Louis A. Sherman

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 Lauden-bach 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.

Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp. strain PCC 6803.

We utilized a full genome cDNA microarray to identify the genes that comprise the peroxide stimulon in the cyanobacterium Synechocystis sp. strain PCC 6803. We determined that a gene (slr1738) encoding a protein similar to PerR in Bacillus subtilis was induced by peroxide. We constructed a PerR knockout strain and used it to help identify components of the PerR regulon, and we found that the regulatory properties were consistent with the hypothesis that PerR functions as a repressor. This effort was guided by finding putative PerR boxes in positions upstream of specific genes and by careful statistical analysis. PerR and sll1621 (ahpC), which codes for a peroxiredoxin, share a divergent promoter that is regulated by PerR. We found that isiA, encoding a Chl protein that is induced under low-iron conditions, was strongly induced by a short-term peroxide stress. Other genes that were strongly induced by peroxide included sigD, sigB, and genes encoding peroxiredoxins and Dsb-like proteins that have not been studied yet in this strain. A gene (slr1894) that encoded a protein similar to MrgA in B. subtilis was upregulated by peroxide, and a strain containing an mrgA knockout mutation was highly sensitive to peroxide. A number of genes were downregulated, including key genes in the chlorophyll biosynthesis pathway and numerous regulatory genes, including those encoding histidine kinases. We used PerR mutants and a thioredoxin mutant (TrxA1) to study differential expression in response to peroxide and determined that neither PerR nor TrxA1 is essential for the peroxide protective response.

Microarray Analysis of the Genome-Wide Response to Iron Deficiency and Iron Reconstitution in the Cyanobacterium Synechocystis sp. PCC 6803

A full-genome microarray of the (oxy)photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used to identify genes that were transcriptionally regulated by growth in iron (Fe)-deficient versus Fe-sufficient media. Transcript accumulation for 3,165 genes in the genome was analyzed using an analysis of variance model that accounted for slide and replicate (random) effects and dye (a fixed) effect in testing for differences in the four time periods. We determined that 85 genes showed statistically significant changes in the level of transcription (P ≤ 0.05/3,165 = 0.0000158) across the four time points examined, whereas 781 genes were characterized as interesting (P ≤ 0.05 but greater than 0.0000158; 731 of these had a fold change >1.25×). The genes identified included those known previously to be Fe regulated, such as isiA that encodes a novel chlorophyll-binding protein responsible for the pigment characteristics of low-Fe (LoFe) cells. ATP synthetase and phycobilisome genes were down-regulated in LoFe, and there were interesting changes in the transcription of genes involved in chlorophyll biosynthesis, in photosystem I and II assembly, and in energy metabolism. Hierarchical clustering demonstrated that photosynthesis genes, as a class, were repressed in LoFe and induced upon the re-addition of Fe. Specific regulatory genes were transcriptionally active in LoFe, including two genes that show homology to plant phytochromes (cph1 and cph2). These observations established the existence of a complex network of regulatory interactions and coordination in response to Fe availability.

A Redox-Responsive Regulator of Photosynthesis Gene Expression in the Cyanobacterium Synechocystis sp. Strain PCC 6803

We have identified genes in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 that are involved with redox control of photosynthesis and pigment-related genes. The genes, rppA (sll0797) and rppB (sll0798), represent a two-component regulatory system that controls the synthesis of photosystem II (PSII) and PSI genes, in addition to photopigment-related genes. rppA (regulator of photosynthesis- and photopigment-related gene expression) and rppB exhibit strong sequence similarity to prokaryotic response regulators and histidine kinases, respectively. In the wild type, the steady-state mRNA levels of PSII reaction center genes increased when the plastoquinone (PQ) pool was oxidized and decreased when the PQ pool was reduced, whereas transcription of the PSI reaction center genes was affected in an opposite fashion. Such results suggested that the redox poise of the PQ pool is critical for regulation of the photosystem reaction center genes. In Delta rppA, an insertion mutation of rppA, the PSII gene transcripts were highly up-regulated relative to the wild type under all redox conditions, whereas transcription of phycobilisome-related genes and PSI genes was decreased. The higher transcription of the psbA gene in Delta rppA was manifest by higher translation of the D1 protein and a concomitant increase in O(2) evolution. The results demonstrated that RppA is a regulator of photosynthesis- and photopigment-related gene expression, is involved in the establishment of the appropriate stoichiometry between the photosystems, and can sense changes in the PQ redox poise.

The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle

Unicellular cyanobacteria have recently been recognized for their contributions to nitrogen fixation in marine environments, a function previously thought to be filled mainly by filamentous cyanobacteria such as Trichodesmium. To begin a systems level analysis of the physiology of the unicellular N2-fixing microbes, we have sequenced to completion the genome of Cyanothece sp. ATCC 51142, the first such organism. Cyanothece 51142 performs oxygenic photosynthesis and nitrogen fixation, separating these two incompatible processes temporally within the same cell, while concomitantly accumulating metabolic products in inclusion bodies that are later mobilized as part of a robust diurnal cycle. The 5,460,377-bp Cyanothece 51142 genome has a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of a linear element in the genome of a photosynthetic bacterium. On the 429,701-bp linear chromosome is a cluster of genes for enzymes involved in pyruvate metabolism, suggesting an important role for the linear chromosome in fermentative processes. The annotation of the genome was significantly aided by simultaneous global proteomic studies of this organism. Compared with other nitrogen-fixing cyanobacteria, Cyanothece 51142 contains the largest intact contiguous cluster of nitrogen fixation-related genes. We discuss the implications of such an organization on the regulation of nitrogen fixation. The genome sequence provides important information regarding the ability of Cyanothece 51142 to accomplish metabolic compartmentalization and energy storage, as well as how a unicellular bacterium balances multiple, often incompatible, processes in a single cell.

Differential Transcriptional Analysis of the Cyanobacterium Cyanothece sp. Strain ATCC 51142 during Light-Dark and Continuous-Light Growth

We analyzed the metabolic rhythms and differential gene expression in the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under N(2)-fixing conditions after a shift from normal 12-h light-12-h dark cycles to continuous light. We found that the mRNA levels of approximately 10% of the genes in the genome demonstrated circadian behavior during growth in free-running (continuous light) conditions. The genes for N(2) fixation displayed a strong circadian behavior, whereas photosynthesis and respiration genes were not as tightly regulated. One of our main objectives was to determine the strategies used by these cells to perform N(2) fixation under normal day-night conditions, as well as under the greater stress caused by continuous light. We determined that N(2) fixation cycled in continuous light but with a lower N(2) fixation activity. Glycogen degradation, respiration, and photosynthesis were also lower; nonetheless, O(2) evolution was about 50% of the normal peak. We also demonstrated that nifH (encoding the nitrogenase Fe protein), nifB, and nifX were strongly induced in continuous light; this is consistent with the role of these proteins during the assembly of the enzyme complex and suggested that the decreased N(2) fixation activity was due to protein-level regulation or inhibition. Many soluble electron carriers (e.g., ferredoxins), as well as redox carriers (e.g., thioredoxin and glutathione), were strongly induced during N(2) fixation in continuous light. We suggest that these carriers are required to enhance cyclic electron transport and phosphorylation for energy production and to maintain appropriate redox levels in the presence of elevated O(2), respectively.

Diurnal rhythms in metabolism: A day in the life of a unicellular, diazotrophic cyanobacterium

N2 fixation and oxygenic photosynthesis are important metabolic processes that are at odds with each other, since the N2-fixing enzyme, nitrogenase, is highly sensitive to oxygen. This review will discuss the strategies devised by the unicellular, diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142, to permit N2 fixation and photosynthesis to coexist in the same cell. This strain, like a number of other unicellular and filamentous (non-heterocystous) cyanobacteria, has developed a type of temporal regulation in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle. For nitrogenase, everyday dawns anew. The nifHDK operon is tightly regulated, such that transcription and translation occur within the first four hours of the dark period; nitrogenase is then proteolytically degraded. Photosynthesis also varies throughout the day reaching a minimum at the peak of nitrogenase activity and a maximum by late afternoon. This review will mainly concentrate on the various changes that occur in the photosynthetic apparatus as the cell modulates O2 evolution. The results indicate that the redox poise of the plastoquinone pool and the overall cellular energy needs are the basic driving forces behind these changes in the photosynthetic apparatus. Throughout the course of the diurnal cycle, Photosystem II becomes very heterogeneous as determined by 77 K fluorescence spectra, PAM fluorescence and O2-flash yield experiments. This system provides some important insight into cyanobacterial state transitions and, especially, on the organization of the photosystems within the membrane. Overall, PS II is altered on both the oxidizing and reducing sides of the photosystem.

Electrophoretic profiles of cyanobacterial membrane polypeptides showing heme-dependent peroxidase activity

Abstract The photosynthetic membranes of Anacystis nidulans R2 were examined electrophoretically following solubilization with lithium dodecyl sulfate. Electrophoresis yielded six prominent chlorophyll-containing bands. In addition, five polypeptides were observed which possessed heme-dependent peroxidase activity, monitored by incubating gels with 3,3′,5,5′-tetramethylbenzidine plus hydrogen peroxide. One such polypeptide, at 105 kdaltons, was removed by repeated washing of the membranes. Four remaining peroxidase-active polypeptides were observed at 7.2, 13.5, 18.5 and 33 kdaltons. Further examination of these four polypeptides yielded the following results. (1) The mobility of the 33 kdalton polypeptide was altered from 29 to 33 kdaltons upon heating (70°C) during membrane solubilization. (2) All four polypeptides showed stable heme-protein associations in the presence of 8 M urea; however, in the presence of urea, alterations in protein mobility were observed for each poly-peptide and only two (at 13.5 and 33 kdaltons) showed peroxidase activity following heating (70°C) during membrane solubilization. (3) The presence of thiols during membrane solubilization at 0°C was required to observe peroxidase activity at 7.2 kdaltons. These results, when compared to known properties of isolated cytochromes, suggest that the four polypeptides characterized here correspond to the subunits of photosynthetic cytochromes. Electrophoretic assessment of maize mutants lacking cytochrome f and b6 activity supports this suggestion.

Isolation and characterization of photosystem I and II membrane particles from the blue-green alga, Synechococcus cedrorum.

Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10--30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose. The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 mumol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains beta-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N2 fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome b-559 ratio of 50 : 1. The Photosystem I particle is highly enriched in P-700, with a chlorophyll to P-700 ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.

The Mechanism of Iron Homeostasis in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803 and Its Relationship to Oxidative Stress

In this article, we demonstrate the connection between intracellular iron storage and oxidative stress response in cyanobacteria. Iron is essential for the survival of all organisms. However, the redox properties that make iron a valuable cofactor also lead to oxidative interactions, resulting in the formation of harmful radicals. Therefore, iron accumulation in cells should be tightly regulated, a process in which ferritin family proteins play an important role. Synechocystis sp. PCC 6803 contains two ferritin-type storage complexes, bacterioferritin and MrgA. Previous studies demonstrated the role of bacterioferritin and MrgA in iron storage. In addition, MrgA was found to play a key role in oxidative stress response. Here, we examined the dual role of the ferritin family proteins using physiological and transcriptomic approaches. Microarray analysis of iron-limited wild-type and ΔmrgA cultures revealed a substantial up-regulation of oxidative stress-related genes in mutant cells. The PerR regulator was found to play an important role in that process. Furthermore, we were able to demonstrate the connection between internal iron quota, the presence of the two storage complexes, and the sensitivity to externally applied oxidative stress. These data suggest a pivotal role for the ferritin-type proteins of Synechocystis sp. PCC 6803 in coordinating iron homeostasis and in oxidative stress response. The combined action of the two complexes allows for the safe accumulation and release of iron from storage by minimizing damage resulting from interactions between reduced iron and the oxygen radicals that are produced in abundance by the photosynthetic apparatus.