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.

Molecular and functional characterization of unique thermo-halophilic thioredoxin from the metagenome of an exotic environment.

The lower convective layer (LCL) at Atlantis II brine pool of the Red sea represents one of the exceptional, unique ecosystems. Thioredoxin is a multi-functional antioxidant redox protein that has a crucial role in various vital cellular processes. In the current study, a functional metagenomics approach was used to isolate and characterize thioredoxin from the LCL of Atlantis II Deep brine pool (Trx-ATII). From the metagenomic DNA of the LCL, the thioredoxin gene was directly retrieved and sequenced. Sequence analysis showed that the gene belonged to thioredoxin-like superfamily with classical Trx motif (-CXXC-). Phylogenetic analysis revealed that Trx-ATII was closely related to Trx of Prochlorococcus marinus with a maximum identity of 86%. Successfully, Trx-ATII was cloned and expressed in E. coli, where the purified protein had M.wt of 16 kDa. Characterization studies revealed that Trx-ATII protein is halophilic; can tolerate up to 2.5 M NaCl and thermostable, where 90% of its activity was retained at 60 °C. Trx-ATII can reduce both DTNB and insulin disulfide- containing substrates. In conclusion, a unique thioredoxin protein was isolated from a harsh environment that can maintain its activity under extreme conditions of salinity and temperature as a promising redox protein for biotechnological applications.

Structural intermediates of a DNA-ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation.

DNA ligases join adjacent 5' phosphate (5'P) and 3' hydroxyl (3'OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase-DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3'OH terminus for nucleophilic attack on the 5'P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

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.

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. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing intraspecific allocation of ecologically important functions. Nitrate assimilation genes are absent in basal lineages 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, gene loss, and homologous recombination. 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.

The Enzymology of Prochlorosin Biosynthesis.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides containing thioether cross-links formed through addition of a cysteine to a dehydroalanine (to form lanthionine) or to a dehydrobutyrine (to form 3-methyllanthionine). Genome sequencing of marine cyanobacteria lead to the discovery of 1.6 million open reading frames encoding lanthipeptides. In many cases, a genome encodes a single lanthipeptide synthetase, but a large number of substrates. The enzymatic modification process in Prochlorococcus MIT9313 has been reconstituted in vitro, and a variety of experimental approaches have been used to try and understand how one enzyme is capable of modifying 30 different substrates. The methods used to characterize this system will be described along with a brief genomic description of the lanthipeptide landscape found in Prochlorococcus and Synechococcus.

Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria.

Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511.

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.

Product Formation by the Promiscuous Lanthipeptide Synthetase ProcM is under Kinetic Control.

Lanthipeptides are natural products that belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain characteristic lanthionine (Lan) or methyllanthionine (MeLan) structures that contribute to their diverse biological activities. Despite its structurally diverse set of 30 substrates, the highly substrate-tolerant lanthipeptide synthetase ProcM is shown to display high selectivity for formation of a single product from selected substrates. Mutation of the active site zinc ligands to alanine or the unique zinc ligand Cys971 to histidine resulted in a decrease of the cyclization rate, especially for the second cyclization of the substrates ProcA1.1, ProcA2.8, and ProcA3.3. Surprisingly, for ProcA3.3 these mutations also altered the regioselectivity of cyclization resulting in a new major product. ProcM was not able to correct the ring topology of incorrectly cyclized intermediates and products, suggesting that thermodynamic control is not operational. Collectively, the data in this study suggest that the high regioselectivity of product formation is governed by the selectivity of the initially formed ring.

Analysis and elucidation of phosphoenolpyruvate carboxylase in cyanobacteria.

Phosphoenolpyruvate carboxylase (PEPC) a cytosolic enzyme of higher plants is also found in bacteria and cyanobacteria. Genetic and biochemical investigations have indicated that there are several isoforms of PEPC belonging to C3; C3/C4 and C4 groups but, the evolution of PEPC in cyanobacteria is not yet understood. The present study opens up an opportunity to understand the isoforms and functions of PEPC in cyanobacteria. The variations observed in PEPC among lower and higher orders of cyanobacteria, suggests convergent evolution of PEPC. There is a specific PEPC phosphorylation residue 'serine' at the N-terminus and PEPC determinant residue 'serine' at the C-terminal that facilitates high affinity for substrate binding. These residues were unique to higher orders of cyanobacteria, but, not in lower orders and other prokaryotes. The different PEPC forms of cyanobacteria were investigated for their kinetic properties with phosphoenolpyruvate as the substrate and the findings corroborated well with the in silico findings. In vitro enzymatic study of cyanobacteria belonging to three different orders demonstrated the role of aspartate as an allosteric effector, which inhibited PEPC by interacting with the highly conserved residues in the active site. The differences in mode of inhibition among the different order, thus, give a fair picture about the cyanobacterial PEPCs. The higher orders appear to possess the sequence coordinates and functionally conserved residues similar to isoforms of C4 type higher plants, whereas isoforms of PEPC of the lower orders did not resemble either that of C3 or C4 plants.

The minimal CO2-concentrating mechanism of Prochlorococcus spp. MED4 is effective and efficient.

As an oligotrophic specialist, Prochlorococcus spp. has streamlined its genome and metabolism including the CO2-concentrating mechanism (CCM), which serves to elevate the CO2 concentration around Rubisco. The genomes of Prochlorococcus spp. indicate that they have a simple CCM composed of one or two HCO3(-) pumps and a carboxysome, but its functionality has not been examined. Here, we show that the CCM of Prochlorococcus spp. is effective and efficient, transporting only two molecules of HCO3(-) per molecule of CO2 fixed. A mechanistic, numerical model with a structure based on the CCM components present in the genome is able to match data on photosynthesis, CO2 efflux, and the intracellular inorganic carbon pool. The model requires the carboxysome shell to be a major barrier to CO2 efflux and shows that excess Rubisco capacity is critical to attaining a high-affinity CCM without CO2 recovery mechanisms or high-affinity HCO3(-) transporters. No differences in CCM physiology or gene expression were observed when Prochlorococcus spp. was fully acclimated to high-CO2 (1,000 µL L(-1)) or low-CO2 (150 µL L(-1)) conditions. Prochlorococcus spp. CCM components in the Global Ocean Survey metagenomes were very similar to those in the genomes of cultivated strains, indicating that the CCM in environmental populations is similar to that of cultured representatives.

Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

Mechanistic studies on the substrate-tolerant lanthipeptide synthetase ProcM.

Lanthipeptides are a class of post-translationally modified peptide natural products. They contain lanthionine (Lan) and methyllanthionine (MeLan) residues, which generate cross-links and endow the peptides with various biological activities. The mechanism of a highly substrate-tolerant lanthipeptide synthetase, ProcM, was investigated herein. We report a hybrid ligation strategy to prepare a series of substrate analogues designed to address a number of mechanistic questions regarding catalysis by ProcM. The method utilizes expressed protein ligation to generate a C-terminal thioester of the leader peptide of ProcA, the substrate of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide. This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide-alkyne cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high substrate tolerance of ProcM. Finally, the enzyme was capable of exchanging protons at the α-carbon of MeLan, suggesting that ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM, which may aid future efforts in lanthipeptide engineering.

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex.

Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP·AlF(3)-stabilized) transition state complex between the DPOR components L(2) and (NB)(2) from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L(2) and (NB)(2). Upon complex formation, substantial ATP-dependent conformational rearrangements of L(2) trigger the protein-protein interactions with (NB)(2) as well as the electron transduction via redox-active [4Fe-4S] clusters. We also present the identification of artificial "small-molecule substrates" of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.

Opposite chirality of α-carotene in unusual cyanobacteria with unique chlorophylls, Acaryochloris and Prochlorococcus.

Among all photosynthetic and non-photosynthetic prokaryotes, only cyanobacterial species belonging to the genera Acaryochloris and Prochlorococcus have been reported to synthesize α-carotene. We reviewed the carotenoids, including their chirality, in unusual cyanobacteria containing diverse Chls. Predominantly Chl d-containing Acaryochloris (two strains) and divinyl-Chl a and divinyl-Chl b-containing Prochlorococcus (three strains) contained ß-carotene and zeaxanthin as well as α-carotene, whereas Chl b-containing Prochlorothrix (one strain) and Prochloron (three isolates) contained only ß-carotene and zeaxanthin but no α-carotene as in other cyanobacteria. Thus, the capability to synthesize α-carotene seemed to have been acquired only by Acaryochloris and Prochlorococcus. In addition, we unexpectedly found that α-carotene in both cyanobacteria had the opposite chirality at C-6': (6'S)-chirality in Acaryochloris and normal (6'R)-chirality in Prochlorococcus, as reported in some green algae and land plants. The results represent the first evidence for the natural occurrence and biosynthesis of (6'S)-α-carotene. All the zeaxanthins in these species were of the usual (3R,3'R)-chirality. Therefore, based on the identification of the carotenoids and genome sequence data, we propose a biosynthetic pathway for the carotenoids, particularly α-carotene, including the participating genes and enzymes.

Temporal orchestration of glycogen synthase (GlgA) gene expression and glycogen accumulation in the oceanic picoplanktonic cyanobacterium Synechococcus sp. strain WH8103.

Glycogen is accumulated during the latter half of the diel cycle in Synechococcus sp. strain WH8103 following a midday maximum in glgA (encoding glycogen synthase) mRNA abundance. This temporal pattern is quite distinct from that of Prochlorococcus and may highlight divergent regulatory control of carbon/nitrogen metabolism in these closely related picocyanobacteria.

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Antisense RNA protects mRNA from RNase E degradation by RNA-RNA duplex formation during phage infection.

The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg(2+), pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

Comparative analysis of carboxysome shell proteins.

Carboxysomes are metabolic modules for CO(2) fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysome's icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO(2) fixation in other organisms or creating novel biological nanostructures.

Prevalence of a calcium-based alkaline phosphatase associated with the marine cyanobacterium Prochlorococcus and other ocean bacteria.

Phosphate plays a key role in regulating primary productivity in several regions of the world's oceans and here dissolved organic phosphate can be an important phosphate source. A key enzyme for utilizing dissolved organic phosphate is alkaline phosphatase and the phoA-type of this enzyme has a zinc cofactor. As the dissolved zinc concentration is low in phosphate depleted environments, this has led to the hypothesis that some phytoplankton may be zinc-P co-limited. Recently, it was shown that many marine bacteria contain an alternative form of alkaline phosphatase called phoX, but it is unclear which marine lineages carry this enzyme. Here, we describe the occurrence in low phosphate environments of phoX that is associated with uncultured Prochlorococcus and SAR11 cells. Through heterologous expression, we demonstrate that phoX encodes an active phosphatase with a calcium cofactor. The enzyme also functions with magnesium and copper, whereas cobalt, manganese, nickel and zinc inhibit enzyme activity to various degrees. We also find that uncultured SAR11 cells and cyanophages contain a different alkaline phosphatase related to a variant present in several Prochlorococcus isolates. Overall, the results suggest that many bacterial lineages including Prochlorococcus and SAR11 may not be subject to zinc-P co-limitation.