RESUMEN

Expression of a thylakoid membrane-associated protein called IdiA (iron-deficiency-induced protein A) is highly elevated and tightly regulated by iron limitation in Synechococcus elongatus PCC 6301 and PCC 7942. Although this protein is not essential for photosystem II (PSII) activity, it plays an important role in protecting the acceptor side of PSII against oxidative damage, especially under iron-limiting growth conditions, by an unknown mechanism. We defined the iron-responsive idiA promoter by using insertional inactivation mutagenesis and reporter gene assays. A 67-bp DNA region was sufficient for full iron deficiency-inducible idiA promoter activity. Within this fragment is a palindromic sequence 4 bp upstream of a putative -35 promoter element, which resembles the binding site of FNR/CAP-type helix-turn-helix transcription factors. The absence of this palindromic sequence or a 3-bp mutation in a putative -10 region eliminated promoter activity completely. A previously identified candidate for a positively acting transcription factor is the IdiB protein, whose gene lies immediately downstream of idiA. IdiB shows strong similarity to helix-turn-helix transcription factors of the FNR/CAP family. A His(6x)-tagged IdiB that was overexpressed in Escherichia coli bound to a 59-bp fragment of the idiA regulatory region that included the palindrome. Although the idiA promoter lacks a consensus binding site for the iron-sensing regulator Fur, we attempted to inactivate fur in order to investigate the potential role of this factor. The resulting merodiploid mutants showed constitutive partial derepression of IdiA expression under iron-sufficient growth conditions. We concluded that IdiB is a specific iron-responsive regulator of idiA and that Fur has an indirect role in influencing idiA expression.

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RESUMEN

Cyanobacteria are abundant throughout most of the world's water bodies and contribute significantly to global primary productivity through oxygenic photosynthesis. This reaction is catalysed by two membrane-bound protein complexes, photosystem I (PSI) and photosystem II (PSII), which both contain chlorophyll-binding subunits functioning as an internal antenna. In addition, phycobilisomes act as peripheral antenna systems, but no additional light-harvesting systems have been found under normal growth conditions. Iron deficiency, which is often the limiting factor for cyanobacterial growth in aquatic ecosystems, leads to the induction of additional proteins such as IsiA (ref. 3). Although IsiA has been implicated in chlorophyll storage, energy absorption and protection against excessive light, its precise molecular function and association to other proteins is unknown. Here we report the purification of a specific PSI-IsiA supercomplex, which is abundant under conditions of iron limitation. Electron microscopy shows that this supercomplex consists of trimeric PSI surrounded by a closed ring of 18 IsiA proteins binding around 180 chlorophyll molecules. We provide a structural characterization of an additional chlorophyll-containing, membrane-integral antenna in a cyanobacterial photosystem.

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RESUMEN

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q(A), gave a decreased O(2) evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage.

RESUMEN

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635-2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion.

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RESUMEN

In the cyanobacteria Synechococcus PCC 6301 and PCC 7942 a protein with an apparent molecular mass of about 34 kDa (called IdiA for iron-deficiency-induced protein A) accumulates under iron and managanese limitation. IdiA from Synechococcus PCC 6301 was partially sequenced, showing that the N-terminal amino acid is an alanine. Moreover, the gene encoding this protein in Synechococcus PCC 6301 has been identified and completely sequenced. The idiA gene codes for a protein starting with valine and consisting of 330 amino acid residues. Thus, IdiA is apparently synthesized as a precursor protein of 36.17 kDa and cleaved to its mature form of 35.01 kDa between two alanine residues at positions 9 and 10. IdiA is a highly basic protein having an isoelectric point of 10.55 (mature protein). Comparison of the amino acid sequence of IdiA with protein sequences in the database revealed that IdiA has similarities to two basic bacterial iron-binding proteins, SfuA from Serratia marcescens and Fbp from Neisseria gonorrhoeae. Insertional inactivation of the idiA gene in Synechococcus PCC 7942 resulted in a mutant which was unable to grow under iron- or manganese-limiting conditions. Manganese limitation of the mutant strain led to a drastic reduction of photosystem II activity (O2 evolution) within less than 48 h, while wild-type cells required a prolonged cultivation in Mn-deficient medium before an effect on photosystem II was observed. Thus, IdiA is a protein involved in the process of providing photosystem II with manganese.

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