Allison Coe

Niche Partitioning Among Prochlorococcus Ecotypes Along Ocean-Scale Environmental Gradients

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.

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

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

Prochlorococcus Ecotype Abundances in the North Atlantic Ocean As Revealed by an Improved Quantitative PCR Method

ABSTRACT The cyanobacterium Prochlorococcus numerically dominates the photosynthetic community in the tropical and subtropical regions of the worlds 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.

Temporal dynamics of Prochlorococcus ecotypes in the Atlantic and Pacific oceans

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.

UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase

Exposure to solar radiation can cause mortality in natural communities of pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV-hyper-resistant variant of high light-adapted strain MED4. The hyper-resistant strain was constitutively upregulated for expression of the mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error-free, in contrast to error-prone SOS dark repair systems that are widespread in bacteria. The UV-hyper-resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant strains from MED4 wild-type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper-resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.

Torn apart and reunited: impact of a heterotroph on the transcriptome of Prochlorococcus

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 spontaneous mutation frequencies of Prochlorococcus strains are commensurate with those of other bacteria

The marine cyanobacterium Prochlorococcus, the smallest and most abundant oxygenic phototroph, has an extremely streamlined genome and a high rate of protein evolution. High-light adapted strains of Prochlorococcus in particular have seemingly inadequate DNA repair systems, raising the possibility that inadequate repair may lead to high mutation rates. Prochlorococcus mutation rates have been difficult to determine, in part because traditional methods involving quantifying colonies on solid selective media are not straightforward for this organism. Here we used a liquid dilution method to measure the approximate number of antibiotic-resistant mutants in liquid cultures of Prochlorococcus strains previously unexposed to antibiotic selection. Several antibiotics for which resistance in other bacteria is known to result from a single base pair change were used. The resulting frequencies of antibiotic resistance in Prochlorococcus cultures allowed us to then estimate maximum spontaneous mutation rates, which were similar to those in organisms such as E. coli (∼5.4 × 10(-7) per gene per generation). Therefore, despite the lack of some DNA repair genes, it appears unlikely that the Prochlorcoccus genomes studied here are currently being shaped by unusually high mutation rates.

Draft Genome Sequence of Alteromonas macleodii Strain MIT1002, Isolated from an Enrichment Culture of the Marine Cyanobacterium Prochlorococcus

ABSTRACT 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.

Heterotroph Interactions Alter Prochlorococcus Transcriptome Dynamics during Extended Periods of Darkness

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

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.