Publication Search Results

2009

Buchko GW, H Robinson, and A Addlagatta. 2009. "Structural characterization of the protein cce_0567 from Cyanothece 51142, a metalloprotein associated with nitrogen fixation in the DUF683 family." Biochimica et Biophysica Acta--Proteins and Proteomics 1794(4):627-633. Abstract The genome of many cyanobacacteria contain the sequence for a small protein (<100 amino acids) with a commom "domain of unknown function" grouped into the DUF683 protein family. While the biological function of DUF683 is still not known, their genomic location within nitrogen fixation clusters suggests that DUF683 proteins may play a role in the process. The diurnal cyanobacterium Cyanothece sp. PCC 51142 contains a gene for a protein that fall into the DUF683 family, cce_0567 (78 aa, 9.0 kDa). In an effort to elucidate the biochemical role DUF683 proteins may play in nitrogen fixation, we have determined the first crystal structure for a protein in this family, cce_0567, to 1.84 Å resolution. Cce_0567 crystallized in space group P21 with two protein molecules and one Ni2+ cation per asymmetric unit. The protein is composed of two α-helices from residues P11 to G41 (α1) and L49-E74 (α2) with the second α-helix containing a short 310-helix (Y46 - N48). A four-residue linker (L42 - D45) between the helices allows them to form an anti-parallel bundle that cross over each other towards their termini. In solution it is likely that two molecules of cce_0567 form a rod-like dimer by the stacking interactions of ~1/2 of the protein. Histidine-36 is highly conserved in all known DUF683 proteins and the N2 nitrogen of the H36 side chain of each molecule in the dimer coordinate with Ni2+ in the crystal structure. The divalent cation Ni2+ was titrated into 15N-labelled cce_0567 and chemical shift perturbations were observed only in the 1H-15N HSQC spectra for residues at, or near, the site of Ni2+ binding observed in the crystal structure. There was no evidence for an increase in the size of cce_0567 upon binding Ni2+, even in large molar excess of Ni2+, indicating that a metal was not required for dimer formation. Circular dichroism spectroscopy indicated that cce_0567 was extremely robust, with a melting temperature of ~62ºC that was reversible.
Toepel J, JE McDermott, T Summerfield, and LA Sherman. 2009. "Transcriptional analysis of the unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 grown under short day/night cycles." Journal of Phycology 45(3):610-620. Abstract Cyanothece sp. strain ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates extensive metabolic periodicities of photosynthesis, respiration and nitrogen fixation when grown under N2-fixing conditions. We have performed a global transcription analysis of this organism using 6 h light/dark cycles in order to determine the response of the cell to these conditions and to differentiate between diurnal and circadian regulated genes. In addition, we used a context-likelihood of relatedness (CLR) analysis with this data and those from two-day light/dark and light-dark plus continuous light experiments to better differentiate between diurnal and circadian regulated genes. Cyanothece sp. adapted in several ways to growth under short light/dark conditions. Nitrogen was fixed in every second dark period and only once in each 24 h period. Nitrogen fixation was strongly correlated to the energy status of the cells and glycogen breakdown and high respiration rates were necessary to provide appropriate energy and anoxic conditions for this process. We conclude that glycogen breakdown is a key regulatory step within these complex processes. Our results demonstrated that the main metabolic genes involved in photosynthesis, respiration, nitrogen fixation and central carbohydrate metabolism have strong (or total) circadian-regulated components. The short light/dark cycles enable us to identify transcriptional differences among the family of psbA genes, as well as the differing patterns of the hup genes, which follow the same pattern as nitrogenase genes, relative to the hox genes which displayed a diurnal, dark-dependent gene expression.

2008

Buchko GW, H Robinson, HB Pakrasi, and MA Kennedy. 2008. "Insights into the structural variation between pentapeptide repeat proteins - Crystal structure of Rfr23 from Cyanothece 51142." Journal of Structural Biology 162(1):184-192. Abstract 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 β-helix called an Rfr-fold. To characterize how structural features of Rfr-folds vary with different amino acid sequences, the crystal structure of Cyanothece Rfr23 (174 residues) was determined at 2.1 Å resolution. The structure is dominated by an Rfr-fold capped at the N-terminus with a nine-residue α-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 β-turns. Second, the pentapeptide repeats are not all tandem in the primary amino acid sequence. Rfr23 contains a 24-residue loop protruding outside one corner of the first complete N-terminal coil of the Rfr-fold (L56 – P79) for which little electron density is observed (24-residue loop). Third, a disulfide bond exists at the corner of one β-turn in the first coil (disulfide bracket). Size exclusion chromatography and NMR and CD spectroscopy indicate that the reduction of the disulfide bracket with the addition of DTT destroys the entire Rfr-fold. Fourth, a single-residue loop in the C-terminal coil perturbs the last coil slightly about one corner of the Rfr-fold (single-residue loop).
Xu M, T Ogawa, HB Pakrasi, and H Mi. 2008. "Identification and Localization of the CupB Protein Involved in Constitutive CO₂ uptake in the Cyanobacterium, Synechocystis sp. Strain PCC 6803." Plant & Cell Physiology 49(6):994-997. doi:10.1093/pcp/pcn074 Abstract The CupB protein was identified in the membranes of Synechocystis sp. strain PCC 6803 in which CupB was tagged with cMyc-6His. Both CupA and NdhH were detected in a highly resolved subcellular fraction containing two protein complexes of about 450 and 550 kDa, obtained after nickel column and gel filtration chromatography of the membranes solubilized with n-dodecyl-β-D-maltoside.
Welsh EA, ML Liberton, J Stockel, T Loh, TR Elvitigala, C Wang, A Wollam, RS Fulton, SW Clifton, JM Jacobs, R Aurora, BK Ghosh, LA Sherman, RD Smith, RK Wilson, and HB Pakrasi. 2008. "The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle." Proceedings of the National Academy of Sciences of the United States of America 105(39):15094-15099. doi:10.1073/pnas.0805418105 Abstract Cyanobacteria are oxygenic photosynthetic bacteria that have significant roles in global biological carbon sequestration and oxygen production. They occupy a diverse range of habitats, from open ocean, to hot springs, deserts, and arctic waters. Cyanobacteria are known as the progenitors of the chloroplasts of plants and algae, and are the simplest known organisms to exhibit circadian behavior4. Cyanothece sp. ATCC 51142 is a unicellular marine cyanobacterium capable of N2-fixation, a process that is biochemically incompatible with oxygenic photosynthesis. To resolve this problem, Cyanothece performs photosynthesis during the day and nitrogen fixation at night, thus temporally separating these processes in the same cell. The genome of Cyanothece 51142 was completely sequenced and found to contain a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of such a linear element in a photosynthetic bacterium. Annotation of the Cyanothece genome was aided by the use of highthroughput proteomics data, enabling the reclassification of 25% of the proteins with no informative sequence homology. Phylogenetic analysis suggests that nitrogen fixation is an ancient process that arose early in evolution and has subsequently been lost in many cyanobacterial strains. In cyanobacterial cells, the circadian clock influences numerous processes, including carbohydrate synthesis, nitrogen fixation, photosynthesis, respiration, and the cell division cycle. During a diurnal period, Cyanothece cells actively accumulate and degrade different storage inclusion bodies for the products of photosynthesis and N2-fixation. This ability to utilize metabolic compartmentalization and energy storage makes Cyanothece an ideal system for bioenergy research, as well as studies of how a unicellular organism balances multiple, often incompatible, processes in the same cell.
Wegener KM, EA Welsh, LE Thornton, NS Keren, JM Jacobs, KK Hixson, ME Monroe, DG Camp, II, RD Smith, and HB Pakrasi. 2008. "High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex." Journal of Biological Chemistry 283(41):27829-27837. doi:10.1074/jbc.M803918200 Abstract Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of the photosynthetic electron transport chain in plants, algae, and cyanobacteria. Utilizing a high-throughput proteomic analysis of isolated PSII complexes from the cyanobacterium Synechocystis sp. PCC 6803, we have identified four PSII associated proteins that are encoded by the cofactor integration operon (cio). This operon contains genes with putative binding domains for chlorophyll, iron-sulfur centers, and bilins. Protein levels of this operon are more abundant in several PSII lumenal mutants, suggesting an accumulation of cio products in partially assembled PSII complexes. This provides a rare example of a bacterial operon whose protein products are translationally coordinated and associated with a single protein complex. Genetic deletion of cio results in decreased oxygen evolution by PSII, suggesting that cio products may function as regulators of PSII complex assembly or degradation, maybe facilitating an uncharacterized step in PSII assembly.
Buchko GW, and HJ Sofia. 2008. "Backbone 1H, 13C, and 15N NMR assignments for the Cyanothece 51142 protein cce_0567: a protein associated with nitrogen fixation in the DUF683 family." Biomolecular NMR Assignments 2:25-28. Abstract The recently sequenced genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 (contig 83.1_1_243_746) contains the sequence for an hypothetical protein that falls into the DUF683 family. As observed for the other 54 DUF683 proteins currently listed in the GenBank database, this 78-residue (9.0 kDa) protein in Cyanothece is also found in a nitrogen fixation gene cluster suggesting that it is involved in the process. To date no structural information exists for any of the proteins in the DUF683 family. In an effort to elucidate the biochemical role DUF683 may play in nitrogen fixation and to obtain structural information for a member of the DUF683 protein family, a construct containing DUF683 from Cyanothece 51142 was generated, expressed, purified, and the solution properties characterized. A total rotational correlation time (tc) of 17.1 ns was estimated by nuclear magnetic resonance (NMR) spectroscopy suggesting a molecular weight of ~ 40 kDa, an observation dictating that DUF683 is a tetramer in solution. Using triple-labeled (2H, 13C, 15N) and residue-specific 15N-labeled amino acids (L, K, V, and E/Q) samples, most of the backbone and side chain resonances for DUF683 were assigned. The 13C alpha chemical shifts and NOESY NMR data indicate that the protein is helical from K18-E75.
Stockel J, EA Welsh, ML Liberton, R Kunnavakkam, R Aurora, and HB Pakrasi. 2008. "Global transcriptomic analysis of Cyanothece 51142 reveals robust diurnal oscillation of central metabolic processes ." Proceedings of the National Academy of Sciences of the United States of America 105(16):6156-6161. doi:10.1073/pnas.0711068105 Abstract Cyanobacteria are oxygenic photosynthetic organisms, and the only prokaryotes known to have a circadian cycle. Unicellular diazotrophic cyanobacteria such as Cyanothece 51142 can fix atmospheric nitrogen, a process exquisitely sensitive to oxygen. Thus, the intracellular environment of Cyanothece oscillates between aerobic and anaerobic conditions during a day-night cycle. This is accomplished by temporal separation of two processes: photosynthesis during the day, and nitrogen fixation at night. While previous studies have examined periodic changes 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 5000 genes in Cyanothece 51142 during two consecutive diurnal periods. We found that ~30% of these genes exhibited robust oscillating expression profiles. Interestingly, this set included genes for almost all central metabolic processes in Cyanothece. A transcriptional network of all genes with significantly oscillating transcript levels revealed that the majority of genes in numerous individual pathways, such as glycolysis, pentose phosphate pathway and glycogen metabolism, were co-regulated and maximally expressed at distinct phases during the diurnal cycle. Our analyses suggest that the demands of nitrogen fixation greatly influence major metabolic activities inside Cyanothece cells and thus drive various cellular activities. These studies provide a comprehensive picture of how a physiologically relevant diurnal light-dark cycle influences the metabolism in a photosynthetic bacterium
Toepel J, EA Welsh, T Summerfield, HB Pakrasi, and LA Sherman. 2008. "Differential Transcriptional Analysis of the Cyanobacterium Cyanothece sp. Strain ATCC 51142 during Light-Dark and Continuous-Light Growth." Journal of Bacteriology 190(11):3904-3913. doi:10.1128/JB.00206-08 Abstract We analyzed the metabolic rhythms and differential gene transcription in the unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC51142 under N₂-fixing conditions with 12h light-12h dark cycles followed by 36 h continuous light. Cultures were grown in a 6-L bioreactor that was specially designed for photosynthetic microorganisms and that permitted continuous monitoring of parameters such as pH and dissolved oxygen. Our main objective was to determine the strategies used by these cells to perform N₂ fixation under normal day-night conditions, as well as under greater stress caused by continuous light. Our results strongly suggested that the level of N₂ fixation is dependent upon respiration for energy production and for removal of intracellular O₂. We determined that N₂ fixation cycled in continuous light, but that the N₂ fixation peak was lower and that glycogen degradation and respiration were also lower under these conditions. We also demonstrated that nifH (the gene encoding the Fe protein) and nifB and nifX were strongly induced in the continuous light; this is consistent with the mode of operation of these proteins relative to the MoFe protein and suggested that any regulation of N₂ fixation was at a posttranscriptional level. Also, many soluble electron carriers (e.g., ferredoxins), as well as redox carriers (e.g., thioredoxin and glutathione) were strongly induced during N₂ fixation in continuous light. We suggest that these carriers were required to generate enhanced cyclic electron transport and phosphorylation for energy production and to maintain appropriate redox levels in the presence of enhanced O₂, respectively.
Gorton I, CS Oehmen, and JE McDermott. 2008. "It Takes Glue to Tango: MeDICi integration framework creates data-intensive computing pipeline." Scientific Computing 25(7):16-24. Abstract Biologists increasingly rely on high-performance computing (HPC) platforms to rapidly process the tsunami of data generated by high throughput genome and metagenome sequencing technology and high-throughput proteomics. Unfortunately, the platforms that produce the massive data sets rarely work smoothly with the interactive analysis and visualization programs used in bioinformatics. This makes it difficult for researchers to exploit the computational power of HPC platforms to speed scientific discovery. At the Department of Energy’s Pacific Northwest National Laboratory in Richland, Wash., researchers are creating computing environments for biologists that seamlessly integrate collections of data and computational resources. These advantages enable users to rapidly analyze high-throughput data. A major goal is to shield the biologist from the complexity of interacting with multiple dissimilar databases and running tasks on HPC platforms and computational clusters. One of those environments the MeDICi Integration Framework is now available for free download. Short for Middleware for Data-Intensive Computing, MeDICi makes it easy to integrate separate codes into complex applications that operate as a data analysis pipeline.

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