Cyanobacterium Cyanothece Sp Research Articles

Structural characterization of the protein cce_0567 from Cyanothece 51142, a metalloprotein associated with nitrogen fixation in the DUF683 family

The genomes of many cyanobacteria contain the sequence for a small protein with a common “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 falls 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 P2 1 with two protein molecules and one Ni 2+ cation per asymmetric unit. The protein is composed of two α-helices, residues P11 to G41 (α1) and L49–E74 (α2), with the second α-helix containing a short 3 10-helix (Y46–N48). A four-residue linker (L42–D45) between the helices allows them to form an anti-parallel bundle and 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 is coordinated with Ni 2+ in the crystal structure. The divalent cation Ni 2+ was titrated into 15N-labeled cce_0567 and chemical shift perturbations were observed only in the 1H– 15N HSQC spectra for residues at, or near, the site of Ni 2+ binding observed in the crystal structure. There was no evidence for an increase in the size of cce_0567 upon binding Ni 2+, even in large molar excess of Ni 2+, 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 ∼ 62 °C that was reversible.

Competition and facilitation between unicellular nitrogen‐fixing cyanobacteria and non—nitrogen‐fixing phytoplankton species

Recent discoveries show that small unicellular nitrogen‐fixing cyanobacteria are more widespread than previously thought and can make major contributions to the nitrogen budget of the oceans. We combined theory and experiments to investigate competition for nitrogen and light between these small unicellular diazotrophs and other phytoplankton species. We developed a competition model that incorporates several physiological processes, including the light dependence of nitrogen fixation, the switch between nitrate assimilation and nitrogen fixation, and the release of fixed nitrogen. Model predictions were tested in nitrogen‐limited and lightlimited chemostat experiments using the unicellular nitrogen‐fixing cyanobacterium Cyanothece sp. Miami BG 043511, the picocyanobacterium Synechococcus bacillaris CCMP 1333, and the small green alga Chlorella cf sp. CCMP 1227. Parameter values of the species were estimated by calibration of the model in monoculture experiments. The model predictions were subsequently tested in a series of competition experiments at different nitrate levels. The model predictions were generally in good agreement with observed population dynamics. As predicted, in experiments with high nitrate input concentrations, the species with lowest critical light intensity (S. bacillaris) competitively excluded the other species. At low nitrate input concentration, nitrogen release by Cyanothece enabled stable coexistence of Cyanothece and S. bacillaris. More specifically, model simulations predicted that fixed nitrogen release by Cyanothece enabled S. bacillaris to become four times more abundant in the species mixture than it would have been in monoculture. This intricate interplay between competition and facilitation is likely to be a major determinant of the relative abundances of unicellular nitrogen‐fixing cyanobacteria and non—nitrogen—fixing phytoplankton species in the oligotrophic ocean.

Capillary LC Coupled with High-Mass Measurement Accuracy Mass Spectrometry for Metabolic Profiling

We have developed an efficient and robust high-pressure capillary LC-MS method for the identification of large numbers of metabolites in biological samples using both positive and negative ESI modes. Initial efforts focused on optimizing the separation conditions for metabolite extracts using various LC stationary phases in conjunction with multiple mobile-phase systems, as applied to the separation of 45 metabolite standards. The optimal mobile and stationary phases of those tested were determined experimentally (in terms of peak shapes, theoretical plates, retention of small, polar compounds, etc.), and both linear and exponential gradients were applied in the study of metabolite extracts from the cyanobacterium Cyanothece sp. ATCC 51142. Finally, an automated dual-capillary LC system was constructed and evaluated for the effectiveness and reproducibility of the chromatographic separations using the above samples. When coupled with a commercial LTQ-orbitrap MS, approximately 900 features were reproducibly detected from Cyanothece sp. ATCC 51142 metabolite extracts. In addition, 12 compounds were tentatively identified, based on accurate mass, isotopic distribution, and MS/MS information.

Analysis of chlorophyll–protein complexes from the cyanobacterium Cyanothece sp. ATCC 51142 by non-denaturing gel electrophoresis

The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 temporally separates N 2 fixation from photosynthesis. We are analyzing the mechanism by which photosynthesis is down-regulated so that O 2 evolution is minimized during N 2 fixation. Previous results suggested changes in photosynthesis that are mediated through the redox poise of the plastoquinone pool (a process involving state transitions, in which the redistribution of excitation energy between the two photosystems helps to optimize photosynthetic yield) and the oligomerization state of the photosystems. Our working hypothesis was that the regulation of photosynthesis involved changes in the oligomerization of the photosystems. To analyze this hypothesis, we utilized a low-ionic strength, non-denaturing gel electrophoresis system to study the Chl–protein complexes. We determined that PSI is mostly trimeric, whereas PSII appears mainly as monomers. We demonstrated that most of the Chl–protein complexes in Cyanothece sp. remained constant throughout the diurnal cycle, except for the transient accumulation of a Chl–protein complex (band C) which appeared only during the late light period. Based on the size of this complex, band C represents either an interaction of PSI and PSII or a PSII dimer. These results provide support for the dynamic nature of the photosystems with respect to the diurnal cycle.

Exopolysaccharide Production by a Marine Cyanobacterium Cyanothece sp.: Application in Dye Removal by Its Gelation Phenomenon

Cyanobacterium Cyanothece sp. ATCC 51142 has been shown to produce an exopolysaccharide (EPS) at a high level. EPS production was found to be influenced by the concentration of salt, pH, and type of nitrogen source. Maximum polysaccharide production was found to occur at a 4.5% (w/v) NaCl salt concentration, pH 7.0, and in the presence of NaNO3 as the nitrogen source. The gelation of EPS in alkaline conditions was employed to remove the dyes from the effluents. The effect of organic molecules and metal ions on the efficiency of dye removal capacity was investigated. A laboratory-scale reactor was prepared to treat artificial textile effluent.

Photosystem II cyclic heterogeneity and photoactivation in the diazotrophic, unicellular cyanobacterium cyanothece species ATCC 51142

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N2-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O2-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (qN) of PSII increased during N2 fixation and persisted after treatments known to induce transitions to state 1. The qN was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, epsilon, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N2 fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O2 evolution, altered dark stability of PSII centers, and substantial changes in qN.

Temporal Changes in State Transitions and Photosystem Organization in the Unicellular, Diazotrophic Cyanobacterium Cyanothece sp. ATCC 51142.

The unicellular Cyanobacterium Cyanothece sp. ATCC 51142, grown under alternating 12-h light/12-h dark conditions, temporally separated N2 fixation from photosynthesis. The regulation of photosynthesis was studied using fluorescence spectra and kinetics to determine changes in state transitions and photosystem organization. The redox poise of the plastoquinone (PQ) pool appeared to be central to this regulation. Respiration supported N2 fixation by oxidizing carbohydrate granules, but reduced the PQ pool. This induced state 2 photosystem II monomers and lowered the capacity for O2 evolution. State 2 favored photosystem I trimers and cyclic electron transport, which could stimulate N2 fixation; the stimulation suggested an ATP limitation to N2 and CO2 fixation. The exhaustion of carbohydrate granules at around 6 h in the dark resulted in reduced respiratory electron flow, which led to a more oxidized PQ pool and produced a sharp transition from state 2 to state 1. This transient state 1 returned to state 2 in the remaining hours of darkness. In the light phase, photosystem II dimerization correlated with increased phycobilisome coupling to photosystem II (state 1) and increased rates of O2 evolution. However, dark adaptation did not guarantee state 2 and left photosystem I centers in a mostly monomeric state at certain times.

Phenotypic variation in exopolysaccharide production in the marine, aerobic nitrogen-fixing unicellular cyanobacterium Cyanothece sp.

The aerobic nitrogen-fixing cyanobacterium, Cyanothece sp. BH68K produces non-mucoid variants defective in exopolysaccharide (EPS) production at a high frequency. The EPS-producing wild-type colonies (EPS(+)) have a characteristic smooth and shiny appearance which allows them to be easily distinguished from the EPS(-) variants. When grown on agar plates lacking a source of combined nitrogen, the EPS(-) variants exhibited a yellow phenotype typical of nitrogen starvation. These EPS(-) variants showed varying degrees of reversion back to the EPS(+) phenotype. After reversion, they exhibited normal diazotrophic growth on agar plates. Alcian blue and ruthenium red staining indicated that the EPS is an acidic polysaccharide, which is present as a loose network around the cell, and which can be completely removed by low speed centrifugation. The accumulation of EPS takes place mainly during the stationary phase. All EPS(-) variants failed to produce any EPS. Analysis of growth of wild-type and EPS(-) variants revealed that EPS production is beneficial for diazotrophic growth on solid medium, but not in liquid medium. In addition, EPS phenotypic alteration may have some advantage in the dispersal of cells from one place to another in the natural environment.