Phosphorus deficiency alleviates iron limitation in Synechocystis cyanobacteria through direct PhoB-mediated gene regulation.

Iron and phosphorus are essential nutrients that exist at low concentrations in surface waters and may be co-limiting resources for phytoplankton growth. Here, we show that phosphorus deficiency increases the growth of iron-limited cyanobacteria (Synechocystis sp. PCC 6803) through a PhoB-mediated regulatory network. We find that PhoB, in addition to its well-recognized role in controlling phosphate homeostasis, also regulates key metabolic processes crucial for iron-limited cyanobacteria, including ROS detoxification and iron uptake. Transcript abundances of PhoB-targeted genes are enriched in samples from phosphorus-depleted seawater, and a conserved PhoB-binding site is widely present in the promoters of the target genes, suggesting that the PhoB-mediated regulation may be highly conserved. Our findings provide molecular insights into the responses of cyanobacteria to simultaneous iron/phosphorus nutrient limitation.

Light Stimulates Copper-Limited Growth of an Oceanic Diatom by Increasing Cellular Copper(II) Reduction─A Rate-Determining Step in Copper Uptake.

Uptake of Cu by Thalassiosira oceanica requires that Cu(II) is reduced to Cu(I) prior to transport across the cell membrane. The reduction step is mediated biochemically by cellular reductases active with a broad range of Cu chemical species. Here, we report on the cellular Cu(II) reduction and Cu(I) uptake of a diatom under saturating and subsaturating irradiance. An increase in growth irradiance, from 50 to 400 µmol photons m-2 s-1, increased the rate of extracellular Cu(II) reduction and steady-state Cu uptake. Under these conditions, Cu-limited cells acquired Cu more efficiently and maintained faster rates of growth than Cu-limited cells in low light. Pseudo-first-order reaction rate constants were about 70-fold faster for Cu(I) uptake than for Cu(II) reduction so that reduction was the rate-determining step in Cu acquisition. Accordingly, steady-state Cu uptake rates predicted from the reduction rate constants agreed well with measured rates of Cu uptake obtained from cultures growing at low nanomolar Cu concentrations. Transcript abundance of putative Cu(II) reductases followed a similar pattern to cupric reductase activity, increasing in Cu-limited cells and with increasing growth irradiance. The results are significant in showing Cu(II) reduction as the rate-determining step in Cu uptake: they suggest that biologically mediated Cu(II) reduction may be an important part of the Cu cycle in surface waters of the open sea.

Transcriptomes of an oceanic diatom reveal the initial and final stages of acclimation to copper deficiency.

Copper (Cu) concentration is greatly reduced in the open sea so that phytoplankton must adjust their uptake systems and acclimate to sustain growth. Acclimation to low Cu involves changes to the photosynthetic apparatus and specific biochemical reactions that use Cu, but little is known how Cu affects cellular metabolic networks. Here we report results of whole transcriptome analysis of a plastocyanin-containing diatom, Thalassiosira oceanica 1005, during its initial stages of acclimation and after long-term adaptation in Cu-deficient seawater. Gene expression profiles, used to identify Cu-regulated metabolic pathways, show downregulation of anabolic and energy-yielding reactions in Cu-limited cells. These include the light reactions of photosynthesis, carbon fixation, nitrogen assimilation and glycolysis. Reduction of these pathways is consistent with reduced growth requirements for C and N caused by slower rates of photosynthetic electron transport. Upregulation of oxidative stress defence systems persists in adapted cells, suggesting cellular damage by increased reactive oxygen species (ROS) occurs even after acclimation. Copper deficiency also alters fatty acid metabolism, possibly in response to an increase in lipid peroxidation and membrane damage driven by ROS. During the initial stages of Cu-limitation the majority of differentially regulated genes are associated with photosynthetic metabolism, highlighting the chloroplast as the primary target of low Cu availability. The results provide insights into the mechanisms of acclimation and adaptation of T. oceanica to Cu deficiency.

Identification of copper-regulated proteins in an oceanic diatom, Thalassiosira oceanica 1005.

Copper (Cu) is an essential cofactor of photosynthetic and respiratory redox proteins in phytoplankton and a scarce resource in parts of the open sea. Although its importance for growth is well recognized, the molecular mechanisms by which phytoplankton respond and acclimate to Cu deficiency are not well known. In this study, we identified the dominant Cu-regulated proteins and measured key physiological traits of Thalassiosira oceanica (CCMP 1005) under Cu-limiting and sufficient conditions. Growth limitation of T. oceanica occurred at environmentally relevant Cu concentrations (1 nM) as a result of decreased photosynthetic efficiency (ΦPSII). In Cu-limited cells, levels of plastocyanin decreased by 3-fold compared to Cu-replete cells and rates of maximum photosynthetic electron transport were reduced. Proteins associated with light harvesting complexes also declined in response to Cu limitation, presumably to adjust to reduced photosynthetic electron flow and to avoid photodamage to the photosystems. Key enzymes involved in carbon and nitrogen assimilation were down-regulated in low-Cu cells, as were steady state rates of C and N uptake. Relatively fewer proteins were up-regulated by Cu limitation, but among them were two enzymes involved in fatty acid oxidation (FAO). The increase in FAO may be a sign of increased turnover of cellular lipids caused by damage from oxidative stress. A putative transcription factor containing three, repetitive methionine motifs (MpgMgggM; MpgMggM) increased significantly in Cu-limited cells. The collective results provide a general description of how plastocyanin-dependent diatoms adjust metabolism to cope with chronic Cu deficiency.

Functional CTR-type Cu(I) transporters in an oceanic diatom.

Copper concentration is so low in some remote parts of the sea it limits phytoplankton growth, but may be high enough in coastal and estuarine regions to be toxic. Acclimation to variations in Cu concentration thus requires a tightly regulated Cu transport system to help maintain Cu homeostasis. In marine species, the molecular mechanisms of Cu transport are not known. We studied Cu-responsive genes and uptake in Thalassiosira oceanica at environmentally relevant Cu concentrations varying between 0.012 and 12 900 pmol Cu' l-1 . Copper uptake rate assessed at high Cu concentration was three-fold faster in Cu-limited than in Cu-replete cells, confirming the existence of an inducible uptake pathway in this diatom. Four putative CTR-type Cu transporters (ToCTR1, ToCTR2, ToCTR3a and ToCTR3b) identified in the transcriptome shared conserved features with known high-affinity Cu(I) transporters. Expression of the CTR genes was upregulated as Cu concentration declined and cells maintained maximum rates of growth. Further decreases in Cu led to decreased growth rate and increased abundance of ToCTR3a/b. Both ToCTR3a and 3b restored growth of a Cu transport mutant, Saccharomyces cerevisiae ctr1Δctr3Δ, in Cu-deficient medium and increased the uptake rates of Cu(I) and Cu(II). Thus, ToCTR3a/3b is a high-affinity Cu(I) transporter that, in conjunction with the other ToCTRs, may enable T. oceanica to survive in Cu-deplete ocean environments and respond to natural variation in Cu availability.

The influence of light on copper-limited growth of an oceanic diatom, Thalassiosira oceanica (Coscinodiscophyceae).

Thalassiosira oceanica (CCMP 1005) was grown over a range of copper concentrations at saturating and subsaturating irradiance to test the hypothesis that Cu and light were interacting essential resources. Growth was a hyperbolic function of irradiance in Cu-replete medium (263 fmol Cu' · L-1 ) with maximum rates achieved at 200 µmol photons · m-2  · s-1 . Lowering the Cu concentration at this irradiance to 30.8 fmol Cu' · L-1 decreased cellular Cu quota by 7-fold and reduced growth rate by 50%. Copper-deficient cells had significantly slower (P < 0.0001) rates of maximum, relative photosynthetic electron transport (rETRmax ) than Cu-sufficient cells, consistent with the role of Cu in photosynthesis in this diatom. In low-Cu medium (30.8 fmol Cu' · L-1 ), growth rate was best described as a positive, linear function of irradiance and reached the maximum value measured in Cu-replete cells when irradiance increased to 400 µmol photons · m-2  · s-1 . Thus, at high light, low-Cu concentration was no longer limiting to growth: Cu concentration and light interacted strongly to affect growth rate of T. oceanica (P < 0.0001). Relative ETRmax and Cu quota of cells grown at low Cu also increased at 400 µmol photons · m-2  · s-1 to levels measured in Cu-replete cells. Steady-state uptake rates of Cu-deficient and sufficient cells were light-dependent, suggesting that faster growth of T. oceanica under high light and low Cu was a result of light-stimulated Cu uptake.

Identification of an iron permease, cFTR1, in cyanobacteria involved in the iron reduction/re-oxidation uptake pathway.

Cyanobacteria are globally important primary producers and abundant in many iron-limited aquatic environments. The ways in which they take up iron are largely unknown, but reduction of Fe3+ is an important step in the process. Here we report a special iron permease in Synechocystis, cFTR1, that is required for Fe3+ uptake following Fe2+ re-oxidation. The expression of cFTR1 is induced by iron starvation, and a mutant lacking the gene is abnormally sensitive to iron starvation. The cFTR1 protein localizes to the plasma membrane and contains the iron-binding motif "REXXE". Point-directed mutagenesis of the REXXE motif results in a sensitivity to Fe-deficiency. Measurements of iron (55 Fe) uptake rate show that cFTR1 takes up Fe3+ rather than Fe2+ . The function of cFTR1 in Synechocystis could be genetically complemented by the iron permease, Ftr1p, of Saccharomyces cerevisiae, that is known to transport Fe3+ produced by the oxidation of Fe2+ via a multicopper oxidase. Unlike yeast Ftr1p, cyanobacterial cFTR1 probably obtains Fe3+ primarily from the oxidation of Fe2+ by oxygen. Growth assays show that the cFTR1 is required during oxygenic, photoautotrophic growth but not when oxygen production is inhibited during photoheterotrophic growth. In cyanobacteria, iron reduction/re-oxidation uptake pathway may represent their adaptation to oxygenated environments.

New insights into iron acquisition by cyanobacteria: an essential role for ExbB-ExbD complex in inorganic iron uptake.

Cyanobacteria are globally important primary producers that have an exceptionally large iron requirement for photosynthesis. In many aquatic ecosystems, the levels of dissolved iron are so low and some of the chemical species so unreactive that growth of cyanobacteria is impaired. Pathways of iron uptake through cyanobacterial membranes are now being elucidated, but the molecular details are still largely unknown. Here we report that the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 contains three exbB-exbD gene clusters that are obligatorily required for growth and are involved in iron acquisition. The three exbB-exbDs are redundant, but single and double mutants have reduced rates of iron uptake compared with wild-type cells, and the triple mutant appeared to be lethal. Short-term measurements in chemically well-defined medium show that iron uptake by Synechocystis depends on inorganic iron (Fe') concentration and ExbB-ExbD complexes are essentially required for the Fe' transport process. Although transport of iron bound to a model siderophore, ferrioxamine B, is also reduced in the exbB-exbD mutants, the rate of uptake at similar total [Fe] is about 800-fold slower than Fe', suggesting that hydroxamate siderophore iron uptake may be less ecologically relevant than free iron. These results provide the first evidence that ExbB-ExbD is involved in inorganic iron uptake and is an essential part of the iron acquisition pathway in cyanobacteria. The involvement of an ExbB-ExbD system for inorganic iron uptake may allow cyanobacteria to more tightly maintain iron homeostasis, particularly in variable environments where iron concentrations range from limiting to sufficient.

Sll1263, a unique cation diffusion facilitator protein that promotes iron uptake in the cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanobacteria are known to survive in iron-deficient environments, but the ways in which they acquire Fe and acclimate are not completely understood. Here we report a novel gene sll1263 that is required for Synechocystis sp. strain PCC 6803 to grow under iron-deficient conditions. sll1263 encodes a putative cation diffusion facilitator protein (CDF) that shows 50% amino acid similarity with ferrous iron efflux protein (FieF) of heterotrophic bacteria. In bacteria, the gene product is involved in metal export from the cell, but in Synechocystis sll1263 plays a role in iron uptake. The results show that expression of sll1263 was induced by iron-deficient conditions and its inactivation significantly decreased the growth rate of an sll1263(-) mutant. Other genes known to be required for Fe acquisition were also strongly up-regulated in the mutant even in the presence of high Fe. Overexpression of sll1263 increased growth under iron deficiency but reduced growth under high-iron stress, suggesting that the gene product was involved in iron uptake rather than detoxification. Expression of FieF in the sll1263(-) mutant was unable to rescue the Fe-deficient phenotype, but Sll1263 completely restored it. Measurements of cellular iron content and the iron uptake rate showed that they were significantly less in the sll1263(-) mutant than in the wild type, consistent with a role for sll1263 in iron uptake. We hypothesize that the low-iron habitats and high-iron requirements of cyanobacteria may be the reason why cyanobacterial CDF protein functions in Fe uptake and not efflux as in non-photosynthetic bacteria.

DIFFERENT PHYSIOLOGICAL RESPONSES OF FOUR MARINE SYNECHOCOCCUS STRAINS (CYANOPHYCEAE) TO NICKEL STARVATION UNDER IRON-REPLETE AND IRON-DEPLETE CONDITIONS(1).

Synechococcus species are important primary producers in coastal and open-ocean ecosystems. When nitrate was provided as the sole nitrogen source, nickel starvation inhibited the growth of strains WH8102 and WH7803, while it had little effect on two euryhaline strains, WH5701 and PCC 7002. Nickel was required for the acclimation of Synechococcus WH7803 to low iron and high light. In WH8102 and WH7803, nickel starvation decreased the linear electron transport activity, slowed down QA reoxidation, but increased the connectivity factor between individual photosynthetic units. Under such conditions, the reduction of their intersystem electron transport chains was expected to increase, and their cyclic electron transport around PSI would be favored. Nickel starvation decreased the total superoxide dismutase (SOD) activity of WH8102 and WH7803 by 30% and 15% of the control, respectively. The protein-bound (63) Ni of the oceanic strain WH8102 comigrated with SOD activity on nondenaturing gels and thus provided additional evidence for the existence of active NiSOD in Synechococcus WH8102. In WH7803, it seems likely that nickel starvation affected other metabolic pathways and thus indirectly affected the total SOD activity.

Copper-containing plastocyanin used for electron transport by an oceanic diatom.

The supply of some essential metals to pelagic ecosystems is less than the demand, so many phytoplankton have slow rates of photosynthetic production and restricted growth. The types and amounts of metals required by phytoplankton depends on their evolutionary history and on their adaptations to metal availability, which varies widely among ocean habitats. Diatoms, for example, need considerably less iron (Fe) to grow than chlorophyll-b-containing taxa, and the oceanic species demand roughly one-tenth the amount of coastal strains. Like Fe, copper (Cu) is scarce in the open sea, but notably higher concentrations of it are required for the growth of oceanic than of coastal isolates. Here we report that the greater Cu requirement in an oceanic diatom, Thalassiosira oceanica, is entirely due to a single Cu-containing protein, plastocyanin, which--until now--was only known to exist in organisms with chlorophyll b and cyanobacteria. Algae containing chlorophyll c, including the closely related coastal species T. weissflogii, are thought to lack plastocyanin and contain a functionally equivalent Fe-containing homologue, cytochrome c6 (ref. 9). Copper deficiency in T. oceanica inhibits electron transport regardless of Fe status, implying a constitutive role for plastocyanin in the light reactions of photosynthesis in this species. The results suggest that selection pressure imposed by Fe limitation has resulted in the use of a Cu protein for photosynthesis in an oceanic diatom. This biochemical switch reduces the need for Fe and increases the requirement for Cu, which is relatively more abundant in the open sea.