Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

Common environmental stress responses in a model marine diatom.

Marine planktonic diatoms are among the most important contributors to phytoplankton blooms and marine net primary production. Their ecological success has been attributed to their ability to rapidly respond to changing environmental conditions. Here, we report common molecular mechanisms used by the model marine diatom Thalassiosira pseudonana to respond to 10 diverse environmental stressors using RNA-Seq analysis. We identify a specific subset of 1076 genes that are differentially expressed in response to stressors that induce an imbalance between energy or resource supply and metabolic capacity, which we termed the diatom environmental stress response (d-ESR). The d-ESR is primarily composed of genes that maintain proteome homeostasis and primary metabolism. Photosynthesis is strongly regulated in response to environmental stressors but chloroplast-encoded genes were predominantly upregulated while the nuclear-encoded genes were mostly downregulated in response to low light and high temperature. In aggregate, these results provide insight into the molecular mechanisms used by diatoms to respond to a range of environmental perturbations and the unique role of the chloroplast in managing environmental stress in diatoms. This study facilitates our understanding of the molecular mechanisms underpinning the ecological success of diatoms in the ocean.

ATR Plays a Direct Antiapoptotic Role at Mitochondria, which Is Regulated by Prolyl Isomerase Pin1.

ATR, a PI3K-like protein kinase, plays a key role in regulating DNA damage responses. Its nuclear checkpoint kinase function is well documented, but little is known about its function outside the nucleus. Here we report that ATR has an antiapoptotic activity at mitochondria in response to UV damage, and this activity is independent of its hallmark checkpoint/kinase activity and partner ATRIP. ATR contains a BH3-like domain that allows ATR-tBid interaction at mitochondria, suppressing cytochrome c release and apoptosis. This mitochondrial activity of ATR is downregulated by Pin1 that isomerizes ATR from cis-isomer to trans-isomer at the phosphorylated Ser428-Pro429 motif. However, UV inactivates Pin1 via DAPK1, stabilizing the pro-survival cis-isomeric ATR. In contrast, nuclear ATR remains in the trans-isoform disregarding UV. This cytoplasmic response of ATR may provide a mechanism for the observed antiapoptotic role of ATR in suppressing carcinogenesis and its inhibition in sensitizing anticancer agents for killing of cancer cells.

Application of Nanomaterials in the Production of Biomolecules in Microalgae: A Review.

Nanomaterials (NMs) are becoming more commonly used in microalgal biotechnology to empower the production of algal biomass and valuable metabolites, such as lipids, proteins, and exopolysaccharides. It provides an effective and promising supplement to the existing algal biotechnology. In this review, the potential for NMs to enhance microalgal growth by improving photosynthetic utilization efficiency and removing reactive oxygen species is first summarized. Then, their positive roles in accumulation, bioactivity modification, and extraction of valuable microalgal metabolites are presented. After the application of NMs in microalgae cultivation, the extracted metabolites, particularly exopolysaccharides, contain trace amounts of NM residues, and thus, the impact of these residues on the functional properties of the metabolites is also evaluated. Finally, the methods for removing NM residues from the extracted metabolites are summarized. This review provides insights into the application of nanotechnology for sustainable production of valuable metabolites in microalgae and will contribute useful information for ongoing and future practice.

Electron-Deficient Polycyclic Molecules via Ring Fusion for n-Type Organic Electrochemical Transistors.

The primary challenge for n-type small-molecule organic electrochemical transistors (OECTs) is to improve their electron mobilities and thus the key figure of merit µC*. Nevertheless, few reports in OECTs have specially proposed to address this issue. Herein, we report a 10-ring-fused polycyclic π-system consisting of the core of naphthalene bis-isatin dimer and the terminal moieties of rhodanine, which features intramolecular noncovalent interactions, high π-delocalization and strong electron-deficient characteristics. We find that this extended π-conjugated system using the ring fusion strategy displays improved electron mobilities up to 0.043 cm2 V-1 s-1 compared to our previously reported small molecule gNR, and thereby leads to a remarkable µC* of 10.3 F cm-1 V-1 s-1 in n-type OECTs, which is the highest value reported to date for small-molecule OECTs. This work highlights the importance of π-conjugation extension in polycyclic-fused molecules for enhancing the performance of n-type small-molecule OECTs.

hDNA2 nuclease/helicase promotes centromeric DNA replication and genome stability.

DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric DNA replication and CENP-A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late-S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.

Conservation and architecture of housekeeping genes in the model marine diatom Thalassiosira pseudonana.

Housekeeping genes (HKGs) are constitutively expressed with low variation across tissues/conditions. They are thought to be highly conserved and fundamental to cellular maintenance, with distinctive genomic features. Here, we identify 1505 HKGs in the unicellular marine diatom Thalassiosira pseudonana based on an RNA-seq analysis of 232 samples taken under 12 experimental conditions over 0-72 h. We identify promising internal reference genes (IRGs) for T. pseudonana from the most stably expressed HKGs. A comparative analysis indicates < 18% of HKGs in T. pseudonana have orthologs in other eukaryotes, including other diatom species. Contrary to work on human tissues, T. pseudonana HKGs are longer than non-HKGs, due to elongated introns. More ancient HKGs tend to be shorter than more recent HKGs, and expression levels of HKGs decrease more rapidly with gene length relative to non-HKGs. Our results indicate that HKGs are highly variable across the tree of life and thus unlikely to be universally fundamental for cellular maintenance. We hypothesize that the distinct genomic features of HKGs of T. pseudonana may be a consequence of selection pressures associated with high expression and low variance across conditions.

ATR prevents Ca2+ overload-induced necrotic cell death through phosphorylation-mediated inactivation of PARP1 without DNA damage signaling.

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+ /ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid-induced necrotic death of cells including SH-SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site-specific phosphorylation inactivates PARP1, inhibiting ionophore-induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR-mediated anti-necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling.

Kovacikia minuta sp. nov. (Leptolyngbyaceae, Cyanobacteria), a new freshwater chlorophyll f-producing cyanobacterium.

A few groups of cyanobacteria have been characterized as having far-red light photoacclimation (FaRLiP) that results from chlorophyll f (Chl f) production. In this study, using a polyphasic approach, we taxonomically transferred the Cf. Leptolyngbya sp. CCNUW1 isolated from a shaded freshwater pond, which produces Chl f under far-red light, to the genus Kovacikia and named this taxon Kovacikia minuta sp. nov. This strain was morphologically similar to Leptolyngbya-like strains. The thin filaments were purplish-brown under white light but became grass green under far-red light. The 31-gene phylogeny grouped K. minuta CCNU0001 into order Synechococcales and family Leptolyngbyaceae. Phylogenetic analysis based on 16S rRNA gene sequences further showed that K. minuta CCNU0001 was clustered into Kovacikia with similarities of 97.2-97.4% to the recently reported type species of Kovacikia muscicola HA7619-LM3. Additionally, the internal transcribed spacer region between 16S-23S rRNA genes had a unique sequence and secondary structure compared with other Kovacikia strains and phylogenetically related taxa. Draft genome sequences of K. minuta CCNU0001 (8,564,336 bp) were assembled into one circular chromosome and two circular plasmids. A FaRLiP 20-gene cluster comprised two operons with the unique organization. In sum, K. minuta was established as a new species, and it is the first species reported to produce Chl f and for which a draft genome was produced in genus Kovacikia. This study expanded our knowledge regarding the diversity of Chl f-producing cyanobacteria in far-red light-enriched environments and provides important foundational information for future investigations of FaRLiP evolution in cyanobacteria.

A unique porin meditates iron-selective transport through cyanobacterial outer membranes.

Cyanobacteria are globally important primary producers and nitrogen fixers with high iron demands. Low ambient dissolved iron concentrations in many aquatic environments mean that these organisms must maintain sufficient and selective transport of iron into the cell. However, the nature of iron transport pathways through the cyanobacterial outer membrane remains obscure. Here we present multiple lines of experimental evidence that collectively support the existence of a novel class of substrate-selective iron porin, Slr1908, in the outer membrane of the cyanobacterium Synechocystis sp. PCC 6803. Elemental composition analysis and short-term iron uptake assays with mutants in Slr1908 reveal that this protein is primarily involved in inorganic iron uptake and contributes less to the accumulation of other metals. Homologues of Slr1908 are widely distributed in both freshwater and marine cyanobacteria, most notably in unicellular marine diazotrophs. Complementary experiments with a homologue of Slr1908 in Synechococcus sp. PCC 7002 restored the phenotype of Synechocystis knockdown mutants, showing that this siderophore producing species also possesses a porin with a similar function in Fe transport. The involvement of a substrate-selective porins in iron uptake may allow cyanobacteria to tightly control iron flux into the cell, particularly in environments where iron concentrations fluctuate.

Telomere and ATM Dynamics in CD4 T-Cell Depletion in Active and Virus-Suppressed HIV Infections.

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection.IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection.

Dynamic Photophysiological Stress Response of a Model Diatom to Ten Environmental Stresses.

Stressful environmental conditions can induce many different acclimation mechanisms in marine phytoplankton, resulting in a range of changes in their photophysiology. Here we characterize the common photophysiological stress response of the model diatom Thalassiosira pseudonana to ten environmental stressors and identify diagnostic responses to particular stressors. We quantify the magnitude and temporal trajectory of physiological parameters including the functional absorption cross-section of PSII (σPSII ), quantum efficiency of PSII, non-photochemical quenching (NPQ), cell volume, Chl a, and carotenoid (Car) content in response to nutrient starvation (nitrogen (N), phosphorus (P), silicon (Si), and iron (Fe)), changes in temperature, irradiance, pH, and reactive oxygen species (ROS) over 5 time points (0, 2, 6, 24, 72 h). We find changes in conditions: temperature, irradiance, and ROS, often result in the most rapid changes in photophysiological parameters (<2 h), and in some cases are followed by recovery. In contrast, nutrient starvation (N, P, Si, Fe) often has slower (6-72 h) but ultimately larger magnitude effects on many photophysiological parameters. Diagnostic changes include large increases in cell volume under Si-starvation, very large increases in NPQ under P-starvation, and large decreases in the σPSII under high light. The ultimate goal of this analysis is to facilitate and enhance the interpretation of fluorescence data and our understanding of phytoplankton photophysiology from laboratory and field studies.

A novel thyroid hormone receptor isoform, TRß2-46, promotes SKP2 expression and retinoblastoma cell proliferation.

Retinoblastoma is a childhood retinal tumor that develops from cone photoreceptor precursors in response to inactivating RB1 mutations and loss of functional RB protein. The cone precursor's response to RB loss involves cell type-specific signaling circuitry that helps to drive tumorigenesis. One component of the cone precursor circuitry, the thyroid hormone receptor ß2 (TRß2), enables the aberrant proliferation of diverse RB-deficient cells in part by opposing the down-regulation of S-phase kinase-associated protein 2 (SKP2) by the more widely expressed and tumor-suppressive TRß1. However, it is unclear how TRß2 opposes TRß1 to enable SKP2 expression and cell proliferation. Here, we show that in human retinoblastoma cells TRß2 mRNA encodes two TRß2 protein isoforms: a predominantly cytoplasmic 54-kDa protein (TRß2-54) corresponding to the well-characterized full-length murine Trß2 and an N-terminally truncated and exclusively cytoplasmic 46-kDa protein (TRß2-46) that starts at Met-79. Whereas TRß2 knockdown decreased SKP2 expression and impaired retinoblastoma cell cycle progression, re-expression of TRß2-46 but not TRß2-54 stabilized SKP2 and restored proliferation to an extent similar to that of ectopic SKP2 restoration. We conclude that TRß2-46 is an oncogenic thyroid hormone receptor isoform that promotes SKP2 expression and SKP2-dependent retinoblastoma cell proliferation.

Photosynthetic adaptation to light availability shapes the ecological success of bloom-forming cyanobacterium Pseudanabaena to iron limitation.

The poorly understood filamentous cyanobacterium Pseudanabaena is commonly epiphytic on Microcystis colonies and their abundances are often highly correlated during blooms. The response and adaptation of Microcystis to iron limitation have been extensively studied, but the strategies Pseudanabaena uses to respond to iron limitation are largely unknown. Here, physiological responses to iron limitation were compared between one Pseudanabaena and two Microcystis strains grown under different light intensities. The results showed that low-intensity light exacerbated, but high-intensity light alleviated, the negative effect of iron limitation on Pseudanabaena growth relative to two Microcystis strains. It was found that robust light-harvesting and photosynthetic efficiency allowed adaptation of Pseudanabaena to low light availability relative to two Microcystis strains only during iron sufficiency. The results also indicated that a larger investment in the photosynthetic antenna probably contributed to light/iron co-limitation of Pseudanabaena relative to two Microcystis strains under both light and iron limitation. Furthermore, the lower antenna pigments/chlorophyll a ratio and photosynthetic efficiency, and higher nonphotochemical quenching and saturation irradiance provided Pseudanabaena photoadaptation and photoprotection advantages over the two Microcystis strains under the high-light condition. The lower investment in antenna pigments of Pseudanabaena than the two Microcystis strains under high-light intensity is likely an efficient strategy for both saving iron quotas and decreasing photosensitivity. Therefore, when compared with Microcystis, the high plasticity of antenna pigments, along with the excellent photoadaptation and photoprotection ability of Pseudanabaena, probably ensures its ecological success under iron limitation when light is sufficient.

Widespread occurrence and unexpected diversity of red-shifted chlorophyll producing cyanobacteria in humid subtropical forest ecosystems.

Discovery of red-shifted chlorophyll d and f in cyanobacteria has opened up new avenues to estimate global carbon fixation driven by far-red light. Shaded habitats in humid subtropical forest ecosystems contain an increased proportion of far-red light components relative to residual white light. After an extensive survey of shaded ecosystems within subtropical forests, wide occurrence of red-shifted chlorophyll-producing cyanobacteria was demonstrated by isolated Chl f-producing and Chl d-containing cyanobacteria. Chl f-producing cyanobacteria were classified into the genera of Aphanocapsa and Chroococcidiopsis and two undescribed genera within Leptolyngbyaceae. Newly isolated Chl d-containing Acaryochloris sp. CCNUM4 showed the closest phylogenetic relationship with Acaryochloris species isolated from marine environments. Acaryochloris sp. CCNUM4 produced Chl d as major photopigment, and Chl f-producing cyanobacteria use Chl a under white light conditions but Chl a + f under far-red light conditions. Their habitats are widely distributed in subtropical forest ecosystems and varied from mosses on limestone to macrophyte and freshwater in the streams and ponds. This study presents a significant advance in the knowledge of distribution and diversity of red-shifted chlorophyll-producing cyanobacteria in terrestrial ecosystems. The results suggest that Chl f-producing and Chl d-containing cyanobacteria might be important primary producers in far-red light dominant niches worldwide.

Quantitative relations between interaction parameter, miscibility and function in organic solar cells.

Although it is known that molecular interactions govern morphology formation and purity of mixed domains of conjugated polymer donors and small-molecule acceptors, and thus largely control the achievable performance of organic solar cells, quantifying interaction-function relations has remained elusive. Here, we first determine the temperature-dependent effective amorphous-amorphous interaction parameter, χaa(T), by mapping out the phase diagram of a model amorphous polymer:fullerene material system. We then establish a quantitative 'constant-kink-saturation' relation between χaa and the fill factor in organic solar cells that is verified in detail in a model system and delineated across numerous high- and low-performing materials systems, including fullerene and non-fullerene acceptors. Our experimental and computational data reveal that a high fill factor is obtained only when χaa is large enough to lead to strong phase separation. Our work outlines a basis for using various miscibility tests and future simulation methods that will significantly reduce or eliminate trial-and-error approaches to material synthesis and device fabrication of functional semiconducting blends and organic blends in general.

Orange and red carotenoid proteins are involved in the adaptation of the terrestrial cyanobacterium Nostoc flagelliforme to desiccation.

The remarkable drought-resistance of the terrestrial cyanobacterium Nostoc flagelliforme (N. flagelliforme) has attracted attention for many years. In this study, we purified a group of red proteins that accumulate in dried field samples of N. flagelliforme. These red proteins contain canthaxanthin as the bound chromophore. Native-PAGE analysis revealed that the purified red proteins resolved into six visible red bands and were composed of four helical carotenoid proteins (HCPs), HCP1, HCP2, HCP3, and HCP6 (homologs to the N-terminal domain of the orange carotenoid protein (OCP)). Seven genes encode homologs of the OCP in the genome of N. flagelliforme: two full-length ocp genes (ocpx1 and ocpx2), four N-terminal domain hcp genes (hcp1, hcp2, hcp3, and hcp6), and one C-terminal domain ccp gene. The expression levels of hcp1, hcp2, and hcp6 were highly dependent on the water status of field N. flagelliforme samples, being downregulated during rehydration and upregulated during subsequent dehydration. Transcripts of ocpx2 were dominant in the dried field samples, which we confirmed by detecting the presence of OCPx2-derived peptides in the purified red proteins. The results shed light on the relationship between carotenoid-binding proteins and the desiccation resistance of terrestrial cyanobacteria, and the physiological functions of carotenoid-binding protein complexes in relation to desiccation are discussed.

Sycrp2 Is Essential for Twitching Motility in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

Outer Membrane Iron Uptake Pathways in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanobacteria are foundational drivers of global nutrient cycling, with high intracellular iron (Fe) requirements. Fe is found at extremely low concentrations in aquatic systems, however, and the ways in which cyanobacteria take up Fe are largely unknown, especially the initial step in Fe transport across the outer membrane. Here, we identified one TonB protein and four TonB-dependent transporters (TBDTs) of the energy-requiring Fe acquisition system and six porins of the passive diffusion Fe uptake system in the model cyanobacterium Synechocystis sp. strain PCC 6803. The results experimentally demonstrated that TBDTs not only participated in organic ferri-siderophore uptake but also in inorganic free Fe (Fe') acquisition. 55Fe uptake rate measurements showed that a TBDT quadruple mutant acquired Fe at a lower rate than the wild type and lost nearly all ability to take up ferri-siderophores, indicating that TBDTs are critical for siderophore uptake. However, the mutant retained the ability to take up Fe' at 42% of the wild-type Fe' uptake rate, suggesting additional pathways of Fe' acquisition besides TBDTs, likely by porins. Mutations in four of the six porin-encoding genes produced a low-Fe-sensitive phenotype, while a mutation in all six genes was lethal to cell survival. These diverse outer membrane Fe uptake pathways reflect cyanobacterial evolution and adaptation under a range of Fe regimes across aquatic systems.IMPORTANCE Cyanobacteria are globally important primary producers and contribute about 25% of global CO2 fixation. Low Fe bioavailability in surface waters is thought to limit the primary productivity in as much as 40% of the global ocean. The Fe acquisition strategies that cyanobacteria have evolved to overcome Fe deficiency remain poorly characterized. We experimentally characterized the key players and the cooperative work mode of two Fe uptake pathways, including an active uptake pathway and a passive diffusion pathway in the model cyanobacterium Synechocystis sp. PCC 6803. Our finding proved that cyanobacteria use ferri-siderophore transporters to take up Fe', and they shed light on the adaptive mechanisms of cyanobacteria to cope with widespread Fe deficiency across aquatic environments.