Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation

Iron deficiency is known to suppress primary productivity in both marine and freshwater ecosystems. In response to iron deficiency, certain cyanobacteria induce a chlorophyll (Chl)-protein complex, CP43', which is encoded by the isiA gene. The deduced amino-acid sequence of CP43' predicts some structural similarity to the CP43 polypeptide of photosystem II, but the function of CP43' remains uncertain. In order to assess its physiological role, the isiA gene of a cyanobacterium, Synechococcus sp. PCC7942, was inactivated by insertion mutagenesis (giving isiA cells). Compared with isiA cells, under iron deprivation, wild-type cells showed both lower rates of photosystem II-mediated O2 evolution at limiting light irradiances and decreased yields of room temperature Chl fluorescence at various irradiances. These observations strongly suggest that the decreased photosystem II activity in wild-type cells with CP43' is attributable to increased non-radiative dissipation of light energy. In agreement with this hypothesis, isiA cells were more susceptible to photoinhibition of photosynthesis than wild-type cells, resulting in much slower growth rates under iron limitation. Based on these results, we suggest that CP43' functions as a non-radiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.     

Iron deficiency in cyanobacteria causes monomerization of photosystem I trimers and reduces the capacity for state transitions and the effective absorption cross section of photosystem I in vivo

The induction of the isiA (CP43') protein in iron-stressed cyanobacteria is accompanied by the formation of a ring of 18 CP43' proteins around the photosystem I (PSI) trimer and is thought to increase the absorption cross section of PSI within the CP43'-PSI supercomplex. In contrast to these in vitro studies, our in vivo measurements failed to demonstrate any increase of the PSI absorption cross section in two strains (Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803) of iron-stressed cells. We report that iron-stressed cells exhibited a reduced capacity for state transitions and limited dark reduction of the plastoquinone pool, which accounts for the increase in PSII-related 685 nm chlorophyll fluorescence under iron deficiency. This was accompanied by lower abundance of the NADP-dehydrogenase complex and the PSI-associated subunit PsaL, as well as a reduced amount of phosphatidylglycerol. Nondenaturating polyacrylamide gel electrophoresis separation of the chlorophyll-protein complexes indicated that the monomeric form of PSI is favored over the trimeric form of PSI under iron stress. Thus, we demonstrate that the induction of CP43' does not increase the PSI functional absorption cross section of whole cells in vivo, but rather, induces monomerization of PSI trimers and reduces the capacity for state transitions. We discuss the role of CP43' as an effective energy quencher to photoprotect PSII and PSI under unfavorable environmental conditions in cyanobacteria in vivo.     

PrxQ-A, a member of the peroxiredoxin Q family, plays a major role in defense against oxidative stress in the cyanobacterium Anabaena sp. strain PCC7120

The genome of the cyanobacterium Anabaena PCC 7120 encodes seven polypeptides showing sequence similarities with peroxiredoxins (Prx-s). One of them, prxQ-A (alr2503), which encodes a Prx Q homologue, is located in the same gene cluster as pkn22, which encodes a Ser/Thr kinase. Here we report that the pkn22-knockout mutant (Mp22) is sensitive to oxidative stress because it fails to synthesize PrxQ-A; the expression of prxQ-A is significantly induced under oxidative stress conditions. The hypersensitivity of the Mp22 mutant to oxidative stress was restored by inducing the expression of the prxQ-A gene in trans. The recombinant PrxQ-A protein shows antioxidant activity protecting the DNA from being degraded by reactive oxygen species, catalyzes the reduction of H2O2 in the presence of DTT, and shows thioredoxin-dependent peroxidase activity in vitro. The conserved Cys47 residue is the peroxide oxidation site, since the replacement of Cys47 by a Ser residue completely abolished the peroxidase activity. All these data suggest that PrxQ-A may efficiently protect this organism from oxidative stress.     

Aggregates of the chlorophyll-binding protein IsiA (CP43') dissipate energy in cyanobacteria

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.     

The induction of CP43' by iron-stress in Synechococcus sp. PCC 7942 is associated with carotenoid accumulation and enhanced fatty acid unsaturation

Comparative lipid analysis demonstrated reduced amount of PG (50%) and lower ratio of MGDG/DGDG in iron-stressed Synechococcus sp. PCC 7942 cells compared to cells grown under iron sufficient conditions. In parallel, the monoenoic (C:1) fatty acids in MGDG, DGDG and PG increased from 46.8%, 43.7% and 45.6%, respectively in control cells to 51.6%, 48.8% and 48.7%, respectively in iron-stressed cells. This suggests increased membrane dynamics, which may facilitate the diffusion of PQ and keep the PQ pool in relatively more oxidized state in iron-stressed compared to control cells. This was confirmed by chlorophyll fluorescence and thermoluminescence measurements. Analysis of carotenoid composition demonstrated that the induction of isiA (CP43') protein in response to iron stress is accompanied by significant increase of the relative abundance of all carotenoids. The quantity of carotenoids calculated on a Chl basis increased differentially with nostoxanthin, cryptoxanthin, zeaxanthin and beta-carotene showing 2.6-, 3.1-, 1.9- and 1.9-fold increases, respectively, while the relative amount of caloxanthin was increased only by 30%. HPLC analyses of the pigment composition of Chl-protein complexes separated by non-denaturating SDS-PAGE demonstrated even higher relative carotenoids content, especially of cryptoxanthin, in trimer and monomer PSI Chl-protein complexes co-migrating with CP43' from iron-stressed cells than in PSI complexes from control cells where CP43' is not present. This implies a carotenoid-binding role for the CP43' protein which supports our previous suggestion for effective energy quenching and photoprotective role of CP43' protein in cyanobacteria under iron stress.     

Structural analysis of the photosystem I supercomplex of cyanobacteria induced by iron deficiency

Here we describe the three-dimensional structure of the newly discovered CP43'-photosystem I (PSI) supercomplex of cyanobacteria calculated by single-particle analysis of images obtained by electron cryomicroscopy (cryo-EM). This large membrane protein complex has a molecular mass of approximately 2 MDa and is found in cyanobacteria when grown in iron deficient media. It is composed of a reaction center trimer surrounded by 18 subunits of the chlorophyll a binding CP43'protein, encoded by the isiA gene, which increases the light harvesting capacity of PSI by approximately 70%. By modeling higher-resolution structural data obtained from X-ray crystallography into the three-dimensional (3D) cryo-EM map, we have been able to gain a better understanding of the structure and functional properties of this supermolecular complex. We have identified three separate clusters of chlorophyll molecules at the periphery of the PSI core which may aid energy transfer from the CP43' antenna ring to the reaction center. Moreover, it is shown that despite the replacement of ferredoxin with flavodoxin as an electron acceptor under iron stress conditions, the 3D map has density to accommodate the extrinsic proteins, PsaC, PsaD, and PsaE. The presence of these three proteins was also confirmed by immunoblotting.     

A pair of iron-responsive genes encoding protein kinases with a Ser/Thr kinase domain and a His kinase domain are regulated by NtcA in the Cyanobacterium Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N(2) when combined nitrogen is not available in the growth medium. It has a family of 13 genes encoding proteins with both a Ser/Thr kinase domain and a His kinase domain. The function of these enzymes is unknown. Two of them are encoded by pkn41 (alr0709) and pkn42 (alr0710). These two genes are separated by only 72 bp on the chromosome, and our results indicate that they are cotranscribed. The expression of pkn41 and pkn42 is induced by iron deprivation irrespective of the nature of the nitrogen source. Mutants inactivating either pkn41, pkn42, or both grow similarly to the wild type under normal conditions, but their growth is impaired either in the presence of an iron chelator or under conditions of nitrogen fixation and iron limitation, two situations where the demand for iron is particularly strong. Consistent with these results, these mutants display lower iron content than the wild type and a higher level of expression for nifJ1 and nifJ2, which encode pyruvate:ferredoxin oxidoreductases. Both nifJ1 and nifJ2 are known to be induced by iron limitation. NtcA, a global regulatory factor for different metabolic pathways, binds to the putative promoter region of pkn41, and the induction of pkn41 in response to iron limitation no longer occurs in an ntcA mutant. Our results suggest that ntcA not only regulates the expression of genes involved in nitrogen and carbon metabolism but also coordinates iron acquisition and nitrogen metabolism by activating the expression of pkn41 and pkn42.     

The highly abundant chlorophyll-protein complex of iron-deficient Synechococcus sp. PCC7942 (CP43') is encoded by the isiA gene.

A chlorophyll (Chl)-protein complex designated CPVI-4 becomes the major pigment-protein complex in Synechococcus sp. PCC7942 cells grown under conditions of iron limitation. Work by Laudenbach et al. (J Bacteriol [1988] 170: 5018-5026) has identified an iron-repressible operon, designated isiAB, containing the flavodoxin gene and a gene predicted to encode a Chl-binding protein resembling CP43 of photosystem II. To test the hypothesis that the CP43-like protein is a component of the CPVI-4 complex, we have inactivated the isiAB operon in Synechococcus sp. PCC7942 using directed insertional mutagenesis. Mutant cells grown under conditions of iron limitation exhibit pronounced changes in their spectroscopic and photosynthetic properties relative to similarly grown wild-type cells. Notably, the strong 77 K fluorescence emission at 685 nm, which dominates the spectrum of iron-deficient wild-type cells, is dramatically reduced in the mutant. The loss of this emission appears to unmask the otherwise obscured photosystem II emissions at 685 and 695 nm. Most importantly, mildly denaturing gel electrophoresis shows that mutant cells no longer express the CPVI-4 complex, indicating that the isiA gene encodes a component of this abundant Chl-protein complex.     

Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures

Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll-carotenoid protein complex known as IsiA or CP43'. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.     

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43' (isiA)

The chlorophyll-protein CP43' (isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the 'energy-dependent non-photochemical quenching' (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence.     

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.     

Three-dimensional model and characterization of the iron stress-induced CP43'-photosystem I supercomplex isolated from the cyanobacterium Synechocystis PCC 6803

The cyanobacterium Synechocystis PCC 6803 has been subjected to growth under iron-deficient conditions. As a consequence, the isiA gene is expressed, and its product, the chlorophyll a-binding protein CP43', accumulates in the cell. Recently, we have shown for the first time that 18 copies of this photosystem II (PSII)-like chlorophyll a-binding protein forms a ring around the trimeric photosystem I (PSI) reaction center (Bibby, T. S., Nield, J., and Barber, J. (2001) Nature, 412, 743-745). Here we further characterize the biochemical and structural properties of this novel CP43'-PSI supercomplex confirming that it is a functional unit of approximately 1900 kDa where the antenna size of PSI is increased by 70% or more. Using electron microscopy and single particle analysis, we have constructed a preliminary three-dimensional model of the CP43'-PSI supercomplex and used it as a framework to incorporate higher resolution structures of PSI and CP43 recently derived from x-ray crystallography. Not only does this work emphasize the flexibility of cyanobacterial light-harvesting systems in response to the lowering of phycobilisome and PSI levels under iron-deficient conditions, but it also has implications for understanding the organization of the related chlorophyll a/b-binding Pcb proteins of oxychlorobacteria, formerly known as prochlorophytes.     

CP43-like chlorophyll binding proteins: structural and evolutionary implications

CP43, encoded by the psbC gene, is a chlorophyll (Chl)-binding protein of Photosystem II (PSII), the water-splitting and oxygen-evolving enzyme of photosynthesis. CP47, encoded by psbB, a Chl-binding protein of PSII, is closely related to CP43. The Chl-binding six transmembrane helical unit typified by CP43, is also structurally related to the N-terminal domains of the PsaA and PsaB proteins of Photosystem I (PSI) as well as to the family of light-harvesting proteins encoded by cyanobacterial isiA genes and prochlorophyte pcb genes. Here we use recent structural information derived for PSII and PSI to review similarities and differences between the various members of the CP43-like class of light-harvesting proteins, exploring both functional and evolutionary implications.     

Supramolecular organization and dual function of the IsiA chlorophyll-binding protein in cyanobacteria

A significant part of global primary productivity is provided by cyanobacteria, which are abundant in most marine and freshwater habitats. In many oceanographic regions, however, the concentration of iron can be so low that it limits growth. Cyanobacteria respond to this condition by expressing a number of iron stress inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. It was recently shown that 18 IsiA proteins encircle trimeric photosystem I (PSI) under iron-deficient growth conditions. We report here that after prolonged growth of Synechocystis PCC 6803 in an iron-deficient medium, the number of bound IsiA proteins can be much higher than previously known. The largest complexes bind 12-14 units in an inner ring and 19-21 units in an outer ring around a PSI monomer. Fluorescence excitation spectra indicate an efficient light harvesting function for all PSI-bound chlorophylls. We also find that IsiA accumulates in cyanobacteria in excess of what is needed for functional light harvesting by PSI, and that a significant part of IsiA builds supercomplexes without PSI. Because the further decline of PSI makes photosystem II (PSII) increasingly vulnerable to photooxidation, we postulate that the surplus synthesis of IsiA shields PSII from excess light. We suggest that IsiA plays a surprisingly versatile role in cyanobacteria, by significantly enhancing the light harvesting ability of PSI and providing photoprotection for PSII.