrients, superior resource acquisition, and other advantages associated with cell size reduction and genome streamlining. In this study, we tested the prediction that Prochlorococcus outcompetes its rivals for scarce nutrients and that this advantage leads to its numerical success in nutrient-limited waters. Strains of Prochlorococcus and its sister genus Synechococcus grew well in both mono- and cocultures when nutrients were replete. However, in nitrogen-limited medium, Prochlorococcus outgrew Synechococcus but only when heterotrophic bacteria were also present. In the nitrogen-limited medium, the heterotroph Alteromonas macleodii outcompeted Synechococcus for nitrogen but only if stimulated by the exudate released by Prochlorococcus or if a proxy organic carbon source was provided. Genetic analysis of Alteromonas suggested that it outcompetes Synechococcus for nitrate and/or nitrite, during which cocultured Prochlorococcus grows on ammonia or other available nitrogen species. We propose that Prochlorococcus can stimulate antagonism between heterotrophic bacteria and potential phytoplankton competitors through a metabolic cross-feeding interaction, and this stimulation could contribute to the numerical success of Prochlorococcus in nutrient-limited regions of the ocean. IMPORTANCE In nutrient-poor habitats, competition for limited resources is thought to select for organisms with an enhanced ability to scavenge nutrients and utilize them efficiently. Such adaptations characterize the cyanobacterium Prochlorococcus , the most abundant photosynthetic organism in the nutrient-limited open ocean. In this study, the competitive superiority of Prochlorococcus over a rival cyanobacterium, Synechococcus , was captured in laboratory culture. Critically, this outcome was achieved only when key aspects of the open ocean were simulated: a limited supply of nitrogen and the presence of heterotrophic bacteria. The results indicate that Prochlorococcus promotes its numerical dominance over Synechococcus by energizing the heterotroph's ability to outcompete Synechococcus for available nitrogen. This study demonstrates how interactions between trophic groups can influence interactions within trophic groups and how these interactions likely contribute to the success of the most abundant photosynthetic microorganism.

The mechanisms driving cyanobacterial harmful algal blooms (HABs) like those caused by Microcystis aeruginosa remain elusive, but improved defense against viral predation has been implicated for success in eutrophic environments. Our genus-level analyses of 139,023 genomes revealed that HAB-forming cyanobacteria carry vastly more restriction modification systems per genome (RMPG) than nearly all other prokaryotic genera, suggesting that viral defense is a cornerstone of their ecological success. In contrast, picocyanobacteria that numerically dominate nutrient-poor systems have the fewest RMPG within the phylum Cyanobacteria . We used classic resource competition models to explore the hypothesis that nutrient enrichments drive ecological selection for high RMPG due to increased host-phage contact rate. These classic models, agnostic to the mechanism of defense, explain how nutrient loading can select for increased RMPG but, importantly, fail to explain the extreme accumulation of these defense systems. However, extreme accumulation of RMPG can be achieved in a novel "memory" model that accounts for a unique activity of restriction modification systems: the accidental methylation of viral DNA by the methyltransferase. The methylated virus "remembers" the RM defenses of its former host and can evade these defenses if they are present in the next host. This viral memory leads to continual RM system devaluation; RMs accumulate extensively because the benefit of each addition is diminished. Our modeling leads to the hypothesis that nutrient loading and virion methylation drive the extreme accumulation of RMPG in HAB-forming cyanobacteria. Finally, our models suggest that hosts with different RMPG values can coexist when hosts have unique sets of RM systems. IMPORTANCE Harmful algal blooms (HABs), caused by cyanobacteria like Microcystis aeruginosa, are a global threat to water quality and use across the planet. Researchers have agreed that nutrient loading is a major contributor to HAB persistence. While we may understand the environmental conditions that cause HABs, we still struggle in identifying the mechanisms that explain why these organisms have a competitive edge against other, less ecologically hazardous organisms. Our interdisciplinary approach in microbiology, mathematical population modeling, and genomics allows us to use nearly 70 years of research in restriction modification systems to show that HAB-forming cyanobacteria are exceptional in their ability to defend against viruses, and this capacity is intimately tied to nutrient loading. Our hypothesis suggests that defense against viral predation is a fundamental pillar of cyanobacterial ecological strategy and an important contributor to HAB dynamics.

Chloroviruses (family Phycodnaviridae ) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing ( aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. Cvi AII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives., (Copyright © 2020 Coy, Gann, Papoulis, Holder, Ajami, Petrosino, Zinser, Van Etten and Wilhelm.)

[This corrects the article DOI: 10.1128/mSystems.00254-18.].

Primary production by Prochlorococcus , the smallest known free-living photosynthetic organism in terms of both physical and genomic size, is thought to have a significant role in global carbon cycles. Despite its small size and low growth rate, Prochlorococcus numerically dominates the phytoplankton community in the nutrient-poor oligotrophic ocean, the largest biome of the Earth's surface. How nutrient limitation, and nitrogen limitation in particular, affects the fate and flux of carbon fixed by Prochlorococcus is currently unknown. To address this gap in knowledge, we compared the bulk rates of photosynthesis and organic carbon release, the concentrations of intracellular metabolites, and the rates of assimilated carbon into the metabolite pools between replete and N-limited chemostat cultures. Total photosynthesis of our N-limited cultures was less than half of those observed in replete cultures, and nitrogen limitation also appears to cause a larger proportion of total fixed carbon to be released to the environment. Our data suggest this occurs in concert with the maintenance of large slow-moving pools of metabolites, including nitrogen-rich molecules such as glutamate. Additionally, we report field data suggesting metabolisms of Prochlorococcus are comparable to results we observe in our laboratory studies. Accounting for these observations, potential metabolic mechanisms utilized by Prochlorococcus are discussed as we build upon our understanding of nutrient-limited photosynthesis and carbon metabolism. IMPORTANCE Photosynthetic microbes are the predominant sources of organic carbon in the sunlit regions of the ocean. During photosynthesis, nitrogen and carbon metabolism are coordinated to synthesize nitrogen-containing organics such as amino acids and nucleic acids. In large regions of the ocean, nitrogen is thought to limit the growth of phytoplankton. The impact of nitrogen limitation on the synthesis of organic carbon is not well understood, especially for the most abundant photosynthetic organism in the nitrogen-limited regions of the ocean, Prochlorococcus . This study compares the carbon metabolism of nitrogen-replete and nitrogen-limited Prochlorococcus spp. to determine how nitrogen availability influences inorganic carbon assimilation into an organic form. Metabolomics and physiological data revealed that cells under nitrogen limitation have reduced metabolic flux and total carbon fixation rates while maintaining elevated metabolite pool levels and releasing a larger proportion of total fixed carbon to the environment.

This review surveys the current state of knowledge of the concentrations, sources and sinks of reactive oxygen species (ROS) in the ocean. Both abiotic and biotic factors contribute to ROS dynamics in seawater, and ROS can feature prominently in marine microbe-microbe interactions. The sun plays a key role in the production of ROS in the ocean, and consequently ROS concentrations are typically maximal in the sun-exposed surface. However, microbes can also contribute significantly to extracellular ROS. Production of superoxide is widespread within the microbial community, and may benefit the producers as antimicrobial agents or perhaps more generally, as a means of nutrient scavenging. Decomposition of hydrogen peroxide is a community-wide activity, though some members may play less significant roles in this process. The more reactive forms of ROS, singlet oxygen and the hydroxyl radical, may be less important as microbial stressors, as they tend to react with the chemicals in seawater before they can contact the cells. However, exceptions may exist for microbes attached to singlet oxygen-generating sinking particulate matter. Extracellular ROS thus plays an important role in the ecology of marine microbes, the full extent to which we are only beginning to appreciate., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Hydrogen peroxide (HOOH) is a reactive oxygen species, derived from molecular oxygen, that is capable of damaging microbial cells. Surprisingly, the HOOH defence systems of some aerobes in the oxygenated marine environments are critically depleted, relative to model aerobes. For instance, the gene encoding catalase is absent in the numerically dominant photosynthetic cyanobacterium, Prochlorococcus. Accordingly, Prochlorococcus is highly susceptible to HOOH when exposed as pure cultures. Pure cultures do not exist in the marine environment, however. Catalase-positive community members can remove HOOH from the seawater medium, thus lowering the threat to Prochlorococcus and any other member that likewise lacks their own catalase. This cross-protection may constitute a loosely defined symbiosis, whereby the catalase-positive helper cells may benefit through the acquisition of nutrients released by the beneficiaries such as Prochlorococcus. Other members of the community that may be helped by the catalase-positive cells may include some lineages of Synechococcus - the sister genus of Prochlorococcus - as well as some lineages of SAR11 and ammonia oxidizing archaea and bacteria. The co-occurrence of catalase-positive and -negative members suggests that cross-protection from HOOH-mediated oxidative stress may play an important role in the construction of the marine microbial community., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Prochlorococcus, the smallest and most abundant phytoplankter in the ocean, is highly sensitive to hydrogen peroxide (HOOH), and co-occurring heterotrophs such as Alteromonas facilitate the growth of Prochlorococcus by scavenging HOOH. Temperature is also a major influence on Prochlorococcus abundance and distribution in the ocean, and studies in other photosynthetic organisms have shown that HOOH and temperature extremes can act together as synergistic stressors. To address potential synergistic effects of temperature and HOOH on Prochlorococcus growth, high- and low-temperature-adapted representative strains were cultured at ecologically relevant concentrations under a range of HOOH concentrations and temperatures. Higher concentrations of HOOH severely diminished the permissive temperature range for growth of both Prochlorococcus strains. At the permissive temperatures, the growth rates of both Prochlorococcus strains decreased as a function of HOOH, and cold temperature increased susceptibility of photosystem II to HOOH-mediated damage. Serving as a proxy for the natural community, co-cultured heterotrophic bacteria increased the Prochlorococcus growth rate under these temperatures, and expanded the permissive range of temperature for growth. These studies indicate that in the ocean, the cross-protective function of the microbial community may confer a fitness increase for Prochlorococcus at its temperature extremes, especially near the ocean surface where oxidative stress is highest. This interaction may play a substantial role in defining the realized thermal niche and habitat range of Prochlorococcus with respect to latitude., (© 2018. International Society for Microbial Ecology.)

Prochlorococcus is a globally important marine cyanobacterium that lacks the gene catalase and relies on 'helper' bacteria such as Alteromonas to remove reactive oxygen species. Increasing atmospheric CO 2 decreases the need for carbon concentrating mechanisms and photorespiration in phytoplankton, potentially altering their metabolism and microbial interactions even when carbon is not limiting growth. Here, Prochlorococcus (VOL4, MIT9312) was co-cultured with Alteromonas (strain EZ55) under ambient (400 p.p.m.) and elevated CO 2 (800 p.p.m.). Under elevated CO 2 , Prochlorococcus had a significantly longer lag phase and greater apparent die-offs after transfers suggesting an increase in oxidative stress. Whole-transcriptome analysis of Prochlorococcus revealed decreased expression of the carbon fixation operon, including carboxysome subunits, corresponding with significantly fewer carboxysome structures observed by electron microscopy. Prochlorococcus co-culture responsive gene 1 had significantly increased expression in elevated CO 2 , potentially indicating a shift in the microbial interaction. Transcriptome analysis of Alteromonas in co-culture with Prochlorococcus revealed decreased expression of the catalase gene, known to be critical in relieving oxidative stress in Prochlorococcus by removing hydrogen peroxide. The decrease in catalase gene expression was corroborated by a significant ~6-fold decrease in removal rates of hydrogen peroxide from co-cultures. These data suggest Prochlorococcus may be more vulnerable to oxidative stress under elevated CO 2 in part from a decrease in ecosystem services provided by heterotrophs like Alteromonas. This work highlights the importance of considering microbial interactions in the context of a changing ocean., (© 2018. International Society for Microbial Ecology.)

Microcystins are secondary metabolites produced by several freshwater, bloom-forming cyanobacterial species. Microcystin-producing cyanobacteria co-occur with a complex community of heterotrophic bacteria. Though conflicting, studies suggest that microcystins affect the physiology of heterotrophic bacteria by inducing oxidative stress and increasing cell envelope permeability. Based on these observations, we hypothesized that exposure to microcystin should induce differential expression in genes responding to oxidative and envelope stress and trigger shifts in metabolite pools. We tested this hypothesis by exposing Escherichia coli MG1655 to 1 and 10 mg/L microcystin-LR and monitored global changes to gene expression, cellular metabolite pools, and lipid composition using RNA-sequencing and UPLC-MS. Contrary to reported studies, we observed no evidence that microcystin-LR induced oxidative or cell envelope stress in E. coli under the tested conditions. Our results suggest a potential difference in mechanism by which microcystin-LR interacts with heterotrophic bacteria vs. cyanobacteria.

The North Pacific Ocean (between approximately 0°N and 50°N) contains the largest continuous ecosystem on Earth. This region plays a vital role in the cycling of globally important nutrients as well as carbon. Although the microbial communities in this region have been assessed, the dynamics of viruses (abundances and production rates) remains understudied. To address this gap, scientific cruises during the winter and summer seasons (2013) covered the North Pacific basin to determine factors that may drive virus abundances and production rates. Along with information on virus particle abundance and production, we collected a spectrum of oceanographic metrics as well as information on microbial diversity. The data suggest that both biotic and abiotic factors affect the distribution of virus particles. Factors influencing virus dynamics did not vary greatly between seasons, although the abundance of viruses was almost an order of magnitude greater in the summer. When considered in the context of microbial community structure, our observations suggest that members of the bacterial phyla Proteobacteria, Planctomycetes, and Bacteroidetes were correlated to both virus abundances and virus production rates: these phyla have been shown to be enriched in particle associated communities. The findings suggest that environmental factors influence virus community functions (e.g., virion particle degradation) and that particle-associated communities may be important drivers of virus activity.

Variability in plankton elemental requirements can be important for global ocean biogeochemistry but we currently have a limited understanding of how ocean temperature influences the plankton C/N/P ratio. Multiple studies have put forward a 'translation-compensation' hypothesis to describe the positive relationship between temperature and plankton N/P or C/P as cells should have lower demand for P-rich ribosomes and associated depressed QP when growing at higher temperature. However, temperature affects many cellular processes beyond translation with unknown outcomes on cellular elemental composition. In addition, the impact of temperature on growth and elemental composition of phytoplankton is likely modulated by the life history and growth rate of the organism. To test the direct and indirect (via growth rate changes) effect of temperature, we here analyzed the elemental composition and ratios in six strains affiliated with the globally abundant marine Cyanobacteria Prochlorococcus. We found that temperature had a significant positive effect on the carbon and nitrogen cell quota, whereas no clear trend was observed for the phosphorus cell quota. The effect on N/P and C/P were marginally significantly positive across Prochlorococcus. The elemental composition and ratios of individual strains were also affected but we found complex interactions between the strain identity, temperature, and growth rate in controlling the individual elemental ratios in Prochlorococcus and no common trends emerged. Thus, the observations presented here does not support the 'translation-compensation' theory and instead suggest unique cellular elemental effects as a result of rising temperature among closely related phytoplankton lineages. Thus, the biodiversity context should be considered when predicting future elemental ratios and how cycles of carbon, nitrogen, and phosphorus may change in a future ocean., Competing Interests: The authors have declared that no competing interests exist.

Biogeography studies that correlate the observed distribution of organisms to environmental variables are typically based on local conditions. However, in cases with substantial translocation, like planktonic organisms carried by ocean currents, selection may happen upstream and local environmental factors may not be representative of those that shaped the local population. Here we use an individual-based model of microbes in the global surface ocean to explore this effect for temperature. We simulate up to 25 million individual cells belonging to up to 50 species with different temperature optima. Microbes are moved around the globe based on a hydrodynamic model, and grow and die based on local temperature. We quantify the role of currents using the "advective temperature differential" metric, which is the optimum temperature of the most abundant species from the model with advection minus that from the model without advection. This differential depends on the location and can be up to 4°C. Poleward-flowing currents, like the Gulf Stream, generally experience cooling and the differential is positive. We apply our results to three global datasets. For observations of optimum growth temperature of phytoplankton, accounting for the effect of currents leads to a slightly better agreement with observations, but there is large variability and the improvement is not statistically significant. For observed Prochlorococcus ecotype ratios and metagenome nucleotide divergence, accounting for advection improves the correlation significantly, especially in areas with relatively strong poleward or equatorward currents., Competing Interests: The authors have declared that no competing interests exist.

The distribution of major clades of Prochlorococcus tracks light, temperature and other environmental variables; yet, the drivers of genomic diversity within these ecotypes and the net effect on biodiversity of the larger community are poorly understood. We examined high light (HL) adapted Prochlorococcus communities across spatial and temporal environmental gradients in the Pacific Ocean to determine the ecological drivers of population structure and diversity across taxonomic ranks. We show that the Prochlorococcus community has the highest diversity at low latitudes, but seasonality driven by temperature, day length and nutrients adds complexity. At finer taxonomic resolution, some 'sub-ecotype' clades have unique, cohesive responses to environmental variables and distinct biogeographies, suggesting that presently defined ecotypes can be further partitioned into ecologically meaningful units. Intriguingly, biogeographies of the HL-I sub-ecotypes are driven by unique combinations of environmental traits, rather than through trait hierarchy, while the HL-II sub-ecotypes appear ecologically similar, thus demonstrating differences among these dominant HL ecotypes. Examining biodiversity across taxonomic ranks reveals high-resolution dynamics of Prochlorococcus evolution and ecology that are masked at phylogenetically coarse resolution. Spatial and seasonal trends of Prochlorococcus communities suggest that the future ocean may be comprised of different populations, with implications for ecosystem structure and function.

The vast majority of the phytoplankton communities in surface mixed layer of the oligotrophic ocean are numerically dominated by one of two ecotypes of Prochlorococcus, eMIT9312 or eMED4. In this study, we surveyed large latitudinal transects in the Atlantic and Pacific Ocean to determine if these ecotypes discretely partition the surface mixed layer niche, or if populations exist as a continuum along key environmental gradients, particularly temperature. Transitions of dominance occurred at approximately 19-21°C, with the eMED4 ecotype dominating the colder, and eMIT9312 ecotype dominating the warmer regions. Within these zones of regional dominance, however, the minority ecotype was not competed to extinction. Rather, a robust log-linear relationship between ecotype ratio and temperature characterized this stabilized coexistence: for every 2.5°C increase in temperature, the eMIT9312:eMED4 ratio increased by an order of magnitude. This relationship was observed in both quantitative polymerase chain reaction and in pyrosequencing assays. Water column stratification also contributed to the ecotype ratio along the basin-scale transects, but to a lesser extent. Finally, instances where the ratio of the eMED4 and eMIT9312 abundances did not correlate well with temperature were identified. Such occurrences are likely due to changes in water temperatures outpacing changes in community structure., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Newly designed primers targeting rbcL (CO2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function.

We investigated the production of hydrogen peroxide (HOOH) in illuminated seawater media containing a variety of zwitterionic buffers. Production rates varied extensively among buffers, with 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) highest and N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) among the lowest. The rate of HOOH accumulation was remarkably consistent over many days, and increased linearly with buffer concentration, natural seawater concentration, and light level. Concentrations of HEPES commonly used in culture media (1-10 mM) generated enough HOOH to kill the axenic Prochlorococcus strain VOL1 during growth in enriched seawater media at lower, environmentally realistic cell concentrations and/or under high light exposure. We also demonstrated that HEPES can be used experimentally to study the biological effects of chronic exposure to sublethal levels of HOOH such as may be experienced by light-exposed microorganisms., (© 2013 Phycological Society of America.)

In the open ocean genetically diverse clades of the unicellular cyanobacteria Prochlorococcus are biogeographically structured along environmental gradients, yet little is known about their in situ activity. To address this gap, here we use the numerically dominant Prochlorococcus clade eHL-II (eMIT9312) as a model organism to develop and apply a method to examine their in situ activity using rRNA content and cell size as metrics of cellular physiology. For two representative isolates (MIT9312 and MIT9215) rRNA cell(-1) increases linearly with specific growth rate but is anticorrelated with cell size indicated by flow cytometrically measured (SSC). Although each strain has a unique relationship between cellular rRNA (or cell size) and growth rate, both strains have the same strong positive correlation between rRNA cell(-1) SSC(-1) and growth rate. We field test this approach and observe distinct patterns of eHL-II clade specific activity (rRNA cell(-1) SSC(-1)) with depth that are consistent with patterns of photosynthetic rates. This molecular technique provides unique insight into the ecology of Prochlorococcus and could potentially be expanded to include other microbes to unravel the ecological and biogeochemical contributions of genetically distinct marine side scatter microbes., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Reductive genomic evolution, driven by genetic drift, is common in endosymbiotic bacteria. Genome reduction is less common in free-living organisms, but it has occurred in the numerically dominant open-ocean bacterioplankton Prochlorococcus and "Candidatus Pelagibacter," and in these cases the reduction appears to be driven by natural selection rather than drift. Gene loss in free-living organisms may leave them dependent on cooccurring microbes for lost metabolic functions. We present the Black Queen Hypothesis (BQH), a novel theory of reductive evolution that explains how selection leads to such dependencies; its name refers to the queen of spades in the game Hearts, where the usual strategy is to avoid taking this card. Gene loss can provide a selective advantage by conserving an organism's limiting resources, provided the gene's function is dispensable. Many vital genetic functions are leaky, thereby unavoidably producing public goods that are available to the entire community. Such leaky functions are thus dispensable for individuals, provided they are not lost entirely from the community. The BQH predicts that the loss of a costly, leaky function is selectively favored at the individual level and will proceed until the production of public goods is just sufficient to support the equilibrium community; at that point, the benefit of any further loss would be offset by the cost. Evolution in accordance with the BQH thus generates "beneficiaries" of reduced genomic content that are dependent on leaky "helpers," and it may explain the observed nonuniversality of prototrophy, stress resistance, and other cellular functions in the microbial world.

We completed a transect through the Western Pacific Warm Pool to examine how environmental variables may influence viral and bacterial abundance and production rates in this globally important oceanic region. Of the variables analyzed, viral abundance and production had the most significant relationship to bacterial cell abundance: viral parameters were not significantly correlated to the measured environmental variables, including temperature. Bacterial production rates were significantly correlated to temperature in open ocean waters, but not in waters close to land masses. Analyses of 16S rRNA gene by pyrosequencing indicated only minor changes in eubacterial community structure across the transect, with α-proteobacteria dominating all sampled populations. Diversity within the prokaryotic community did not correlate directly with viral abundance or activity. Comparisons to two other ocean-scale transects (> 8000 km of open ocean in total) in the Atlantic Ocean indicated that correlations between viral and bacterial abundance and production relative to environmental variables are regime dependent. In particular, correlations to temperature showed remarkable differences across the three transects. Collectively, our observations suggest that seemingly similar oceanic regions may have very different microbial community responses to environmental variables. Our observations and analyses demonstrate that ocean-scale generalizations may not apply in the case of viral ecology., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The phytoplankton community in the oligotrophic open ocean is numerically dominated by the cyanobacterium Prochlorococcus, accounting for approximately half of all photosynthesis. In the illuminated euphotic zone where Prochlorococcus grows, reactive oxygen species are continuously generated via photochemical reactions with dissolved organic matter. However, Prochlorococcus genomes lack catalase and additional protective mechanisms common in other aerobes, and this genus is highly susceptible to oxidative damage from hydrogen peroxide (HOOH). In this study we showed that the extant microbial community plays a vital, previously unrecognized role in cross-protecting Prochlorococcus from oxidative damage in the surface mixed layer of the oligotrophic ocean. Microbes are the primary HOOH sink in marine systems, and in the absence of the microbial community, surface waters in the Atlantic and Pacific Ocean accumulated HOOH to concentrations that were lethal for Prochlorococcus cultures. In laboratory experiments with the marine heterotroph Alteromonas sp., serving as a proxy for the natural community of HOOH-degrading microbes, bacterial depletion of HOOH from the extracellular milieu prevented oxidative damage to the cell envelope and photosystems of co-cultured Prochlorococcus, and facilitated the growth of Prochlorococcus at ecologically-relevant cell concentrations. Curiously, the more recently evolved lineages of Prochlorococcus that exploit the surface mixed layer niche were also the most sensitive to HOOH. The genomic streamlining of these evolved lineages during adaptation to the high-light exposed upper euphotic zone thus appears to be coincident with an acquired dependency on the extant HOOH-consuming community. These results underscore the importance of (indirect) biotic interactions in establishing niche boundaries, and highlight the impacts that community-level responses to stress may have in the ecological and evolutionary outcomes for co-existing species.

To better understand the temporal and spatial dynamics of Prochlorococcus populations, and how these populations co-vary with the physical environment, we followed monthly changes in the abundance of five ecotypes-two high-light adapted and three low-light adapted-over a 5-year period in coordination with the Bermuda Atlantic Time Series (BATS) and Hawaii Ocean Time-series (HOT) programs. Ecotype abundance displayed weak seasonal fluctuations at HOT and strong seasonal fluctuations at BATS. Furthermore, stable 'layered' depth distributions, where different Prochlorococcus ecotypes reached maximum abundance at different depths, were maintained consistently for 5 years at HOT. Layered distributions were also observed at BATS, although winter deep mixing events disrupted these patterns each year and produced large variations in ecotype abundance. Interestingly, the layered ecotype distributions were regularly reestablished each year after deep mixing subsided at BATS. In addition, Prochlorococcus ecotypes each responded differently to the strong seasonal changes in light, temperature and mixing at BATS, resulting in a reproducible annual succession of ecotype blooms. Patterns of ecotype abundance, in combination with physiological assays of cultured isolates, confirmed that the low-light adapted eNATL could be distinguished from other low-light adapted ecotypes based on its ability to withstand temporary exposure to high-intensity light, a characteristic stress of the surface mixed layer. Finally, total Prochlorococcus and Synechococcus dynamics were compared with similar time series data collected a decade earlier at each location. The two data sets were remarkably similar-testimony to the resilience of these complex dynamic systems on decadal time scales.

The marine cyanobacterium Prochlorococcus MED4 has the smallest genome and cell size of all known photosynthetic organisms. Like all phototrophs at temperate latitudes, it experiences predictable daily variation in available light energy which leads to temporal regulation and partitioning of key cellular processes. To better understand the tempo and choreography of this minimal phototroph, we studied the entire transcriptome of the cell over a simulated daily light-dark cycle, and placed it in the context of diagnostic physiological and cell cycle parameters. All cells in the culture progressed through their cell cycles in synchrony, thus ensuring that our measurements reflected the behavior of individual cells. Ninety percent of the annotated genes were expressed, and 80% had cyclic expression over the diel cycle. For most genes, expression peaked near sunrise or sunset, although more subtle phasing of gene expression was also evident. Periodicities of the transcripts of genes involved in physiological processes such as in cell cycle progression, photosynthesis, and phosphorus metabolism tracked the timing of these activities relative to the light-dark cycle. Furthermore, the transitions between photosynthesis during the day and catabolic consumption of energy reserves at night- metabolic processes that share some of the same enzymes--appear to be tightly choreographed at the level of RNA expression. In-depth investigation of these patterns identified potential regulatory proteins involved in balancing these opposing pathways. Finally, while this analysis has not helped resolve how a cell with so little regulatory capacity, and a 'deficient' circadian mechanism, aligns its cell cycle and metabolism so tightly to a light-dark cycle, it does provide us with a valuable framework upon which to build when the Prochlorococcus proteome and metabolome become available.

Axenic (pure) cultures of marine unicellular cyanobacteria of the Prochlorococcus genus grow efficiently only if the inoculation concentration is large; colonies form on semisolid medium at low efficiencies. In this work, we describe a novel method for growing Prochlorococcus colonies on semisolid agar that improves the level of recovery to approximately 100%. Prochlorococcus grows robustly at low cell concentrations, in liquid or on solid medium, when cocultured with marine heterotrophic bacteria. Once the Prochlorococcus cell concentration surpasses a critical threshold, the "helper" heterotrophs can be eliminated with antibiotics to produce axenic cultures. Our preliminary evidence suggests that one mechanism by which the heterotrophs help Prochlorococcus is the reduction of oxidative stress.

Prochlorococcus is the numerically dominant phytoplankter in the oligotrophic oceans, accounting for up to half of the photosynthetic biomass and production in some regions. Here, we describe how the abundance of six known ecotypes, which have small subunit ribosomal RNA sequences that differ by less than 3%, changed along local and basin-wide environmental gradients in the Atlantic Ocean. Temperature was significantly correlated with shifts in ecotype abundance, and laboratory experiments confirmed different temperature optima and tolerance ranges for cultured strains. Light, nutrients, and competitor abundances also appeared to play a role in shaping different distributions.

The cyanobacterium Prochlorococcus numerically dominates the photosynthetic community in the tropical and subtropical regions of the world's oceans. Six evolutionary lineages of Prochlorococcus have been described, and their distinctive physiologies and genomes indicate that these lineages are "ecotypes" and should have different oceanic distributions. Two methods recently developed to quantify these ecotypes in the field, probe hybridization and quantitative PCR (QPCR), have shown that this is indeed the case. To facilitate a global investigation of these ecotypes, we modified our QPCR protocol to significantly increase its speed, sensitivity, and accessibility and validated the method in the western and eastern North Atlantic Ocean. We showed that all six ecotypes had distinct distributions that varied with depth and location, and, with the exception of the deeper waters at the western North Atlantic site, the total Prochlorococcus counts determined by QPCR matched the total counts measured by flow cytometry. Clone library analyses of the deeper western North Atlantic waters revealed ecotypes that are not represented in the culture collections with which the QPCR primers were designed, explaining this discrepancy. Finally, similar patterns of relative ecotype abundance were obtained in QPCR and probe hybridization analyses of the same field samples, which could allow comparisons between studies.

The process of evolution by natural selection has been known for a century and a half, yet the mechanics of selection are still poorly understood. In most cases where natural selection has been studied, the genetic and physiological bases of fitness variation that result in population changes were not identified, leaving only a partial understanding of selection. Starved cultures of the bacterium Escherichia coli present a model system with which to address the genetic and physiological bases of natural selection. This is a model system that also reflects the prevalent state of bacteria in the natural world; due to intense competition for nutrients, microorganisms spend the majority of their lives under starvation conditions. Genetic analyses of a single survivor of starvation identified four adaptive mutations(1). Investigation of these mutations has revealed insights into the molecular and physiological bases of evolution during prolonged starvation stress.

The marine unicellular cyanobacterium Prochlorococcus is the smallest-known oxygen-evolving autotroph. It numerically dominates the phytoplankton in the tropical and subtropical oceans, and is responsible for a significant fraction of global photosynthesis. Here we compare the genomes of two Prochlorococcus strains that span the largest evolutionary distance within the Prochlorococcus lineage and that have different minimum, maximum and optimal light intensities for growth. The high-light-adapted ecotype has the smallest genome (1,657,990 base pairs, 1,716 genes) of any known oxygenic phototroph, whereas the genome of its low-light-adapted counterpart is significantly larger, at 2,410,873 base pairs (2,275 genes). The comparative architectures of these two strains reveal dynamic genomes that are constantly changing in response to myriad selection pressures. Although the two strains have 1,350 genes in common, a significant number are not shared, and these have been differentially retained from the common ancestor, or acquired through duplication or lateral transfer. Some of these genes have obvious roles in determining the relative fitness of the ecotypes in response to key environmental variables, and hence in regulating their distribution and abundance in the oceans.

The loss of preexisting genes or gene activities during evolution is a major mechanism of ecological specialization. Evolutionary processes that can account for gene loss or inactivation have so far been restricted to one of two mechanisms: direct selection for the loss of gene activities that are disadvantageous under the conditions of selection (i.e., antagonistic pleiotropy) and selection-independent genetic drift of neutral (or nearly neutral) mutations (i.e., mutation accumulation). In this study we demonstrate with an evolved strain of Escherichia coli that a third, distinct mechanism exists by which gene activities can be lost. This selection-dependent mechanism involves the expropriation of one gene's upstream regulatory element by a second gene via a homologous recombination event. Resulting from this genetic exchange is the activation of the second gene and a concomitant inactivation of the first gene. This gene-for-gene expression tradeoff provides a net fitness gain, even if the forfeited activity of the first gene can play a positive role in fitness under the conditions of selection.

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes.

Starved cultures of Escherichia coli undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the rpoS819 allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). Here we have identified three new GASP loci from an aged rpoS819 strain: sgaA, sgaB, and sgaC. Each locus is capable of conferring GASP on the rpoS819 parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells.