Paul M. Berube

Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria

The discovery of ammonia oxidation by mesophilic and thermophilic Crenarchaeota and the widespread distribution of these organisms in marine and terrestrial environments indicated an important role for them in the global nitrogen cycle. However, very little is known about their physiology or their contribution to nitrification. Here we report oligotrophic ammonia oxidation kinetics and cellular characteristics of the mesophilic crenarchaeon ‘Candidatus Nitrosopumilus maritimus’ strain SCM1. Unlike characterized ammonia-oxidizing bacteria, SCM1 is adapted to life under extreme nutrient limitation, sustaining high specific oxidation rates at ammonium concentrations found in open oceans. Its half-saturation constant (Km = 133 nM total ammonium) and substrate threshold (≤10 nM) closely resemble kinetics of in situ nitrification in marine systems and directly link ammonia-oxidizing Archaea to oligotrophic nitrification. The remarkably high specific affinity for reduced nitrogen (68,700 l per g cells per h) of SCM1 suggests that Nitrosopumilus-like ammonia-oxidizing Archaea could successfully compete with heterotrophic bacterioplankton and phytoplankton. Together these findings support the hypothesis that nitrification is more prevalent in the marine nitrogen cycle than accounted for in current biogeochemical models.

Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes

The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in oligotrophic regions of the oceans. The inability to assimilate nitrate is considered an important factor underlying the distribution of Prochlorococcus, and thought to explain, in part, low abundance of Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe the widespread occurrence of a genomic island containing nitrite and nitrate assimilation genes in uncultured Prochlorococcus cells from marine surface waters. These genes are characterized by low GC content, form a separate phylogenetic clade most closely related to marine Synechococcus, and are located in a different genomic region compared with an orthologous cluster found in marine Synechococcus strains. This sequence distinction suggests that these genes were not transferred recently from Synechococcus. We demonstrate that the nitrogen assimilation genes encode functional proteins and are expressed in the ocean. Also, we find that their relative occurrence is higher in the Caribbean Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean, which may be related to the nitrogen availability in each region. Our data suggest that the ability to assimilate nitrite and nitrate is associated with microdiverse lineages within high- and low-light (LL) adapted Prochlorococcus ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The potential for previously unrecognized productivity by Prochlorococcus in the presence of oxidized nitrogen species has implications for understanding the biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen cycles.

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

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

Physiology and evolution of nitrate acquisition in Prochlorococcus.

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

Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus

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

Nitrogen cost minimization is promoted by structural changes in the transcriptome of N-deprived Prochlorococcus cells

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.

Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments

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.

Emergence of trait variability through the lens of nitrogen assimilation in Prochlorococcus

Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. The marine cyanobacterium Prochlorococcus is a useful model system for understanding how trait variability emerges within microbial species: Its functional diversity is overlaid on measurable environmental gradients, providing a powerful lens into large-scale evolutionary processes. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing the allocation of ecologically important functions within microbial species. We find that nitrate assimilation genes are absent in basal lineages of Prochlorococcus 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, stochastic gene loss, and homologous recombination among closely related cells. 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.

Marine microbial metagenomes sampled across space and time

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.