Klaus-Peter Michel

A giant chlorophyll–protein complex induced by iron deficiency in cyanobacteria

Cyanobacteria are abundant throughout most of the worlds 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.

Unusual Regulatory Elements for Iron Deficiency Induction of the idiA Gene of Synechococcus elongatus PCC 7942

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.

Metabolic and Transcriptomic Phenotyping of Inorganic Carbon Acclimation in the Cyanobacterium Synechococcus elongatus PCC 7942

The amount of inorganic carbon is one of the main limiting environmental factors for photosynthetic organisms such as cyanobacteria. Using Synechococcus elongatus PCC 7942, we characterized metabolic and transcriptomic changes in cells that had been shifted from high to low CO2 levels. Metabolic phenotyping indicated an activation of glycolysis, the oxidative pentose phosphate cycle, and glycolate metabolism at lowered CO2 levels. The metabolic changes coincided with a general reprogramming of gene expression, which included not only increased transcription of inorganic carbon transporter genes but also genes for enzymes involved in glycolytic and photorespiratory metabolism. In contrast, the mRNA content for genes from nitrogen assimilatory pathways decreased. These observations indicated that cyanobacteria control the homeostasis of the carbon-nitrogen ratio. Therefore, results obtained from the wild type were compared with the MP2 mutant of Synechococcus 7942, which is defective for the carbon-nitrogen ratio-regulating PII protein. Metabolites and genes linked to nitrogen assimilation were differentially regulated, whereas the changes in metabolite concentrations and gene expression for processes related to central carbon metabolism were mostly similar in mutant and wild-type cells after shifts to low-CO2 conditions. The PII signaling appears to down-regulate the nitrogen metabolism at lowered CO2, whereas the specific shortage of inorganic carbon is recognized by different mechanisms.

IdiA, a 34 kDa protein in the cyanobacteria Synechococcus sp. strains PCC 6301 and PCC 7942, is required for growth under iron and manganese limitations

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.

Localization and function of the IdiA homologue Slr1295 in the cyanobacterium Synechocystis sp. strain PCC 6803.

Slr1295 (and Slr0513) in the cyanobacterium Synechocystis sp. PCC 6803 has amino acid similarity to the bacterial FbpA protein family and also to IdiA of Synechococcus PCC 6301/PCC 7942. To determine whether Slr1295 is the periplasm-located component of an iron transporter, or has a function in protecting photosystem (PS) II, subcellular localization and Deltaslr1295 mutant characterization studies were performed. Localization of Slr1295 provided evidence that it has an intracellular function, since virtually no Slr1295 was detected in the soluble protein fraction of the periplasm or in the cytoplasmic membrane. Characterization of a Deltaslr1295 Synechocystis PCC 6803 mutant indicated that PS II is more susceptible to inactivation in the mutant than in the wild-type (WT). Under mild iron limitation, modification of PS I to the PS I-IsiA complex is more advanced in the Deltaslr1295 mutant, indicating that iron deficiency leads more rapidly to changes in the photosynthetic apparatus in the mutant than in the WT. Biochemical fractionation procedures provide evidence that Slr1295 co-purifies with PS II. These results suggest a function of Slr1295 that is comparable to the function of IdiA in Synechococcus PCC 6301/PCC 7942 being a protein that protects PS II under iron limitation in an as yet unknown way.

Transcript Profiling Reveals New Insights into the Acclimation of the Mesophilic Fresh-Water Cyanobacterium Synechococcus elongatus PCC 7942 to Iron Starvation

The regulatory network for acclimation of the obligate photoautotrophic fresh water cyanobacterium Synechococcus elongatus PCC 7942 to iron (Fe) limitation was studied by transcript profiling with an oligonucleotide whole genome DNA microarray. Six regions on the chromosome with several Fe-regulated genes each were identified. The irpAB and fut region encode putative Fe uptake systems, the suf region participates in [Fe-sulfur] cluster assembly under oxidative stress and Fe limitation, the isiAB region encodes CP43′ and flavodoxin, the idiCB region encodes the NuoE-like electron transport associated protein IdiC and the transcriptional activator IdiB, and the ackA/pgam region encodes an acetate kinase and a phosphoglycerate mutase. We also investigated the response of two S. elongatus PCC 7942 mutants to Fe starvation. These were mutant K10, lacking IdiB but containing IdiC, and mutant MuD, representing a idiC-merodiploid mutant with a strongly reduced amount of IdiC as well as IdiB. The absence of IdiB in mutant K10 or the strongly reduced amount of IdiB in mutant MuD allowed for the identification of additional members of the Fe-responsive IdiB regulon. Besides idiA and the irpAB operon somB(1), somA(2), ftr1, ackA, pgam, and nat also seem to be regulated by IdiB. In addition to the reduced amount of IdiB in MuD, the low concentration of IdiC may be responsible for a number of additional changes in the abundance of mainly photosynthesis-related transcripts as compared to the wild type and mutant K10. This fact may explain why it has been impossible to obtain a fully segregated IdiC-free mutant, whereas it was possible to obtain a fully segregated IdiB-free mutant.

Molecular characterization of idiA and adjacent genes in the cyanobacteria Synechococcus sp. strains PCC 6301 and PCC 7942

IdiA (iron-deficiency-induced protein A) is a protein expressed at highly elevated levels in Synechococcus sp. strains PCC 6301 and PCC 7942 under Fe- or Mn-limiting growth conditions. Besides being similar to two bacterial Fe-binding proteins, SfuA and FbpA, IdiA shows similarity to two ORFs (slr0513 and sir1295) of Synechocystis sp. PCC 6803. Northern blot analysis detected one transcript of about 1300 nt in RNA extracted from Synechococcus sp. PCC 6301 and PCC 7942 grown under Fe deficiency. The intensity of this transcript was considerably reduced in Fe-sufficient culture. It could be further shown that the regulation of IdiA expression is at the transcriptional level and that transcription and translation of IdiA are closely linked. Primer extension analysis indicated a single transcriptional start site 193 nt upstream of the first presumed translational start codon. Moreover, molecular characterization of the entire 5.8 kb chromosomal HindIII DNA fragment carrying the idiA gene from Synechococcus sp. PCC 6301 led to the identification of six long ORFs in addition to idiA. The two genes adjacent to idiA, and dpsA located 2018 nt downstream of idiA, were insertionally inactivated in Synechococcus sp. PCC 7942 and the corresponding mutants were partially characterized. These experiments provide evidence that the gene products of idiB, located immediately downstream of idiA, and of dpsA are involved in the activation of IdiA expression, since the absence of each of these two gene products prevents the greatly elevated expression of IdiA under nutrient deficiency.

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Abstract. 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.

Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

BackgroundSo far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis.ResultsWe have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i) an L-arginine decarboxylase pathway, (ii) an L-arginine deiminase pathway, and (iii) an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 μmol photons m-2 s-1 showed that the transcripts for the first enzyme(s) of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase.ConclusionThe evaluation of 24 cyanobacterial genomes revealed that five different L-arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L-arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways were constitutively expressed with the exception of the transcript for the carbamate kinase, which was substantially up-regulated in cells grown with L-arginine.

Adaptation to iron deficiency: a comparison between the cyanobacterium Synechococcus elongatus PCC 7942 wild-type and a DpsA-free mutant

To learn more about the adaptive response of Synechococcus elongatus PCC 7942 to iron starvation and the role of DpsA, presumably a protein protecting chromosomal DNA against oxidative damage, we performed a comparative analysis of S. elongatus PCC 7942 wild-type and a DpsA-free mutant, called K11. Relative to wild-type, the DpsA-free mutant had significantly higher amounts of phycocyanin and allophycocyanin, even upon iron limitation. While the Photosystem I activity in mutant K11 remained high under iron deficiency, the Photosystem II activity dropped severely with respect to wild-type. The DpsA content in wild-type was already fairly high under regular growth conditions and did not significantly increase under iron deficiency nor in the presence of 0.3 mM 2′2′-dipyridyl in iron-sufficient BG11 medium. Nevertheless, the absence of DpsA in K11 resulted in a significantly altered transcriptional/translational activity of genes known to be involved in adaptation to iron starvation. The amount of isiA/B transcript was about two-fold lower than in wild-type, resulting in a lower 77 K chlorophyll a fluorescence at 685 nm, implying a lower concentration of Photosystem I-IsiA supercomplexes. While in wild-type idiA, idiB, and irpA transcripts were highly up-regulated, hardly any were detectable in mutant K11 under iron limitation. The concentration of mapA transcript, however, was greatly increased in K11 compared to wild-type. Measurements of acridine yellow fluorescence with intact wild-type and K11 cells revealed that iron deficiency caused an increased contribution of cyclic electron transport to membrane energisation and ATP synthesis being in agreement with the formation of the Photosystem I-IsiA supercomplex. In addition, mutant K11 had a much higher respiratory activity compared to wild-type under iron limitation.