Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is ¹³C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a ¹³C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes.

As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps ². First, we grow cells with ¹³C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways^{3, 4}. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active ². Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.

¹³C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism¹. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

The Journal of Biological Chemistry

Elemental manganese is essential for the production of molecular oxygen by cyanobacteria, plants, and algae. In the cyanobacterium Synechocystis sp. PCC 6803, transcription of the mntCAB operon, encoding a high affinity Mn transporter, occurs under Mn starvation (nm Mn) conditions but not in Mn-sufficient (microm Mn) growth medium. Using a strain in which the promoter of this operon directs the transcription of the luxAB reporter genes, we determined that inactivation of the slr0640 gene, which encodes a histidine kinase sensor protein component of a two-component signal transduction system, resulted in constitutive high levels of lux luminescence. Systematic targeted inactivation mutagenesis also identified slr1837 as the gene encoding the corresponding response regulator protein. We have named these two genes manS (manganese-sensor) and manR (manganese-regulator), respectively. A polyhistidine-tagged form of the ManS protein was localized in the Synechocystis 6803 cell membrane. Directed replacement of the conserved catalytic His-205 residue of this protein by Leu abolished its activity, although the mutated protein was present in cyanobacterial membrane. This mutant also showed suboptimal rates of Mn uptake under either Mn-starved or Mn-sufficient growth condition. These data suggest that the ManS/ManR two-component system plays a central role in the homeostasis of manganese in Synechocystis 6803 cells.

Biochemistry

A highly active oxygen-evolving photosystem II (PSII) complex was purified from the HT-3 strain of the widely used cyanobacterium Synechocystis sp. PCC 6803, in which the CP47 polypeptide has been genetically engineered to contain a polyhistidine tag at its carboxyl terminus [Bricker, T. M., Morvant, J., Masri, N., Sutton, H. M., and Frankel, L. K. (1998) Biochim. Biophys. Acta 1409, 50-57]. These purified PSII centers had four manganese atoms, one calcium atom, and two cytochrome b(559) hemes each. Optical absorption and fluorescence emission spectroscopy as well as western immunoblot analysis demonstrated that the purified PSII preparation was devoid of any contamination with photosystem I and phycobiliproteins. A comprehensive proteomic analysis using a system designed to enhance resolution of low-molecular-weight polypeptides, followed by MALDI mass spectrometry and N-terminal amino acid sequencing, identified 31 distinct polypeptides in this PSII preparation. We propose a new nomenclature for the polypeptide components of PSII identified after PsbZ, which proceeds sequentially from Psb27. During this study, the polypeptides PsbJ, PsbM, PsbX, PsbY, PsbZ, Psb27, and Psb28 proteins were detected for the first time in a purified PSII complex from Synechocystis 6803. Five novel polypeptides were also identified in this preparation. They included the Sll1638 protein, which shares significant sequence similarity to PsbQ, a peripheral protein of PSII that was previously thought to be present only in chloroplasts. This work describes newly identified proteins in a highly purified cyanobacterial PSII preparation that is being widely used to investigate the structure, function, and biogenesis of this photosystem.

Biochemistry

The rfrA gene was identified in a suppressor screen of a Synechocystis sp. PCC 6803 strain deficient in both mntC, encoding a component of an ABC transport system for manganese, and psbO, encoding the extrinsic manganese stabilizing protein of photosystem II (PSII). A spontaneous suppressor mutant (DeltaCDeltaO rfrA-Sup) has a point mutation in rfrA, which restores photosynthetic activity to the DeltamntCDeltapsbO double mutant. Manganese transport and photosynthesis are related in that manganese is essential to the function of PSII, and the state of cellular manganese availability influences the rate of oxygen evolution mediated by PSII. Oxygen evolution experiments with the DeltaCDeltaO rfrA-Sup mutant revealed that the mechanism of suppression is not through a direct modification of PSII. Instead, radioactive manganese uptake experiments indicated that RfrA is a regulator of a high affinity manganese transport system different from the more thoroughly characterized manganese ABC transport system in Synechocystis 6803. RfrA was named for the repeated five-residues domain in the amino terminus of the protein. The RFR domain defines a 16-member family in Synechocystis 6803. Predicted proteins with RFR domains have also been identified in other organisms, but RfrA is the first member of this family to be linked to a physiological process.

Plant & Cell Physiology

Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.

Biochemistry

Manganese is an essential micronutrient for many organisms. Because of its unique role in the water oxidizing activity of photosystem II, manganese is required for photosynthetic growth in plants and cyanobacteria. Here we report on the mechanism of manganese uptake in the cyanobacterium Synechocystis sp. PCC 6803. Cells grown in 9 microM manganese-containing medium accumulate up to 1 x 10(8) manganese atoms/cell, bound to the outer membrane (pool A). This pool could be released by EDTA treatment. Accumulation of manganese in pool A was energized by photosynthetic electron flow. Moreover, collapsing the membrane potential resulted in the immediate release of this manganese pool. The manganese in this pool is mainly Mn(II) in a six-coordinate distorted environment. A distinctly different pool of manganese, pool B ( approximately 1.5 x 10(6) atoms/cell), could not be extracted by EDTA. Transport into pool B was light-independent and could be detected only under limiting manganese concentrations (1 nM). Evidently, manganese uptake in Synechocystis 6803 cells occurs in two steps. First, manganese accumulates in the outer membrane (pool A) in a membrane potential-dependent process. Next, manganese is transported through the inner membrane into pool B. We propose that pool A serves as a store that allows the cells to overcome transient limitations in manganese in the environment.

Molecular & Cellular Proteomics : MCP

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.

The Biochemical Journal

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Biochemistry

PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], we investigated the function of PsbU using a psbU deletion mutant (DeltaPsbU) of Synechocystis 6803. In contrast to the wild type, DeltaPsbU did not grow when both Ca2+ and Cl- were eliminated from the growth medium. When only Ca2+ was eliminated, DeltaPsbU grew well, whereas when Cl- was eliminated, the growth rate was highly suppressed. Although DeltaPsbU grew normally in the presence of both ions under moderate light, PS II-related disorders were observed as follows. (1) The mutant cells were highly susceptible to photoinhibition. (2) Both the efficiency of light utilization under low irradiance and the chlorophyll-specific maximum rate of oxygen evolution in DeltaPsbU cells were 60% lower than those of the wild type. (3) The decay of the S2 state in DeltaPsbU cells was decelerated. (4) In isolated PS II complexes from DeltaPsbU cells, the amounts of the other three lumenal extrinsic proteins and the electron donation rate were drastically decreased, indicating that the water oxidation system became significantly labile without PsbU. Furthermore, oxygen-evolving activity in DeltaPsbU thylakoid membranes was highly suppressed in the absence of Cl-, and 60% of the activity was restored by NO3- but not by SO4(2-), indicating that PsbU had functions other than stabilizing Cl-. On the basis of these results, we conclude that PsbU is crucial for the stable architecture of the water-splitting system to optimize the efficiency of the oxygen evolution process.

The Plant Cell

The mechanism of oxygen evolution by photosystem II (PSII) has remained highly conserved during the course of evolution from ancestral cyanobacteria to green plants. A cluster of manganese, calcium, and chloride ions, whose binding environment is optimized by PSII extrinsic proteins, catalyzes this water-splitting reaction. The accepted view is that in plants and green algae, the three extrinsic proteins are PsbO, PsbP, and PsbQ, whereas in cyanobacteria, they are PsbO, PsbV, and PsbU. Our previous proteomic analysis established the presence of a PsbQ homolog in the cyanobacterium Synechocystis 6803. The current study additionally demonstrates the presence of a PsbP homolog in cyanobacterial PSII. Both psbP and psbQ inactivation mutants exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl(2)-depleted conditions. Moreover, purified PSII complexes from each mutant had significantly reduced activity. In cyanobacteria, one PsbQ is present per PSII complex, whereas PsbP is significantly substoichiometric. These findings indicate that both PsbP and PsbQ proteins are regulators that are necessary for the biogenesis of optimally active PSII in Synechocystis 6803. The new picture emerging from these data is that five extrinsic PSII proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV, are present in cyanobacteria, two of which, PsbU and PsbV, have been lost during the evolution of green plants.

The Journal of Biological Chemistry

Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated DeltactpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor Y(Z) was accessible to exogenous Mn(2+) ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in DeltactpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis.

The Journal of Biological Chemistry

Cyanobacterial cells have two autonomous internal membrane systems, plasma membrane and thylakoid membrane. In these oxygenic photosynthetic organisms the assembly of the large membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in thylakoid membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the thylakoid membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the thylakoid membrane.

Journal of Molecular Biology

A number of bacterial metal transporters belong to the cluster 9 family of ABC transporters. The residues in the periplasmic domain thought to be involved in metal binding seem highly conserved and yet the transporters have varying metal specificity. To solve this seeming paradox and ascertain how metal specificity is exacted, the structure of ZnuA, the periplasmic domain of a zinc transporter from Synechocystis 6803, has been determined to a resolution of 1.9A. In previously determined structures of homologous proteins, four residues chelate the bound metal. From sequence alignments of the cluster 9 metal transporters, the fourth residue in this metal-binding site, an aspartate, is also present in the appropriate position in the ZnuA sequence. However, this result is misleading, since our structural data indicate that zinc binds via only three histidine residues and the aspartate is replaced by a large hydrophobic cavity. We propose that ZnuA binds zinc over manganese by providing only three ligating residues. ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal-binding site. Similar loops are found in other types of zinc transporters but not manganese transporters. Therefore, we propose that the function of this domain is to act as a zinc chaperone to facilitate acquisition. Therefore, while Mn2+ transporters can bind Zn2+ in vitro they may not be able to acquire it in vivo without this structure because of the low concentration of free Zn2+.

Plant & Cell Physiology

Exposure to methyl viologen in the presence of light facilitates the production of superoxide that gives severe damage on photosynthetic apparatus as well as many cellular processes in cyanobacteria and plants. The effects of methyl viologen on global gene expression of a unicellular cyanobacterium Synechocystis sp. strain PCC 6803 were determined by DNA microarray. The ORFs sll1621, slr1738, slr0074, slr0075, and slr0589 were significantly induced by treatment of methyl viologen for 15 min commonly under conditions of normal and high light. One of these genes, slr1738, which encodes a ferric uptake repressor (Fur)-type transcriptional regulator, is located divergently next to another induced gene, sll1621, in the genome. We deleted slr1738, and compared the global gene expression patterns of this mutant to that of wild type under non-stressed conditions. It was found that sll1621 was derepressed to the greatest extent, while many other genes including slr0589 but not slr0074 or slr0075 were derepressed to lesser extent in the mutant. Genetic disruption of sll1621, which encodes a putative type 2 peroxiredoxin, indicates that it is essential for aerobic phototrophic growth in both liquid and solid media in high light and on solid medium even in low light. Slr1738 was prepared as a His-tagged recombinant protein and shown to specifically bind to the intergenic region between sll1621 and slr1738. The binding was enhanced by dithiothreitol and abolished by hydrogen peroxide. We concluded that the Fur homolog, Slr1738, plays a regulatory role in the induction of a potent antioxidant gene, sll1621, in response to oxidative stress.

Plant Physiology

Cyanobacteria are key contributors to global photosynthetic productivity, and iron availability is essential for cyanobacterial proliferation. While iron is abundant in the earth's crust, its unique chemical properties render it a limiting factor for photoautotrophic growth. As compared to other nonphotosynthetic organisms, oxygenic photosynthetic organisms such as cyanobacteria, algae, and green plants need large amounts of iron to maintain functional PSI complexes in their photosynthetic apparatus. Ferritins and bacterioferritins are ubiquitously present iron-storage proteins. We have found that in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803), bacterioferritins are responsible for the storage of as much as 50% of cellular iron. Synechocystis 6803, as well as many other cyanobacterial species, have two bacterioferritins, BfrA and BfrB, in which either the heme binding or di-iron center ligating residues are absent. Purified bacterioferritin complex from Synechocystis 6803 has both BfrA and BfrB proteins. Targeted mutagenesis of each of the two bacterioferritin genes resulted in poor growth under iron-deprived conditions. Inactivation of both genes did not result in a more severe phenotype. These results support the presence of a heteromultimeric structure of Synechocystis bacterioferritin, in which one subunit ligates a di-iron center while the other accommodates heme binding. Notably, the reduced internal iron concentrations in the mutant cells resulted in a lower content of PSI. In addition, they triggered iron starvation responses even in the presence of normal levels of external iron, thus demonstrating a central role of bacterioferritins in iron homeostasis in these photosynthetic organisms.

Omics : a Journal of Integrative Biology

Oxygenic photosynthetic organisms require light for their growth and development. However, exposure to high light is detrimental to them. Using time series microarray data from a model cyanobacterium, Synechocystis 6803 transferred from low to high light, we generated a gene co-expression network. The network has twelve sub-networks connected hierarchically, each consisting of an interconnected hub-and-spoke architecture. Within each sub-network, edges formed between genes that recapitulate known pathways. Analysis of the expression profiles shows that the cells undergo a phase transition 6-hours post-shift to high light, characterized by core sub-network. The core sub-network is enriched in proteins that (putatively) bind Fe-S clusters and proteins that mediate iron and sulfate homeostasis. At the center of this core is a sulfate permease, suggesting sulfate is rate limiting for cells grown in high light. To validate this novel finding, we demonstrate the limited ability of cell growth in sulfate-depleted medium in high light. This study highlights how understanding the organization of the networks can provide insights into the coordination of physiologic responses.

Journal of Structural Biology

Cyanothece sp. PCC 51142 contains 35 pentapeptide repeat proteins (PRPs), proteins that contain a minimum of eight tandem repeated five-residues (Rfr) of the general consensus sequence A[N/D]LXX. Published crystal structures of PRPs show that the tandem pentapeptide repeats adopt a type of right-handed quadrilateral beta-helix called an Rfr-fold. To characterize how structural features of Rfr-folds might vary with different amino acid sequences, the crystal structure of Cyanothece Rfr23 (174 residues) was determined at 2.4A resolution. The structure is dominated by an Rfr-fold capped at the N-terminus with a nine-residue alpha-helix (M26(*)-E34). The Rfr-fold of Rfr23 contains four structural features previously unobserved in Rfr-folds. First, Rfr23 is composed entirely of type II beta-turns. Second, the pentapeptide repeats are not consecutive in the primary amino acid sequence. Instead, Rfr23 contains 24-residues protruding outside one corner of the first complete N-terminal coil of the Rfr-fold (L56-P79) (24-residue insertion). Third, a disulfide bond between C39 and C42 bridges the beta-turn between the first and second pentapeptide repeats in the first coil (disulfide bracket). NMR spectroscopy indicates that the reduction of the disulfide bracket with the addition of DTT destroys the entire Rfr-fold. Fourth, a single-residue perturbs the Rfr-fold slightly in the last coil between the C-terminal two pentapeptide repeats (single-residue bulge).

Biotechnology and Bioengineering

Small-scale photobioreactors for cultivation of photoautotrophic microbes are required for precise characterization of the growth parameters of wild-type and engineered strains of these organisms, for their screening, and for optimization of culture conditions. Here, we describe the design and use of a flat-cuvette photobioreactor that allows accurate control of culture irradiance, temperature, pH, and gas composition combined with real-time monitoring by a built-in fluorometer and densitometer. The high-power LED light source generates precise irradiance levels that are programmed by user-designed protocols. The irradiance, temperature, and gas composition may be static or dynamically modulated, while optical density and pH may be stabilized in turbidostat and pH-stat modes, respectively. We demonstrate that the instrument is able to detect minute variations of growth caused, for example, by sudden dilution or by circadian rhythms. The sensitivity of the instrument is sufficient to monitor suspension optical density as low as 10(-2). This newly designed photobioreactor can significantly contribute to the study and use of photoautotrophic microbes in systems biology and biotechnology.

The Journal of Biological Chemistry

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO2 over O2, cyanobacteria have developed highly efficient CO2-concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here, we have described the structure of the bicarbonate-binding protein CmpA in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket, whereas bicarbonate binds in nearly an identical location compared with nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca2+ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal appears to be highly cooperative and suggests that CmpA may co-transport bicarbonate and calcium or that calcium acts a cofactor in bicarbonate transport.

Photosynthesis Research

Years of genetic, biochemical, and structural work have provided a number of insights into the oxygen evolving complex (OEC) of Photosystem II (PSII) for a variety of photosynthetic organisms. However, questions still remain about the functions and interactions among the various subunits that make up the OEC. After a brief introduction to the individual subunits Psb27, PsbP, PsbQ, PsbR, PsbU, and PsbV, a current picture of the OEC as a whole in cyanobacteria, red algae, green algae, and higher plants will be presented. Additionally, the role that these proteins play in the dynamic life cycle of PSII will be discussed.

Biochemistry

A number of bacterial metal transporters belong to the ABC transporter family. To better understand the structural determinants of metal selectivity of one such transporter, we previously determined the structure of the periplasmic domain of a zinc transporter, ZnuA, from Synechocystis 6803 and found that ZnuA binds zinc via three histidines. Unique to these ABC zinc transporters, ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal binding site that we had suggested might facilitate zinc acquisition. To further examine the function of this loop, the structure and zinc binding properties of two ZnuA variants were determined. When the loop is entirely deleted, zinc still binds to the three histidines. However, unlike what was suggested from the structure of a similar solute binding protein, TroA, release of zinc occurs concomitantly with large conformational changes in two of the three chelating histidines. These structural results combined with isothermal titration calorimetry data demonstrate that there are at least two classes of zinc binding sites: the high-affinity site in the cleft between the two domains and at least one additional site on the flexible loop. This loop has approximately 100-fold weaker affinity for zinc than the high-affinity zinc binding site, and its deletion does not affect the high-affinity site. From these results, we suggest that this region might be a sensor for high periplasmic levels of zinc.

The Journal of Biological Chemistry

Cyanobacteria account for a significant percentage of aquatic primary productivity even in areas where the concentrations of essential micronutrients are extremely low. To better understand the mechanism of iron selectivity and transport, the structure of the solute binding domain of an ATP binding cassette iron transporter, FutA1, was determined in the presence and absence of iron. The iron ion is bound within the "C-clamp" structure via four tyrosine and one histidine residues. There are extensive interactions between these ligating residues and the rest of the protein such that the conformations of the side chains remain relatively unchanged as the iron is released by the opening of the metal binding cleft. This is in stark contrast to the zinc-binding protein, ZnuA, where the domains of the metal-binding protein remain relatively fixed, whereas the ligating residues rotate out of the binding pocket upon metal release. The rotation of the domains in FutA1 is facilitated by two flexible beta-strands running along the back of the protein that act like a hinge during domain motion. This motion may require relatively little energy since total contact area between the domains is the same whether the protein is in the open or closed conformation. Consistent with the pH dependence of iron binding, the main trigger for iron release is likely the histidine in the iron-binding site. Finally, neither FutA1 nor FutA2 binds iron as a siderophore complex or in the presence of anions, and both preferentially bind ferrous over ferric ions.

The Journal of Biological Chemistry

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

Protoplasma

Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells.

Proceedings of the National Academy of Sciences of the United States of America

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of all aquatic food chains by fixing carbon and nitrogen into cellular biomass. The single most important nutrient for photosynthesis and growth is nitrate, which is severely limiting in many aquatic environments particularly the open ocean. It is therefore not surprising that NrtA, the solute-binding component of the high-affinity nitrate ABC transporter, is the single-most abundant protein in the plasma membrane of these bacteria. Here, we describe the structure of a nitrate-specific receptor, NrtA from Synechocystis sp. PCC 6803, complexed with nitrate and determined to a resolution of 1.5 A. NrtA is significantly larger than other oxyanion-binding proteins, representing a previously uncharacterized class of transport proteins. From sequence alignments, the only other solute-binding protein in this class is CmpA, a bicarbonate-binding protein. Therefore, these organisms created a solute-binding protein for two of the most important nutrients: inorganic nitrogen and carbon. The electrostatic charge distribution of NrtA appears to force the protein off the membrane while the flexible tether facilitates the delivery of nitrate to the membrane pore. The structure not only details the determinants for nitrate selectivity in NrtA but also the bicarbonate specificity in CmpA. Nitrate and bicarbonate transport are regulated by the cytoplasmic proteins NrtC and CmpC, respectively. Interestingly, the residues lining the ligand binding pockets suggest that they both bind nitrate. This implies that the nitrogen and carbon uptake pathways are synchronized by intracellular nitrate and nitrite.

Protein Science : a Publication of the Protein Society

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.

Proceedings of the National Academy of Sciences of the United States of America

Light-induced conversion of water to molecular oxygen by Photosystem II (PSII) is one of the most important enzymatic reactions in the biosphere. PSII is a multisubunit membrane protein complex with numerous associated cofactors, but it continually undergoes assembly and disassembly due to frequent light-mediated damage as a result of its normal function. Thus, at any instant, there is heterogeneity in the subunit compositions of PSII complexes within the cell. In particular, cyanobacterial PSII complexes have five associated extrinsic proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV. However, little is known about the interactions of the more recently identified PsbQ protein with other components in cyanobacterial PSII. Here we show that PSII complexes can be isolated from the cyanobacterium Synechocystis sp. PCC 6803 on the basis of the presence of a polyhistidine-tagged PsbQ protein. Purification of PSII complexes using a tagged extrinsic protein has not been previously described, and this work conclusively demonstrates that PsbQ is present in combination with the PsbO, PsbU, and PsbV proteins in cyanobacterial PSII. Moreover, PsbQ-associated PSII complexes have higher activity and stability relative to those isolated using histidine-tagged CP47, an integral membrane protein. Therefore, we conclude that the presence of PsbQ defines the fully assembled and optimally active form of the enzyme.

The Plant Cell

Photosystem II (PSII), the enzyme responsible for photosynthetic oxygen evolution, is a rapidly turned over membrane protein complex. However, the factors that regulate biogenesis of PSII are poorly defined. Previous proteomic analysis of the PSII preparations from the cyanobacterium Synechocystis sp PCC 6803 detected a novel protein, Psb29 (Sll1414), homologs of which are found in all cyanobacteria and vascular plants with sequenced genomes. Deletion of psb29 in Synechocystis 6803 results in slower growth rates under high light intensities, increased light sensitivity, and lower PSII efficiency, without affecting the PSII core electron transfer activities. A T-DNA insertion line in the PSB29 gene in Arabidopsis thaliana displays a phenotype similar to that of the Synechocystis mutant. This plant mutant grows slowly and exhibits variegated leaves, and its PSII activity is light sensitive. Low temperature fluorescence emission spectroscopy of both cyanobacterial and plant mutants shows an increase in the proportion of uncoupled proximal antennae in PSII as a function of increasing growth light intensities. The similar phenotypes observed in both plant and cyanobacterial mutants demonstrate that the function of Psb29 has been conserved throughout the evolution of oxygenic photosynthetic organisms and suggest a role for the Psb29 protein in the biogenesis of PSII.

Cell Biology Education

This inquiry-based lab is designed around genetic diseases with a focus on protein structure and function. To allow students to work on their own investigatory projects, 10 projects on 10 different proteins were developed. Students are grouped in sections of 20 and work in pairs on each of the projects. To begin their investigation, students are given a cDNA sequence that translates into a human protein with a single mutation. Each case results in a genetic disease that has been studied and recorded in the Online Mendelian Inheritance in Man (OMIM) database. Students use bioinformatics tools to investigate their proteins and form a hypothesis for the effect of the mutation on protein function. They are also asked to predict the impact of the mutation on human physiology and present their findings in the form of an oral report. Over five laboratory sessions, students use tools on the National Center for Biotechnology Information (NCBI) Web site (BLAST, LocusLink, OMIM, GenBank, and PubMed) as well as ExPasy, Protein Data Bank, ClustalW, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the structure-viewing program DeepView. Assessment results showed that students gained an understanding of the Web-based databases and tools and enjoyed the investigatory nature of the lab.

The Journal of Biological Chemistry

We have previously reported that cyanobacterial photosystem II (PS II) contains a protein homologous to PsbQ, the extrinsic 17-kDa protein found in higher plant and green algal PS II (Kashino, Y., Lauber, W. M., Carroll, J. A., Wang, Q., Whitmarsh, J., Satoh, K., and Pakrasi, H. B. (2002) Biochemistry 41, 8004-8012) and that it has regulatory role(s) on the water oxidation machinery (Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175). In this work, the localization and the function of PsbQ were assessed using the cyanobacterium Synechocystis sp. PCC 6803. From the predicted sequence, cyanobacterial PsbQ is expected to be a lipoprotein on the luminal side of the thylakoid membrane. Indeed, experiments in this work show that upon Triton X-114 fractionation of thylakoid membranes, PsbQ partitioned in the hydrophobic phase, and trypsin digestion revealed that PsbQ was highly exposed to the luminal space of thylakoid membranes. Detailed functional assays were conducted on the psbQ deletion mutant (DeltapsbQ) to analyze its water oxidation machinery. PS II complexes purified from DeltapsbQ mutant cells had impaired oxygen evolution activity and were remarkably sensitive to NH(2)OH, which indicates destabilization of the water oxidation machinery. Additionally, the cytochrome c(550) (PsbV) protein partially dissociated from purified DeltapsbQ PS II complexes, suggesting that PsbQ contributes to the stability of PsbV in cyanobacterial PS II. Therefore, we conclude that the major function of PsbQ is to stabilize the PsbV protein, thereby contributing to the protection of the catalytic Mn(4)-Ca(1)-Cl(x) cluster of the water oxidation machinery.

Photosynthesis Research

With the discovery of targeted gene replacement, moss biology has been rapidly advancing over the last 10 years. This study demonstrates the usefulness of moss as a model organism for plant photosynthesis research. The two mosses examined in this study, Physcomitrella patens and Ceratodon purpureus, are easily cultured through vegetative propagation. Growth tests were conducted to determine carbon sources suitable for maintaining heterotrophic growth while photosynthesis was blocked. Photosynthetic parameters examined in these plants indicated that the photosynthetic activity of Ceratodon and Physcomitrella is more similar to vascular plants than cyanobacteria or green algae. Ceratodon plants grown heterotrophically appeared etiolated in that the plants were taller and plastids did not differentiate thylakoid membranes. After returning to the light, the plants developed green, photosynthetically active chloroplasts. Furthermore, UV-induced mutagenesis was used to show that photosynthesis-deficient mutant Ceratodon plants could be obtained. After screening approximately 1000 plants, we obtained a number of mutants, which could be arranged into the following categories: high fluorescence, low fluorescence, fast and slow fluorescence quenching, and fast and slow greening. Our results indicate that in vivo biophysical analysis of photosynthetic activity in the mosses can be carried out which makes both mosses useful for photosynthesis studies, and Ceratodon best sustains perturbations in photosynthetic activity.

BMC Systems Biology

ABSTRACT:

Proceedings of the National Academy of Sciences of the United States of America

Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn(4)Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K(63)↔CP43D(321) (trypsin) and CP43K(215)↔Psb27D(58)AGGLK(63)↔CP43D(321) (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.

Journal of Proteome Research

Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a (13)C(15)N-l-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen-sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 414 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly, and degradation showed higher levels of isotope incorporation, suggesting that these biochemical pathways are important for growth under continuous light. Calculation of relative isotope abundances (RIA) values allowed the measurement of actual active protein synthesis over time for different biochemical pathways under high light exposure. Overall results demonstrated the utility of "non-steady state" pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

Plant & Cell Physiology

Antibody against cMyc cross-reacted strongly with the CupB protein tagged with His6-cMyc (HM) in thylakoid membrane of Synechocystis sp. strain PCC 6803 but only faintly with the cytoplasmic membrane fraction. The protein was not detected in the membranes of the DeltandhD4 and DeltandhF4 mutants in which CupB was tagged with HM. We concluded that a CupB complex containing NdhD4 and NdhF4 is largely, if not exclusively, confined to the thylakoid membrane. Both CupB and NdhH were detected in a fraction containing protein complexes of > 450 kDa, obtained after nickel column and gel filtration chromatography of the membranes solubilized with n-dodecyl-beta-maltoside.

Molecular Plant

Photosystem II (PSII), a membrane protein complex, catalyzes the photochemical oxidation of water to molecular oxygen. This enzyme complex consists of approximately 20 stoichiometric protein components. However, due to the highly energetic reactions it catalyzes as part of its normal activity, PSII is continuously damaged and repaired. With advances in protein detection technologies, an increasing number of sub-stoichiometric PSII proteins have been identified, many of which aid in the biogenesis and assembly of this protein complex. Psb32 (Sll1390) has previously been identified as a protein associated with highly active purified PSII preparations from the cyanobacterium Synechocystis sp. PCC 6803. To investigate its function, the subcellular localization of Psb32 and the impact of deletion of the psb32 gene on PSII were analyzed. Here, we show that Psb32 is an integral membrane protein, primarily located in the thylakoid membranes. Although not required for cell viability, Psb32 protects cells from oxidative stress and additionally confers a selective fitness advantage in mixed culture experiments. Specifically, Psb32 protects PSII from photodamage and accelerates its repair. Thus, the data suggest that Psb32 plays an important role in minimizing the effect of photoinhibition on PSII.

Molecular BioSystems

Systems biology attempts to reconcile large amounts of disparate data with existing knowledge to provide models of functioning biological systems. The cyanobacterium Cyanothece sp. ATCC 51142 is an excellent candidate for such systems biology studies because: (i) it displays tight functional regulation between photosynthesis and nitrogen fixation; (ii) it has robust cyclic patterns at the genetic, protein and metabolomic levels; and (iii) it has potential applications for bioenergy production and carbon sequestration. We have represented the transcriptomic data from Cyanothece 51142 under diurnal light/dark cycles as a high-level functional abstraction and describe development of a predictive in silico model of diurnal and circadian behavior in terms of regulatory and metabolic processes in this organism. We show that incorporating network topology into the model improves performance in terms of our ability to explain the behavior of the system under new conditions. The model presented robustly describes transcriptomic behavior of Cyanothece 51142 under different cyclic and non-cyclic growth conditions, and represents a significant advance in the understanding of gene regulation in this important organism.

MBio

The genus Cyanothece comprises unicellular cyanobacteria that are morphologically diverse and ecologically versatile. Studies over the last decade have established members of this genus to be important components of the marine ecosystem, contributing significantly to the nitrogen and carbon cycle. System-level studies of Cyanothece sp. ATCC 51142, a prototypic member of this group, revealed many interesting metabolic attributes. To identify the metabolic traits that define this class of cyanobacteria, five additional Cyanothece strains were sequenced to completion. The presence of a large, contiguous nitrogenase gene cluster and the ability to carry out aerobic nitrogen fixation distinguish Cyanothece as a genus of unicellular, aerobic nitrogen-fixing cyanobacteria. Cyanothece cells can create an anoxic intracellular environment at night, allowing oxygen-sensitive processes to take place in these oxygenic organisms. Large carbohydrate reserves accumulate in the cells during the day, ensuring sufficient energy for the processes that require the anoxic phase of the cells. Our study indicates that this genus maintains a plastic genome, incorporating new metabolic capabilities while simultaneously retaining archaic metabolic traits, a unique combination which provides the flexibility to adapt to various ecological and environmental conditions. Rearrangement of the nitrogenase cluster in Cyanothece sp. strain 7425 and the concomitant loss of its aerobic nitrogen-fixing ability suggest that a similar mechanism might have been at play in cyanobacterial strains that eventually lost their nitrogen-fixing ability. IMPORTANCE: The unicellular cyanobacterial genus Cyanothece has significant roles in the nitrogen cycle in aquatic and terrestrial environments. Cyanothece sp. ATCC 51142 was extensively studied over the last decade and has emerged as an important model photosynthetic microbe for bioenergy production. To expand our understanding of the distinctive metabolic capabilities of this cyanobacterial group, we analyzed the genome sequences of five additional Cyanothece strains from different geographical habitats, exhibiting diverse morphological and physiological attributes. These strains exhibit high rates of N(2) fixation and H(2) production under aerobic conditions. They can generate copious amounts of carbohydrates that are stored in large starch-like granules and facilitate energy-intensive processes during the dark, anoxic phase of the cells. The genomes of some Cyanothece strains are quite unique in that there are linear elements in addition to a large circular chromosome. Our study provides novel insights into the metabolism of this class of unicellular nitrogen-fixing cyanobacteria.

Plant Signaling & Behavior

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is left-handed in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.

Applied and Environmental Microbiology

Understanding mechanisms of antibiotic resistance is important to the fields of biology and medicine. We find that glutathione contributes to antibiotic resistance in the cyanobacterium Synechocystis sp. PCC 6803. Our results also suggest that glutathione protects photosystem I from oxidative damage resulting from growth in the presence of gentamicin.

The Journal of Biological Chemistry

Photosystem II (PSII) is a large membrane bound molecular machine that catalyzes light-driven oxygen evolution from water. PSII constantly undergoes assembly and disassembly because of the unavoidable damage that results from its normal photochemistry. Thus, under physiological conditions, in addition to the active PSII complexes, there are always PSII subpopulations incompetent of oxygen evolution, but are in the process of undergoing elaborate biogenesis and repair. These transient complexes are difficult to characterize because of their low abundance, structural heterogeneity, and thermodynamic instability. In this study, we show that a genetically tagged Psb27 protein allows for the biochemical purification of two monomeric PSII assembly intermediates, one with an unprocessed form of D1 (His27ΔctpAPSII) and a second one with a mature form of D1 (His27PSII). Both forms were capable of light-induced charge separation, but unable to photooxidize water, largely because of the absence of a functional tetramanganese cluster. Unexpectedly, there was a significant amount of the extrinsic lumenal PsbO protein in the His27PSII, but not in the His27ΔctpAPSII complex. In contrast, two other lumenal proteins, PsbU and PsbV, were absent in both of these PSII intermediate complexes. Additionally, the only cytoplasmic extrinsic protein, Psb28 was detected in His27PSII complex. Based on these data, we have presented a refined model of PSII biogenesis, illustrating an important role of Psb27 as a gate-keeper during the complex assembly process of the oxygen-evolving centers in PSII.

Plant Signaling & Behavior

Glutathione (GSH) is a low molecular weight thiol compound that plays many roles in photosynthetic organisms. We utilized a ∆gshB (glutathione synthetase) mutant strain as a tool to evaluate the role of GSH in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), a model photosynthetic organism. The ∆gshB mutant does not synthesize glutathione, but instead accumulates the GSH precursor, gamma-glutamylcysteine (gamma-EC), to millimolar levels. We found that gamma-EC was sufficient to permit cellular proliferation during optimal conditions, but not when cells were exposed to conditions promoting oxidative stress. Furthermore, we found that many factors affecting growth rate and photosynthetic activities strongly influenced cellular thiol content. Here, we are providing some additional insights into the role of GSH and gamma-EC in Synechocystis 6803 during conditions promoting oxidative stress.

PloS One

Cyanothece sp. ATCC 51142 is a diazotrophic cyanobacterium notable for its ability to perform oxygenic photosynthesis and dinitrogen fixation in the same single cell. Previous transcriptional analysis revealed that the existence of these incompatible cellular processes largely depends on tightly synchronized expression programs involving ∼30% of genes in the genome. To expand upon current knowledge, we have utilized sensitive proteomic approaches to examine the impact of diurnal rhythms on the protein complement in Cyanothece 51142. We found that 250 proteins accounting for ∼5% of the predicted ORFs from the Cyanothece 51142 genome and 20% of proteins detected under alternating light/dark conditions exhibited periodic oscillations in their abundances. Our results suggest that altered enzyme activities at different phases during the diurnal cycle can be attributed to changes in the abundance of related proteins and key compounds. The integration of global proteomics and transcriptomic data further revealed that post-transcriptional events are important for temporal regulation of processes such as photosynthesis in Cyanothece 51142. This analysis is the first comprehensive report on global quantitative proteomics in a unicellular diazotrophic cyanobacterium and uncovers novel findings about diurnal rhythms.

BMC Genomics

Life on earth is strongly affected by alternating day and night cycles. Accordingly, many organisms have evolved an internal timekeeping system with a period of approximately 24 hours. Cyanobacteria are the only known prokaryotes with robust rhythms under control of a central clock. Numerous studies have been conducted to elucidate components of the circadian clock and to identify circadian-controlled genes. However, the complex interactions between endogenous circadian rhythms and external cues are currently not well understood, and a direct and mathematical based comparison between light-mediated and circadian-controlled gene expression is still outstanding. Therefore, we combined and analyzed data from two independent microarray experiments, previously performed under alternating light-dark and continuous light conditions in Cyanothece sp. ATCC 51142, and sought to classify light responsive and circadian controlled genes.

Microbiology

The unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Cyanothece 51142) is able to grow aerobically under nitrogen-fixing conditions with alternating light-dark cycles or continuous illumination. This study investigated the effects of carbon and nitrogen sources on Cyanothece 51142 metabolism via (13)C-assisted metabolite analysis and biochemical measurements. Under continuous light (50 mumol photons m(-2) s(-1)) and nitrogen-fixing conditions, we found that glycerol addition promoted aerobic biomass growth (by twofold) and nitrogenase-dependent hydrogen production [up to 25 mumol H(2) (mg chlorophyll)( -1) h(-1)], but strongly reduced phototrophic CO(2) utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically, and the activity of light-dependent reactions (e.g. oxygen evolution) was not significantly reduced. In contrast, Synechocystis sp. PCC 6803 showed apparent mixotrophic metabolism under similar growth conditions. Isotopomer analysis also detected that Cyanothece 51142 was able to fix CO(2) via anaplerotic pathways, and to take up glucose and pyruvate for mixotrophic biomass synthesis.

BMC Systems Biology

Cyanobacteria are the only known prokaryotes capable of oxygenic photosynthesis. They play significant roles in global biogeochemical cycles and carbon sequestration, and have recently been recognized as potential vehicles for production of renewable biofuels. Synechocystis sp. PCC 6803 has been extensively used as a model organism for cyanobacterial studies. DNA microarray studies in Synechocystis have shown varying degrees of transcriptome reprogramming under altered environmental conditions. However, it is not clear from published work how transcriptome reprogramming affects pre-existing networks of fine-tuned cellular processes.

IEEE/ACM Transactions on Computational Biology and Bioinformatics / IEEE, ACM

Behavior of living organisms is strongly modulated by the day and night cycle giving rise to a cyclic pattern of activities. Such a pattern helps the organisms to coordinate their activities and maintain a balance between what could be performed during the "day" and what could be relegated to the "night." This cyclic pattern, called the "Circadian Rhythm," is a biological phenomenon observed in a large number of organisms. In this paper, our goal is to analyze transcriptome data from Cyanothece for the purpose of discovering genes whose expressions are rhythmic. We cluster these genes into groups that are close in terms of their phases and show that genes from a specific metabolic functional category are tightly clustered, indicating perhaps a "preferred time of the day/night" when the organism performs this function. The proposed analysis is applied to two sets of microarray experiments performed under varying incident light patterns. Subsequently, we propose a model with a network of three phase oscillators together with a central master clock and use it to approximate a set of "circadian-controlled genes" that can be approximated closely.

Plant Physiology

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.

Nature Communications

Among the emerging renewable and green energy sources, biohydrogen stands out as an appealing choice. Hydrogen can be produced by certain groups of microorganisms that possess functional nitrogenase and/or bidirectional hydrogenases. In particular, the potential of photobiological hydrogen production by oxygenic photosynthetic microbes has attracted significant interest. However, nitrogenase and hydrogenase are generally oxygen sensitive, and require protective mechanisms to function in an aerobic extracellular environment. Here, we describe Cyanothece sp. ATCC 51142, a unicellular, diazotrophic cyanobacterium with the capacity to generate high levels of hydrogen under aerobic conditions. Wild-type Cyanothece 51142 can produce hydrogen at rates as high as 465 μmol per mg of chlorophyll per hour in the presence of glycerol. Hydrogen production in this strain is mediated by an efficient nitrogenase system, which can be manipulated to convert solar energy into hydrogen at rates that are several fold higher, compared with any previously described wild-type hydrogen-producing photosynthetic microbe.

Journal of Biomedicine & Biotechnology

Metabolic flux analysis is a vital tool used to determine the ultimate output of cellular metabolism and thus detect biotechnologically relevant bottlenecks in productivity. ¹³C-based metabolic flux analysis (¹³C-MFA) and flux balance analysis (FBA) have many potential applications in biotechnology. However, noteworthy hurdles in fluxomics study are still present. First, several technical difficulties in both ¹³C-MFA and FBA severely limit the scope of fluxomics findings and the applicability of obtained metabolic information. Second, the complexity of metabolic regulation poses a great challenge for precise prediction and analysis of metabolic networks, as there are gaps between fluxomics results and other omics studies. Third, despite identified metabolic bottlenecks or sources of host stress from product synthesis, it remains difficult to overcome inherent metabolic robustness or to efficiently import and express nonnative pathways. Fourth, product yields often decrease as the number of enzymatic steps increases. Such decrease in yield may not be caused by rate-limiting enzymes, but rather is accumulated through each enzymatic reaction. Fifth, a high-throughput fluxomics tool hasnot been developed for characterizing nonmodel microorganisms and maximizing their application in industrial biotechnology. Refining fluxomics tools and understanding these obstacles will improve our ability to engineer highly efficient metabolic pathways in microbial hosts.

Biochemistry

Sll1252 was identified as a novel protein in photosystem II complexes from Synechocystis sp. PCC 6803. To investigate the function of Sll1252, the corresponding gene, sll1252, was deleted in Synechocystis 6803. Despite the homology of Sll1252 to YlmH, which functions in the cell division machinery in Streptococcus, the growth rate and cell morphology of the mutant were not affected in normal growth medium. Instead, it seems that cells lacking this polypeptide have increased sensitivity to Cl(-) depletion. The growth and oxygen evolving activity of the mutant cells was highly suppressed compared with those of wild-type cells when Cl(-) and/or Ca(2+) was depleted from the medium. Recovery of photosystem II from photoinhibition was suppressed in the mutant. Despite the defects in photosystem II, in the light, the acceptor side of photosystem II was more reduced and the donor side of photosystem I was more oxidized compared with wild-type cells, suggesting that functional impairments were also present in cytochrome b(6)/f complexes. The amounts of cytochrome c(550) and cytochrome f were smaller in the mutant in the Ca(2+)- and Cl(-)-depleted medium. Furthermore, the amount of IsiA protein was increased in the mutant, especially in the Cl(-)-depleted medium, indicating that the mutant cells perceive environmental stress to be greater than it is. The amount of accompanying cytochrome c(550) in purified photosystem II complexes was also smaller in the mutant. Overall, the Sll1252 protein appears to be closely related to redox sensing of the plastoquinone pool to balance the photosynthetic electron flow and the ability to cope with global environmental stresses.

Molecular & Cellular Proteomics : MCP

Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen.

Plant Physiology

Glutathione, a nonribosomal thiol tripeptide, has been shown to be critical for many processes in plants. Much less is known about the roles of glutathione in cyanobacteria, oxygenic photosynthetic prokaryotes that are the evolutionary precursor of the chloroplast. An understanding of glutathione metabolism in cyanobacteria is expected to provide novel insight into the evolution of the elaborate and extensive pathways that utilize glutathione in photosynthetic organisms. To investigate the function of glutathione in cyanobacteria, we generated deletion mutants of glutamate-cysteine ligase (gshA) and glutathione synthetase (gshB) in Synechocystis sp. PCC 6803. Complete segregation of the ΔgshA mutation was not achieved, suggesting that GshA activity is essential for growth. In contrast, fully segregated ΔgshB mutants were isolated and characterized. The ΔgshB strain lacks reduced glutathione (GSH) but instead accumulates the precursor compound γ-glutamylcysteine (γ-EC). The ΔgshB strain grows slower than the wild-type strain under favorable conditions and exhibits extremely reduced growth or death when subjected to conditions promoting oxidative stress. Furthermore, we analyzed thiol contents in the wild type and the ΔgshB mutant after subjecting the strains to multiple environmental and redox perturbations. We found that conditions promoting growth stimulate glutathione biosynthesis. We also determined that cellular GSH and γ-EC content decline following exposure to dark and blue light and during photoheterotrophic growth. Moreover, a rapid depletion of GSH and γ-EC is observed in the wild type and the ΔgshB strain, respectively, when cells are starved for nitrate or sulfate.

Plant & Cell Physiology

A mutant (Delta5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO(2) or HCO(3)(-) and was unable to grow in air with or without glucose. The Delta4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO(3)(-) uptake and grew under these conditions but more slowly than the wild-type strain. The Delta5 mutant required 1.7% CO(2) to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the Delta5 mutant more susceptible to high light than the Delta4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.

Plant Physiology

Photosynthetic organisms experience changes in light quantity and light quality in their natural habitat. In response to changes in light quality, these organisms redistribute excitation energy and adjust photosystem stoichiometry to maximize the utilization of available light energy. However, the response of other cellular processes to changes in light quality is mostly unknown. Here, we report a systematic investigation into the adaptation of cellular processes in Synechocystis species PCC 6803 to light that preferentially excites either photosystem II or photosystem I. We find that preferential excitation of photosystem II and photosystem I induces massive reprogramming of the Synechocystis transcriptome. The rewiring of cellular processes begins as soon as Synechocystis senses the imbalance in the excitation of reaction centers. We find that Synechocystis utilizes the cyclic photosynthetic electron transport chain for ATP generation and a major part of the respiratory pathway to generate reducing equivalents and carbon skeletons during preferential excitation of photosystem I. In contrast, cytochrome c oxidase and photosystem I act as terminal components of the photosynthetic electron transport chain to produce sufficient ATP and limited amounts of NADPH and reduced ferredoxin during preferential excitation of photosystem II. To overcome the shortage of NADPH and reduced ferredoxin, Synechocystis preferentially activates transporters and acquisition pathways to assimilate ammonia, urea, and arginine over nitrate as a nitrogen source. This study provides a systematic analysis of cellular processes in cyanobacteria in response to preferential excitation and shows that the cyanobacterial cell undergoes significant adjustment of cellular processes, many of which were previously unknown.

Microbiology

Cyanothece sp. ATCC 51142 is an aerobic N(2)-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids revealed an identical labelling profile for leucine and isoleucine when Cyanothece 51142 was grown mixotrophically using 2-(13)C-labelled glycerol as the main carbon source. This indicated that Cyanothece 51142 employs the atypical alternative citramalate pathway for isoleucine synthesis, with pyruvate and acetyl-CoA as precursors. Utilization of the citramalate pathway was confirmed by an enzyme assay and LC-MS/MS analysis. Furthermore, the genome sequence of Cyanothece 51142 shows that the gene encoding the key enzyme (threonine ammonia-lyase) in the normal isoleucine pathway is missing. Instead, the cce_0248 gene in Cyanothece 51142 exhibits 53 % identity to the gene encoding citramalate synthase (CimA, GSU1798) from Geobacter sulfurreducens. Reverse-transcription PCR indicated that the cce_0248 gene is expressed and its transcriptional level is lower in medium with isoleucine than in isoleucine-free medium. Additionally, a blast search for citramalate synthase and threonine ammonia-lyase implies that this alternative isoleucine synthesis pathway may be present in other cyanobacteria, such as Cyanothece and Synechococcus. This suggests that the pathway is more widespread than originally thought, as previous identifications of the citramalate pathway are limited to mostly anaerobic bacteria or archaea. Furthermore, this discovery opens the possibility that such autrotrophic micro-organisms may be engineered for robust butanol and propanol production from 2-ketobutyrate, which is an intermediate in the isoleucine biosynthesis pathway.

Advances in Experimental Medicine and Biology

Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium with a versatile metabolism and very pronounced diurnal rhythms. Since nitrogen fixation is exquisitely sensitive to oxygen, Cyanotheceutilizes temporal regulation to accommodate these incompatible processes in a single cell. When grown under 12 h light-dark (LD) periods, it performs photosynthesis during the day and N(2) fixation and respiration at night. Genome sequences of Cyanothece sp. ATCC 51142 and that of five other Cyanothece species have been completed and have produced some surprises. Analysis at both the transcriptomic and the proteomic levels in Cyanothece sp. ATCC 51142 has demonstrated the relationship of the metabolic synchrony with gene expression and has given us insights into diurnal and circadian regulation throughout a daily cycle. We are particularly interested in the regulation of metabolic processes, such as H(2) evolution, and the way in which these organisms respond to environmental cues, such as light, the lack of combined nitrogen, and changing O(2) levels. Cyanothece strains produce copious amounts of H(2) under different types of physiological conditions. Nitrogenase produces far more H(2) than the hydrogenase, in part because the nitrogenase levels are extremely high under N(2)-fixing conditions. With Cyanothece 51142 cultures grown in NO(3)-free media, either photoautotrophically or mixotrophically with glycerol, we have obtained H(2) production rates over 150 mumol/mg Chl/h.

Proceedings of the National Academy of Sciences of the United States of America

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 N(2)-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.

Plant Physiology

Light drives the production of chemical energy and reducing equivalents in photosynthetic organisms required for the assimilation of essential nutrients. This process also generates strong oxidants and reductants that can be damaging to the cellular processes, especially during absorption of excess excitation energy. Cyanobacteria, like other oxygenic photosynthetic organisms, respond to increases in the excitation energy, such as during exposure of cells to high light (HL) by the reduction of antenna size and photosystem content. However, the mechanism of how Synechocystis sp. PCC 6803, a cyanobacterium, maintains redox homeostasis and coordinates various metabolic processes under HL stress remains poorly understood. In this study, we have utilized time series transcriptome data to elucidate the global responses of Synechocystis to HL. Identification of differentially regulated genes involved in the regulation, protection, and maintenance of redox homeostasis has offered important insights into the optimized response of Synechocystis to HL. Our results indicate a comprehensive integrated homeostatic interaction between energy production (photosynthesis) and energy consumption (assimilation of carbon and nitrogen). In addition, measurements of physiological parameters under different growth conditions showed that integration between the two processes is not a consequence of limitations in the external carbon and nitrogen levels available to the cells. We have also discovered the existence of a novel glycosylation pathway, to date known as an important nutrient sensor only in eukaryotes. Up-regulation of a gene encoding the rate-limiting enzyme in the hexosamine pathway suggests a regulatory role for protein glycosylation in Synechocystis under HL.

The Journal of Biological Chemistry

Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of photosynthesis in cyanobacteria, algae, and plants. The unusual redox reactions in PSII often lead to damage, degradation, and reassembly of this molecular machine. To identify novel assembly factors, high sensitivity proteomic analysis of PSII purified from the cyanobacterium Synechocystis sp. PCC 6803 was performed. This analysis identified six PSII-associated proteins that are encoded by an operon containing nine genes, slr0144 to slr0152. This operon encodes proteins that are not essential components of the PSII holocomplex but accumulate to high levels in pre-complexes lacking any of the lumenal proteins PsbP, PsbQ, or PsbV. The operon contains genes with putative binding domains for chlorophylls and bilins, suggesting these proteins may function as a reservoir for cofactors needed during the PSII lifecycle. Genetic deletion of this operon shows that removal of these protein products does not alter photoautotrophic growth or PSII fluorescence properties. However, the deletion does result in decreased PSII-mediated oxygen evolution and an altered distribution of the S states of the catalytic manganese cluster. These data demonstrate that the proteins encoded by the genes in this operon are necessary for optimal function of PSII and function as accessory proteins during assembly of the PSII complex. Thus, we have named the products of the slr0144-slr0152 operon Pap (Photosystem II assembly proteins).

The Journal of Biological Chemistry

The evolution of oxygenic photosynthesis in cyanobacteria nearly three billion years ago provided abundant reducing power and facilitated the elaboration of numerous oxygen-dependent reactions in our biosphere. Cyanobacteria contain an internal thylakoid membrane system, the site of photosynthesis, and a typical Gram-negative envelope membrane system. Like other organisms, the extracytoplasmic space in cyanobacteria houses numerous cysteine-containing proteins. However, the existence of a biochemical system for disulfide bond formation in cyanobacteria remains to be determined. Extracytoplasmic disulfide bond formation in non-photosynthetic organisms is catalyzed by coordinated interaction between two proteins, a disulfide carrier and a disulfide generator. Here we describe a novel gene, SyndsbAB, required for disulfide bond formation in the extracytoplasmic space of cyanobacteria. The SynDsbAB orthologs are present in most cyanobacteria and chloroplasts of higher plants with fully sequenced genomes. The SynDsbAB protein contains two distinct catalytic domains that display significant similarity to proteins involved in disulfide bond formation in Escherichia coli and eukaryotes. Importantly, SyndsbAB complements E. coli strains defective in disulfide bond formation. In addition, the activity of E. coli alkaline phosphatase localized to the periplasm of Synechocystis 6803 is dependent on the function of SynDsbAB. Deletion of SyndsbAB in Synechocystis 6803 causes significant growth impairment under photoautotrophic conditions and results in hyper-sensitivity to dithiothreitol, a reductant, whereas diamide, an oxidant had no effect on the growth of the mutant strains. We conclude that SynDsbAB is a critical protein for disulfide bond formation in oxygenic photosynthetic organisms and required for their optimal photoautotrophic growth.

Proceedings of the National Academy of Sciences of the United States of America

Cyanobacteria are photosynthetic organisms and are the only prokaryotes known to have a circadian lifestyle. Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 produce oxygen and can also fix atmospheric nitrogen, a process exquisitely sensitive to oxygen. To accommodate such antagonistic processes, the intracellular environment of Cyanothece oscillates between aerobic and anaerobic conditions during a day-night cycle. This is accomplished by temporal separation of the two processes: photosynthesis during the day and nitrogen fixation at night. Although previous studies have examined periodic changes in transcript levels for a limited number of genes in Cyanothece and other unicellular diazotrophic cyanobacteria, a comprehensive study of transcriptional activity in a nitrogen-fixing cyanobacterium is necessary to understand the impact of the temporal separation of photosynthesis and nitrogen fixation on global gene regulation and cellular metabolism. We have examined the expression patterns of nearly 5,000 genes in Cyanothece 51142 during two consecutive diurnal periods. Our analysis showed that approximately 30% of these genes exhibited robust oscillating expression profiles. Interestingly, this set included genes for almost all central metabolic processes in Cyanothece 51142. A transcriptional network of all genes with significantly oscillating transcript levels revealed that the majority of genes encoding enzymes in numerous individual biochemical pathways, such as glycolysis, oxidative pentose phosphate pathway, and glycogen metabolism, were coregulated and maximally expressed at distinct phases during the diurnal cycle. These studies provide a comprehensive picture of how a physiologically relevant diurnal light-dark cycle influences the metabolism in a photosynthetic bacterium.

Journal of Bacteriology

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.

Plant Physiology

Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large membrane extrinsic phycobilisome antenna in addition to membrane bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess thylakoid membranes that house photosystems I and II that drive the oxidation of water and the reduction of NADP+, respectively. While thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of the two photosystems within the thylakoid membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis sp. PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the photosystem I to photosystem II titer is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of PSI and PSII under light-limiting conditions.