Marine Cyanobacterium Prochlorococcus Research Articles

Direct interaction between marine cyanobacteria mediated by nanotubes.

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

Structure and assembly of the α-carboxysome in the marine cyanobacterium Prochlorococcus.

Carboxysomes are bacterial microcompartments that encapsulate the enzymes RuBisCO and carbonic anhydrase in a proteinaceous shell to enhance the efficiency of photosynthetic carbon fixation. The self-assembly principles of the intact carboxysome remain elusive. Here we purified α-carboxysomes from Prochlorococcus and examined their intact structures using single-particle cryo-electron microscopy to solve the basic principles of their shell construction and internal RuBisCO organization. The 4.2 Å icosahedral-like shell structure reveals 24 CsoS1 hexamers on each facet and one CsoS4A pentamer at each vertex. RuBisCOs are organized into three concentric layers within the shell, consisting of 72, 32 and up to 4 RuBisCOs at the outer, middle and inner layers, respectively. We uniquely show how full-length and shorter forms of the scaffolding protein CsoS2 bind to the inner surface of the shell via repetitive motifs in the middle and C-terminal regions. Combined with previous reports, we propose a concomitant 'outside-in' assembly principle of α-carboxysomes: the inner surface of the self-assembled shell is reinforced by the middle and C-terminal motifs of the scaffolding protein, while the free N-terminal motifs cluster to recruit RuBisCO in concentric, three-layered spherical arrangements. These new insights into the coordinated assembly of α-carboxysomes may guide the rational design and repurposing of carboxysome structures for improving plant photosynthetic efficiency.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Contrasting Marine Microbial Communities of the Fram Strait with the First Confirmed Record of Cyanobacteria Prochlorococcus marinus in the Arctic Region.

The seawater microbiome is crucial in marine ecosystems because of its role in food chains and biogeochemical cycles; thus, we studied the composition of the pelagic marine microbiome collected in the upper 50 m on the opposite sides of Fram Strait: Spitsbergen and Greenland shelves. We found out that it differed significantly, with salinity being the main environmental variable responsible for these differences. The Spitsbergen shelf was dominated by Atlantic Waters, with a rather homogenous water column in terms of salinity and temperature down to 300 m; hence, the marine microbial community was also homogenous at all sampled depths (0, 25, 50 m). On the contrary, stations on the Greenland shelf were exposed to different water masses of both Arctic and Atlantic origin, which resulted in a more diverse microbial community there. Unexpectedly, for the very first time, we identified cyanobacterium Prochlorococcus marinus in Arctic waters (Spitsbergen shelf, 75-77° N). Till now, the distribution of this cyanobacteria in oceans has been described only between 40° N and 40° S. Considering the accelerated rate of climate warming in the Arctic, our results indicated that the seawater microbiome can be viewed as an amplifier of global change and that the Atlantification is in progress.

Phytoplankton Producer Species and Transformation of Released Compounds over Time Define Bacterial Communities following Phytoplankton Dissolved Organic Matter Pulses.

Phytoplankton-bacterium interactions are mediated, in part, by phytoplankton-released dissolved organic matter (DOMp). Two factors that shape the bacterial community accompanying phytoplankton are (i) the phytoplankton producer species, defining the initial composition of released DOMp, and (ii) the DOMp transformation over time. We added phytoplankton DOMp from the diatom Skeletonema marinoi and the cyanobacterium Prochlorococcus marinus MIT9312 to natural bacterial communities from the eastern Mediterranean and determined the bacterial responses over a time course of 72 h in terms of cell numbers, bacterial production, alkaline phosphatase activity, and changes in active bacterial community composition based on rRNA amplicon sequencing. Both DOMp types were demonstrated to serve the bacterial community as carbon and, potentially, phosphorus sources. Bacterial communities in diatom-derived DOM treatments maintained higher Shannon diversities throughout the experiment and yielded higher bacterial production and lower alkaline phosphatase activity compared to cyanobacterium-derived DOM after 24 h of incubation (but not after 48 and 72 h), indicating greater bacterial usability of diatom-derived DOM. Bacterial communities significantly differed between DOMp types as well as between different incubation times, pointing to a certain bacterial specificity for the DOMp producer as well as a successive utilization of phytoplankton DOM by different bacterial taxa over time. The highest differences in bacterial community composition with DOMp types occurred shortly after DOMp additions, suggesting a high specificity toward highly bioavailable DOMp compounds. We conclude that phytoplankton-associated bacterial communities are strongly shaped by the phytoplankton producer as well as the transformation of its released DOMp over time. IMPORTANCE Phytoplankton-bacterium interactions influence biogeochemical cycles of global importance. Phytoplankton photosynthetically fix carbon dioxide and subsequently release the synthesized compounds as dissolved organic matter (DOMp), which becomes processed and recycled by heterotrophic bacteria. Yet the importance of phytoplankton producers in combination with the time-dependent transformation of DOMp compounds on the accompanying bacterial community has not been explored in detail. The diatom Skeletonema marinoi and the cyanobacterium Prochlorococcus marinus MIT9312 belong to globally important phytoplankton genera, and our study revealed that DOMp of both species was selectively incorporated by the bacterial community. The producer species had the highest impact shortly after DOMp appropriation, and its effect diminished over time. Our results improve the understanding of the dynamics of organic matter produced by phytoplankton in the oceans as it is utilized and modified by cooccurring bacteria.

Production, homology modeling and mutagenesis studies on GlcH glucose transporter from Prochlorococcus sp. strain SS120

The marine cyanobacterium Prochlorococcus is one of the main primary producers on Earth, which can take up glucose by using the high affinity, multiphasic transporter GlcH. We report here the overexpression of glcH from Prochlorococcus marinus strain SS120 in Escherichia coli. Modeling studies of GlcH using the homologous MelB melibiose transporter from Salmonella enterica serovar Typhimurium showed high conservation at the overall fold. We observed that an important structural interaction, mediated by a strong hydrogen bond between D8 and R141, is conserved in Prochlorococcus, although the corresponding amino acids in MelB from Salmonella are different. Biased docking studies suggested that when glucose reaches the pocket of the transporter and interacts with D8 and R141, the hydrogen bond network in which these residues are involved could be disrupted, favoring a conformational change with the subsequent translocation of the glucose molecule towards the cytoplasmic region of the pmGlcH structure. Based on these theoretical predictions and on the conservation of N117 and W348 in other MelB structures, D8, N117, R141 and W348 were mutated to glycine residues. Their key role in glucose transport was evaluated by glucose uptake assays. N117G and W348G mutations led to 17 % decrease in glucose uptake, while D8G and R141G decreased the glucose transport by 66 % and 92 % respectively. Overall, our studies provide insights into the Prochlorococcus 3D-structure of GlcH, paving the way for further analysis to understand the features which are involved in the high affinity and multiphasic kinetics of this transporter.

Differential Timing for Glucose Assimilation in Prochlorococcus and Coexistent Microbial Populations in the North Pacific Subtropical Gyre.

ABSTRACTThe marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here, we investigated whole microbial community and group-specific primary production and glucose assimilation using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis. Our results showed a diel variation for glucose assimilation in Prochlorococcus, with maximum assimilation at midday and minimum at midnight (~2-fold change), which was different from that of the total microbial community. This suggests that the timing in glucose assimilation in Prochlorococcus is coupled to photosynthetic light reactions producing energy, it being more convenient for Prochlorococcus to show maximum glucose uptake precisely when the rest of microbial populations have their minimum glucose uptake. Many transcriptional responses to glucose enrichment occurred after 12- and 24-h periods, but community composition did not change. High-light Prochlorococcus strains were the most impacted by glucose addition, with transcript-level increases observed for genes in pathways for glucose metabolism, such as the pentose phosphate pathway, the Entner-Doudoroff pathway, glycolysis, respiration, and glucose transport. While Prochlorococcus C assimilation from glucose represented less than 0.1% of the bacterium’s photosynthetic C fixation, increased assimilation during the day and glcH gene upregulation upon glucose enrichment indicate an important role of mixotrophic C assimilation by natural populations of Prochlorococcus.IMPORTANCE Several studies have demonstrated that Prochlorococcus, the most abundant photosynthetic organism on Earth, can assimilate organic molecules, such as amino acids, amino sugars, ATP, phosphonates, and dimethylsulfoniopropionate. This autotroph can also assimilate small amounts of glucose, supporting the hypothesis that Prochlorococcus is mixotrophic. Our results show, for the first time, a diel variability in glucose assimilation by natural populations of Prochlorococcus with maximum assimilation during midday. Based on our previous results, this indicates that Prochlorococcus could maximize glucose uptake by using ATP made during the light reactions of photosynthesis. Furthermore, Prochlorococcus showed a different timing of glucose assimilation from the total population, which may offer considerable fitness advantages over competitors “temporal niches.” Finally, we observed transcriptional changes in some of the genes involved in carbon metabolism, suggesting that Prochlorococcus can use both pathways previously proposed in cyanobacteria to metabolize glucose.

Prochlorococcus extracellular vesicles: molecular composition and adsorption to diverse microbes.

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

Investigating the Impact of Cerium Oxide Nanoparticles Upon the Ecologically Significant Marine Cyanobacterium Prochlorococcus

Cerium oxide nanoparticles (nCeO2) are used at an ever-increasing rate, however, their impact within the aquatic environment remains uncertain. Here, we expose the ecologically significant marine cyanobacterium Prochlorococcus sp. MED4 to nCeO2 at a wide range of concentrations (1 μg L–1 to 100 mg L–1) under simulated natural and nutrient rich growth conditions. Flow cytometric analysis of cyanobacterial populations displays the potential of nCeO2 (100 μg L–1) to significantly reduce Prochlorococcus cell density in the short-term (72 h) by up to 68.8% under environmentally relevant conditions. However, following longer exposure (240 h) cyanobacterial populations are observed to recover under simulated natural conditions. In contrast, cell-dense cultures grown under optimal conditions appear more sensitive to exposure during extended incubation, likely as a result of increased rate of encounter between cyanobacteria and nanoparticles at high cell densities. Exposure to supra-environmental nCeO2 concentrations (i.e., 100 mg L–1) resulted in significant declines in cell density up to 95.7 and 82.7% in natural oligotrophic seawater and nutrient enriched media, respectively. Observed cell decline is associated with extensive aggregation behaviour of nCeO2 upon entry into natural seawater, as observed by dynamic light scattering (DLS), and hetero-aggregation with cyanobacteria, confirmed by fluorescent microscopy. Hence, the reduction of planktonic cells is believed to result from physical removal due to co-aggregation and co-sedimentation with nCeO2 rather than by a toxicological and cell death effect. The observed recovery of the cyanobacterial population under simulated natural conditions, and likely reduction in nCeO2 bioavailability as nanoparticles aggregate and undergo sedimentation in saline media, means that the likely environmental risk of nCeO2 in the marine environment appears low.

Clocks, Hourglasses, and History‐dependent Clocks

Many organisms have free-running circadian clocks to anticipate the time of the day, including the Kai protein-based clock in the cyanobacterium Synechococcus elongatus. However, other species have an ‘hourglass’ timer, such as the cyanobacterium Prochlorococcus marinus. What conditions favor an hourglass timer or a free running clock over the other? We argue that this evolutionary choice is influenced by the noise levels in the external environment (e.g., light fluctuations due to weather) relative to noise levels in the internal environment of the cell (e.g., molecular fluctuations). Besides different clock dynamics in different species, we find that the clock of a single species, S. elongatus, exploits a clock-metabolic coupling to tune its clock dynamics towards an hourglass timer to deal with changing noise levels.

Dynamic Allocation of Carbon Storage and Nutrient-Dependent Exudation in a Revised Genome-Scale Model of Prochlorococcus.

Microbial life in the oceans impacts the entire marine ecosystem, global biogeochemistry and climate. The marine cyanobacterium Prochlorococcus, an abundant component of this ecosystem, releases a significant fraction of the carbon fixed through photosynthesis, but the amount, timing and molecular composition of released carbon are still poorly understood. These depend on several factors, including nutrient availability, light intensity and glycogen storage. Here we combine multiple computational approaches to provide insight into carbon storage and exudation in Prochlorococcus. First, with the aid of a new algorithm for recursive filling of metabolic gaps (ReFill), and through substantial manual curation, we extended an existing genome-scale metabolic model of Prochlorococcus MED4. In this revised model (iSO595), we decoupled glycogen biosynthesis/degradation from growth, thus enabling dynamic allocation of carbon storage. In contrast to standard implementations of flux balance modeling, we made use of forced influx of carbon and light into the cell, to recapitulate overflow metabolism due to the decoupling of photosynthesis and carbon fixation from growth during nutrient limitation. By using random sampling in the ensuing flux space, we found that storage of glycogen or exudation of organic acids are favored when the growth is nitrogen limited, while exudation of amino acids becomes more likely when phosphate is the limiting resource. We next used COMETS to simulate day-night cycles and found that the model displays dynamic glycogen allocation and exudation of organic acids. The switch from photosynthesis and glycogen storage to glycogen depletion is associated with a redistribution of fluxes from the Entner–Doudoroff to the Pentose Phosphate pathway. Finally, we show that specific gene knockouts in iSO595 exhibit dynamic anomalies compatible with experimental observations, further demonstrating the value of this model as a tool to probe the metabolic dynamic of Prochlorococcus.

Novel Integrative Elements and Genomic Plasticity in Ocean Ecosystems

Horizontal gene transfer accelerates microbial evolution, promoting diversification and adaptation. The globally abundant marine cyanobacterium Prochlorococcus has a highly streamlined genome with frequent gene exchange reflected in its extensive pangenome. The source of its genomic variability, however, remains elusive since most cells lack the common mechanisms that enable horizontal gene transfer, including conjugation, transformation, plasmids and prophages. Examining 623 genomes, we reveal a diverse system of mobile genetic elements – cargo-carrying transposons we named tycheposons – that shape Prochlorococcus’ genomic plasticity. The excision and integration of tycheposons at seven tRNA genes drive the remodeling of larger genomic islands containing most of Prochlorococcus’ flexible genes. Most tycheposons carry genes important for niche differentiation through nutrient acquisition; others appear similar to phage parasites. Tycheposons are highly enriched in extracellular vesicles and phage particles in ocean samples, suggesting efficient routes for their dispersal, transmission and propagation. Supported by evidence for similar elements in other marine microbes, our work underpins the role of vesicle- and virus-mediated transfer of mobile genetic elements in the diversification and adaptation of microbes in dilute aquatic environments – adding a significant piece to the puzzle of what governs microbial evolution in the planet’s largest habitat.

Mechanisms of silver nanoparticle toxicity on the marine cyanobacterium Prochlorococcus under environmentally-relevant conditions

Global demand for silver nanoparticles (AgNPs), and their inevitable release into the environment, is rapidly increasing. AgNPs display antimicrobial properties and have previously been recorded to exert adverse effects upon marine phytoplankton. However, ecotoxicological research is often compromised by the use of non-ecologically relevant conditions, and the mechanisms of AgNP toxicity under environmental conditions remains unclear. To examine the impact of AgNPs on natural marine communities, a natural assemblage was exposed to citrate-stabilised AgNPs. Here, investigation confirmed that the marine dominant cyanobacteria Prochlorococcus is particularly sensitive to AgNP exposure. Whilst Prochlorococcus represents the most abundant photosynthetic organism on Earth and contributes significantly to global primary productivity, little ecotoxicological research has been carried out on this cyanobacterium. To address this, Prochlorococcus was exposed to citrate-stabilised AgNPs, as well as silver in its ionic form (Ag2SO4), under simulated natural conditions. Both AgNPs and ionic silver were observed to reduce Prochlorococcus populations by over 90% at concentrations ≥10 μg L−1, representing the upper limit of AgNP concentrations predicted in the environment (10 μg L−1). Longer-term assessment revealed this to be a perturbation which was irreversible. Through use of quenching agents for superoxide and hydrogen peroxide, alongside incubations with ionic silver, it was revealed that AgNP toxicity likely arises from synergistic effects of toxic superoxide species generation and leaching of ionic silver. The extent of toxicity was strongly dependent on cell density, and completely mitigated in more cell-dense cultures. Hence, the calculation and reporting of the particle-to-cell ratio reveals that this parameter is effective for standardisation of experimental work, and allows for direct comparison between studies where cell density may vary. Given the key role that marine cyanobacteria play in global primary production and biogeochemical cycling, their higher susceptibility to AgNP exposure is a concern in hotspots of pollution.

Prochlorococcus phage ferredoxin: structural characterization and electron transfer to cyanobacterial sulfite reductases

Marine cyanobacteria are infected by phages whose genomes encode ferredoxin (Fd) electron carriers. These Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, but it is unclear how the biophysical properties and partner specificities of phage Fds relate to those of photosynthetic organisms. Here, results of a bioinformatics analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects the cyanobacterium Prochlorococcus marinus, revealed high levels of similarity to cyanobacterial Fds (root mean square deviations of ≤0.5 Å). Additionally, pssm2-Fd exhibited a low midpoint reduction potential (-336 mV versus a standard hydrogen electrode), similar to other photosynthetic Fds, although it had lower thermostability (Tm = 28 °C) than did many other Fds. When expressed in an Escherichia coli strain deficient in sulfite assimilation, pssm2-Fd complemented bacterial growth when coexpressed with a P. marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high levels of structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterially encoded oxidoreductases.

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

BackgroundAldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability.ResultsHere, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR-based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo.ConclusionThis work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

Bio-Aerosols Negatively Affect Prochlorococcus in Oligotrophic Aerosol-Rich Marine Regions

The marine cyanobacterium Prochlorococcus is a dominant photoautotroph in many oligotrophic Low-Nutrients-Low-Chlorophyll (LNLC) regions. While the chemical impact of aerosols upon interaction with surface seawater was documented in numerous studies, we show that Prochlorococcus cells are affected also by bio-aerosols (potentially biological agents in the dust/aerosols such as membrane-bound extracellular vesicles, small-size bacteria and/or viruses), resulting in lower surface seawater abundances in the oligotrophic Mediterranean Sea. We conducted experimental amendments of ‘live’ aerosol/dust particles and aerosol filtrates (<0.22-µm) to surface Southeastern Mediterranean seawater or to pure Prochlorococcus cultures (MED4). Results show a significant decline in cell biomass (<90%), while UV-sterilized aerosols elicited a much weaker and non-significant response (~10%). We suggest that the difference is due to a negative effect of bio-aerosols specific to Prochlorococcus. Accordingly, the dominance of Synechococcus over Prochlorococcus throughout the surface Mediterranean Sea (observed mainly in spring when atmospheric aerosol levels are relatively high) and the lack of spatial westward gradient in Prochlorococcus biomass as typically observed for chlorophyll-a or other cyanobacteria may be attributed, at least to some extent, to the impact of bio-aerosol deposition across the basin. Predictions for enhanced desertification and increased dust emissions may intensify the transport and potential impact of bio-aerosols in LNLC marine systems.

Clocks, Hourglasses, and History‐dependent Clocks

Many organisms have free‐running circadian clocks to anticipate the time of the day, including the Kai protein‐based clock in the cyanobacterium Synechococcus elongatus. However, other species have an ‘hourglass’ timer, such as the cyanobacterium Prochlorococcus marinus. What conditions favor an hourglass timer or a free running clock over the other? We argue that this evolutionary choice is influenced by the noise levels in the external environment (e.g., light fluctuations due to weather) relative to noise levels in the internal environment of the cell (e.g., molecular fluctuations). Besides different clock dynamics in different species, we find that the clock of a single species, S. elongatus, exploits a clock‐metabolic coupling to tune its clock dynamics towards an hourglass timer to deal with changing noise levels.Support or Funding InformationK.H. thanks the James S. McDonnell Foundation for support via a Postdoctoral Fellowship. A.M. thanks the Simons Foundation for support.

Toward a genetic system in the marine cyanobacterium Prochlorococcus.

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

Multi‐year dynamics of fine‐scale marine cyanobacterial populations are more strongly explained by phage interactions than abiotic, bottom‐up factors

Currently defined ecotypes in marine cyanobacteria Prochlorococcus and Synechococcus likely contain subpopulations that themselves are ecologically distinct. We developed and applied high-throughput sequencing for the 16S-23S rRNA internally transcribed spacer (ITS) to examine ecotype and fine-scale genotypic community dynamics for monthly surface water samples spanning 5 years at the San Pedro Ocean Time-series site. Ecotype-level structure displayed regular seasonal patterns including succession, consistent with strong forcing by seasonally varying abiotic parameters (e.g. temperature, nutrients, light). We identified tens to thousands of amplicon sequence variants (ASVs) within ecotypes, many of which exhibited distinct patterns over time, suggesting ecologically distinct populations within ecotypes. Community structure within some ecotypes exhibited regular, seasonal patterns, but not for others, indicating other more irregular processes such as phage interactions are important. Network analysis including T4-like phage genotypic data revealed distinct viral variants correlated with different groups of cyanobacterial ASVs including time-lagged predator-prey relationships. Variation partitioning analysis indicated that phage community structure more strongly explains cyanobacterial community structure at the ASV level than the abiotic environmental factors. These results support a hierarchical model whereby abiotic environmental factors more strongly shape niche partitioning at the broader ecotype level while phage interactions are more important in shaping community structure of fine-scale variants within ecotypes.

Two aspartate residues close to the lesion binding site of Agrobacterium (6-4) photolyase are required for Mg2+ stimulation of DNA repair.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg2+ . We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA-binding site are essential for the effect of Mg2+ . Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+ . In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron-sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron-sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment.