Seawater contains small biomolecules, or metabolites, that are highly labile components of dissolved organic matter (DOM). Marine microbes interact by exchanging metabolites, thus shaping marine microbial ecology, DOM composition, and global carbon cycling. To better constrain one set of microbe-metabolite interactions, we cultured the marine gammaproteobacterium Alteromonas macleodii MIT1002 on a range of compounds excreted by a sympatric cyanobacterium, Prochlorococcus. Alteromonas macleodii MIT1002 could metabolize the branched-chain amino acids leucine, isoleucine, and valine, as well as 3-methyl-2-oxobutanoic acid (a branched-chain ketoacid intermediate of valine metabolism), but not thymidine, kynurenine, 4-hydroxybenzoic acid, nor the other branched-chain ketoacids. The utilization of branched-chain amino acids indicates that A. macleodii MIT1002 can metabolize each corresponding ketoacid, suggesting that transporter specificity underlies the observed substrate specificity for 3-methyl-2-oxobutanoic acid. These experiments show that even subtle changes in chemical structure can result in different microbial interactions and different fates for dissolved metabolites.

Picocyanobacteria Prochlorococcus and Synechococcus coexist with lytic phages and heterotrophic bacteria in the oceans. These phages are a significant cause of mortality, while heterotrophic bacteria can increase Prochlorococcus fitness by reducing oxidative stress and cross-feeding during extended darkness. Studies of Prochlorococcus-phage interactions are often done with xenic cultures, as it has been historically difficult to obtain and maintain heterotroph-free cultures. Here, we examine how heterotrophic bacteria affect phage infection dynamics in Prochlorococcus and Synechococcus by comparing infections in cultures with and without heterotrophs. We found that Prochlorococcus populations resumed growth following infection only when heterotrophs were present, independent of phage-host or heterotroph-host ratios. In Synechococcus phage-host pairings, outcomes varied, suggesting that heterotrophic effects depend on the phage-host interaction. When hosts recovered from infection, heterotrophs appeared to facilitate this by mitigating oxidative stress and possibly supplying organic carbon sources that support post-infection growth. Recovered Prochlorococcus and Synechococcus populations were resistant to reinfection when transferred to fresh media. However, comparisons of host sequences before and after infection argue against genetic change as the mechanism of tolerance. Instead, we infer that hosts undergo non-genetic adaptations during recovery in the presence of heterotrophs, likely driven by heterotroph-derived organic compounds that reshape host metabolism and confer protection against future lysis. During this process, heterotrophic community composition shifted dynamically across sequential transfers, yet host recovery remained consistent, suggesting that either community function or host adaptations maintained support for growth. IMPORTANCE:This study uncovers a previously unrecognized role of heterotrophic bacteria in shaping phage infection outcomes in marine cyanobacteria. Our findings demonstrate that the presence of heterotrophs can enable Prochlorococcus to recover from phage infection, irrespective of phage:host or heterotroph:host ratios. In contrast, Synechococcus exhibited variable outcomes, suggesting that the impact of heterotrophs is dependent on the heterotroph-host pairing. Recovery after phage infection appears to be driven by heterotroph-facilitated mitigation of oxidative stress and provision of organic carbon, which may alter host metabolism and contribute to protection against future phage infection. These results highlight the importance of the microbial community, particularly heterotrophs, in shaping cyanobacteria-phage dynamics and highlight the need to reframe host-phage interactions within a broader ecological framework.

Prochlorococcus and Synechococcus are abundant marine picocyanobacteria that contribute significantly to ocean primary production. Recent genome sequencing efforts, including those presented here, have yielded a large number of high-quality reference genomes, enabling the classification of these picocyanobacteria in marine metagenomic sequence data at high phylogenetic resolution. When combined with environmental data, these classifications can guide cluster/clade/grade assignments and offer insights into niche differentiation within these populations. Here we present ProSynTax, a curated protein sequence dataset and accompanying classification workflow aimed at enhancing the taxonomic resolution of Prochlorococcus and Synechococcus classification. ProSynTax includes proteins from 1,260 genomes of Prochlorococcus and Synechococcus, including single-amplified genomes, high-quality draft genomes, and newly closed genomes. Additionally, ProSynTax incorporates proteins from 41,753 genomes of marine heterotrophic bacteria, archaea, and viruses to assess microbial and viral communities surrounding Prochlorococcus and Synechococcus. This resource enables accurate classification of picocyanobacterial clusters/clades/grades in metagenomic data - even when present at 0.15% of reads for Prochlorococcus or 0.03% of reads for Synechococcus.

Prochlorococcus and Synechococcus are abundant marine picocyanobacteria that contribute significantly to ocean primary production. Recent genome sequencing efforts, including those presented here, have yielded a large number of high-quality reference genomes, enabling the classification of these picocyanobacteria in marine metagenomic sequence data at high phylogenetic resolution. When combined with environmental data, these classifications can guide cluster/clade/grade assignments and offer insights into niche differentiation within these populations. Here we present ProSynTaxDB, a curated protein sequence database and accompanying workflow aimed at enhancing the taxonomic resolution of Prochlorococcus and Synechococcus classification. ProSynTaxDB includes proteins from 1,260 genomes of Prochlorococcus and Synechococcus , including single-amplified genomes, high-quality draft genomes, and newly closed genomes. Additionally, ProSynTaxDB incorporates proteins from 27,799 genomes of marine heterotrophic bacteria, archaea, and viruses to assess microbial and viral communities surrounding Prochlorococcus and Synechococcus . This resource enables accurate classification of picocyanobacterial clusters/clades/grades in metagenomic data – even when present at 0.60% of reads for Prochlorococcus or 0.09% of reads for Synechococcus . ### Competing Interest Statement The authors have declared no competing interest.

Prochlorococcus is the world’s most abundant photosynthetic organism with over 1027 cells distributed across much of Earth’s oceans, and is collectively responsible for almost 10% of marine carbon fixation. Naturally co-occurring heterotrophic bacteria at roughly 105-106 cells mL-1 in the oceans have been shown to increase Prochlorococcus fitness and productivity. Despite this massive scale, our understanding of these globally important interactions remains limited, with past research largely focused on single Prochlorococcus -heterotroph pairings involving only a few species. In this study, we extend this perspective by using synthetic communities containing multiple diverse heterotrophic strains isolated from Prochlorococcus enrichment cultures. Specifically, we isolated the four most abundant co-occurring heterotroph species and examined both individual Prochlorococcus –heterotroph interactions and interactions within a synthetic community comprising Prochlorococcus and all four heterotrophs. Using absolute quantification of RNA, DNA, and cell counts over the course of Prochlorococcus growth curves, we find that Prochlorococcus has a modest, species-independent transcriptional response to heterotrophs, whereas each heterotroph displays a markedly different transcriptional response to the community and fulfills distinct metabolic roles. Transcriptional analyses reveal several potential crossfeeding interactions and indicate that community dynamics are influenced not only by metabolic activity but also antagonistic mechanisms, defense responses, and coordinated group behaviors. By pairing synthetic community approaches with absolute abundance measurements, we can gain deeper insight into the forces that shape microbial community assembly in the oceans and their role in driving the global carbon cycle. ### Competing Interest Statement The authors have declared no competing interest. Simons Foundation, https://ror.org/01cmst727, 337262, 721246, LS-FMME-00003951

Prochlorococcus is found throughout the euphotic zone in the oligotrophic open ocean. Deep mixing and sinking while attached to particles can, however, transport Prochlorococcus cells below this sunlit zone, depriving them of light for extended periods of time. Previous work has shown that Prochlorococcus by itself cannot survive extended periods of darkness. However, when co-cultured with a heterotrophic microbe and subjected to repeated periods of extended darkness, Prochlorococcus cells develop an epigenetically inherited dark-tolerant phenotype that can survive longer periods of darkness. Here we examine the metabolic and physiological changes underlying this adaptation using co-cultures of dark-tolerant and parental strains of Prochlorococcus, each grown with the heterotroph Alteromonas under diel light:dark conditions. The relative abundance of Alteromonas was higher in dark-tolerant than parental co-cultures, while dark-tolerant Prochlorococcus cells were larger, contained less chlorophyll, and were less synchronized to the light:dark cycle. Meta-transcriptome analysis revealed that dark-tolerant co-cultures undergo a joint change, in which Prochlorococcus undergoes a relative shift from photosynthesis to respiration, while Alteromonas shifts toward using more organic acids instead of sugars. Furthermore, the transcriptome data suggested enhanced biosynthesis of amino acids and purines in dark-tolerant Prochlorococcus and enhanced degradation of these compounds in Alteromonas. Collectively, our results demonstrate that dark adaptation involves a strengthening of the metabolic coupling between Prochlorococcus and Alteromonas, presumably mediated by an enhanced, and compositionally modified, carbon exchange between the two species.

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 b2 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.IMPORTANCEThe 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:a2. 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.

Extracellular vesicles are small (∼50-250 nm diameter) membrane-bound structures released by cells into their surrounding environment. Vesicles are abundant in the global oceans and likely play a number of ecological roles in these microbially dominated ecosystems, yet we know nothing about what influences their production and distributions. Here we examine how vesicle production varies among different strains of cultivated marine microbes and explore the degree to which this is influenced by some key environmental variables. We show that vesicle production rates – the number of vesicles produced per cell per generation – vary across an order of magnitude in cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Vesicle production rates further differ among strains of the cyanobacterium Prochlorococcus , and vary across temperature and light gradients. These data suggest that both community composition and local environmental conditions 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 broadly consistent with culture observations: highest vesicle abundances are found near the surface, where light irradiances and temperatures are greatest, and then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans – essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems. Importance Bacteria secrete extracellular vesicles containing 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. Differences in vesicle abundances likely affect their functional impacts within microbial ecosystems, but the factors influencing vesicle distributions in the environment remain poorly understood. Using 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. Our data indicate that different marine taxa release vesicles at rates varying across an order of magnitude, and that vesicle production can change dynamically as a function of environmental conditions. Taken together with direct measurements of vesicle concentrations in the oceans, these culture-based measurements further provide a window into estimating vesicle loss rates. These findings represent a step forward in our understanding of marine vesicle distributions and provide a basis for quantitatively exploring vesicle dynamics in natural ecosystems.

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.

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.

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.

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 zone, depriving them of light for extended periods of time. Here we demonstrate that Prochlorococcus and Synechococcus can adapt to tolerate repeated periods of light energy deprivation. Cyanobacterial cultures kept in the dark for 3 days and then returned to the light initially required 18-26 days to resume growth, but after multiple rounds of dark exposure the strains began to regrow after only 1-2 days. This dark-tolerant phenotype was stable and heritable; cultures retained the trait across at least 18-21 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 responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation – an epigenetic mechanism in bacteria – occurs in Prochlorococcus . LC-MS/MS analysis showed that while DNA methylations, including 6mA and 5mC, are found in some other Prochlorococcus strains, no methylations were detected in either the parental or dark-tolerant strain used in our experiments –i.e. the NATL2A strain. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation.

Background: Inflammatory arthritis (IA) is a complex life-long disease with negative consequences on occupational performance and significant impact on meaningful life roles. International guidelines for the clinical management of IA consistently recommend early intervention and a multidisciplinary (MDT) approach as the optimum method to address the medical, symptom and broader life impacts, however, barriers to timely access to the MDT is a common issue in clinical practice. Limited research has explored how clinical services support broader life impacts and participation restrictions associated with early disease as part of routine healthcare. Four rheumatology services in Ireland have recently reconfigured to provide an MDT-led early arthritis service which operates in parallel to traditional medical clinics. Staffed by nurses, occupational therapists, and physiotherapists this service provides assessment and management to clients with a new diagnosis of IA along a model of care within four to six weeks of diagnosis with mechanisms to follow the client over their first year of diagnosis. Objectives: To explore how a novel MDT-led early arthritis service addresses client-identified participation restrictions associated with early IA. Methods: A qualitative description (QD) study was used to explore the perspectives of service providers and service users of this MDT-led early arthritis service. Data were gathered using one-off focus groups with service providers, and individual interviews with service users. Results: All staff (n=15) currently working in these services participated in the focus groups. Forty-three service users with IA participated in individual interviews (males n=12: females n=31); diagnosis duration ranged from 5 to 24 months. QD principles and thematic analysis were used in data analysis. Extensive limitations in everyday activity and restriction in participation in meaningful, age-appropriate life roles in early IA were outlined by service users and service providers. Participants described how the MDT-led model provided automatic and immediate access to services focussed on identification and management of occupation-based participation restrictions early in the disease. Participants described how the model used a flexible approach that ensured; ease of early access to a full MDT, prolonged support from the MDT, and a person-centred approach incorporating social prescribing and self-management principles. Service users emphasised the strength of the MDT approach of providing ‘the right advice, in the right way and at the right time’ as the most influential feature that assisted them to address participation restrictions and support positive health outcomes. This was accomplished by the service providers acting in a coordinated and interdisciplinary manner with a clear remit to address participation restrictions alongside the traditional symptom management approach. Findings highlight the feasibility of the MDT-led early arthritis model as an approach to address participation-based restrictions as part of routine healthcare that delivers on the rheumatology guidelines and was positively regarded by both service users and service providers. Conclusion: Findings provide important insights into the role of an innovative early MDT intervention approach in addressing client-identified participation needs in early IA. This model maps onto the Irish rheumatology model of care (HSE, 2018), ‘right place, right time’ approach but importantly includes a ‘right way’ approach which is identified as influential in the effective delivery of client-focussed and client-centred care. References: [1]HSE. (2018). Model of care for rheumatology in Ireland. Retrieved 15/09/2019, https://www.hse.ie/eng/about/who/cspd/ncps/rheumatology/achievements/model-of-care-for-rheumatology-in-ireland.pdf Acknowledgements: The authors, thank you to the research sites who facilitated this study & the service providers and service users who partook in the research. Thank you to Kildare Branch of Arthritis Ireland who provided funding support for the research Disclosure of Interests: None declared

Due to a typesetting error, 25 rows were omitted from Table 3 in the original version of this Data Descriptor. These missing rows correspond to the following sample names:

ABSTRACTBacteria and eukaryotes produce the reactive oxygen species superoxide both within and outside the cell. Although superoxide is typically associated with the detrimental and sometimes fatal effects of oxidative stress, it has also been shown to be involved in a range of essential biochemical processes, including cell signaling, growth, differentiation, and defense. Light‐independent extracellular superoxide production has been shown to be widespread among many marine heterotrophs and phytoplankton, but the extent to which this trait is relevant to marine microbial physiology and ecology throughout the global ocean is unknown. Here, we investigate the dark extracellular superoxide production of five groups of organisms that are geographically widespread and represent some of the most abundant organisms in the global ocean. These include Prochlorococcus, Synechococcus, Pelagibacter, Phaeocystis, and Geminigera. Cell‐normalized net extracellular superoxide production rates ranged seven orders of magnitude, from undetectable to 14,830 amol cell−1 h−1, with the cyanobacterium Prochlorococcus being the lowest producer and the cryptophyte Geminigera being the most prolific producer. Extracellular superoxide production exhibited a strong inverse relationship with cell number, pointing to a potential role in cell signaling. We demonstrate that rapid, cell‐number–dependent changes in the net superoxide production rate by Synechococcus and Pelagibacter arose primarily from changes in gross production of extracellular superoxide, not decay. These results expand the relevance of dark extracellular superoxide production to key marine microbes of the global ocean, suggesting that superoxide production in marine waters is regulated by a diverse suite of marine organisms in both dark and sunlit waters.

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.

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 co-culture, 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.