Revisit to the biodesulfurization capability of hyperthermophilic archeon Sulfolobus Solfataricus P2 revealed DBT consumption by the organism in an oil/water two-phase liquid system at high temperatures

The ability of hyperthermophilic archaeon, Sulfolobus solfataricus P2, to grow on organic and inorganic sulfur sources was investigated. A sulfur free mineral medium has been employed with different sources of carbon. Results showed that inorganic sulfur sources display growth curve patterns significantly different from the curves obtained with organic sulfur sources. Solfataricus has an ability to utilize DBT and its derivatives, but it lacks BT utilization. Solfataricus utilizes DBT at a rate of 1.23 μmol 2-HBP h-1 g DCW-1 even at 78 o40 C, at which DBT is known to be unstable. After enabling DBT stabilization using a two-phase culture system, stable microbial growth was achieved showing a desulfurization rate of 0.34 μM DBT g DCW-1 h-143 . Solfataricus offers beneficial properties compared to the other desulfurizing mesophilic/moderate thermophilic bacteria due to its capacity to utilize DBT and its derivatives under hyperthermophilic conditions

Biodesulfurization of DBT by a hyperthermophilic archaeon sulfolobus solfataricus P2

Combustion of fossil fuels leads to the atmospheric emission of sulfur oxides that contribute to acid rain. Significant quantities of sulfur (up to 70%) in petroleum is found as recalcitrant heterocyclic organosulfurs [dibenzothiophene (DBT) and substituted DBTs]. Current sulfur removal processes involve hydrodesulfurization, which requires high temperatures and pressure for efficient catalysis. Biodesulfurization is an alternative method for oil and coal industry offering low costs and relatively easy operating conditions. The present study describes the usage of a hyperthermoacidophilic archaeon S. sulfotaricus P2 in the utilization of sulfur compounds from variety of organic and inorganic compounds at high temperatures, between 75 and 85◦C and low pH, 3.0. To establish optimal sulfur free conditions, carbon sources containing arabinose, ethanol, glucose, mannose and mannitol have been employed to find the most suitable sources. Growths on the sulfur sources such as DBT, dibenzothiophene sulfone, BT, 4,6- dimethyldibenzothiophene, sodium sulfite, potassium disulphite, and potassium persulphate were investigated. 0.3mm of all sulfur supplements except DBT led to increases in the growth rate from 1.35 to 2.0-fold. Further investigations revealed the maximum DBT tolerance as 0.1mm. It was found that 88.5% of DBT was consumed and maximum desulfurization rate was obtained as 1.23mol 2-HBP hour−1 gDCW−1 within 16.5 hours. Our results also revealed that DBT sulfone consumption is the rate limiting step in the overall DBT usage process for S. solfataricus. To the best of our knowledge,this is the first report showing the DBT desulfurization kinetics analysis of S. solfataricus

Biodesulphurized subbituminous coal by different fungi and bacteria studied by reductive pyrolysis. Part 1: Initial coal

One of the perspective methods for clean solid fuels production is biodesulphurization. In order to increase the effect of this approach it is necessary to apply the advantages of more informative analytical techniques. Atmospheric pressure temperature programming reduction (AP-TPR) coupled with different detection systems gave us ground to attain more satisfactory explanation of the effects of biodesulphurization on the treated solid products. Subbituminous high sulphur coal from ‘‘Pirin” basin (Bulgaria) was selected as a high sulphur containing sample. Different types of microorganisms were chosen and maximal desulphurization of 26% was registered. Biodesulphurization treatments were performed with three types of fungi: ‘‘Trametes Versicolor” – ATCC No. 200801, ‘‘Phanerochaeta Chrysosporium” – ME446, Pleurotus Sajor-Caju and one Mixed Culture of bacteria – ATCC No. 39327. A high degree of inorganic sulphur removal (79%) with Mixed Culture of bacteria and consecutive reduction by 13% for organic sulphur (Sorg) decrease with ‘‘Phanerochaeta Chrysosporium” and ‘‘Trametes Versicolor” were achieved. To follow the Sorg changes a set of different detection systems i.e. AP-TPR coupled ‘‘on-line” with mass spectrometry (AP-TPR/MS), on-line with potentiometry (AP-TPR/pot) and by the ‘‘off-line” AP-TPR/GC/MS analysis was used. The need of applying different atmospheres in pyrolysis experiments was proved and their effects were discussed. In order to reach more precise total sulphur balance, oxygen bomb combustion followed by ion chromatography was used

Purification and characterization of recombinant durum metallothionein domain constructs

Metallothioneins (MTs) are small proteins with high cysteine content and high binding capacity for metals like Zn, Cu and Cd. MTs exist in a wide range of organisms and are classified in one super- family according to the distribution of cysteine motifs in their sequences. Type 1 plant MTs, similar to their mammalian counterparts, have the cysteine motifs clustered in the N-and C-termini constituting the β- and -domains respectively. The two domains are connected by an unusually long (about 42 amino acids) hinge region whose structural and functional properties are unclear. Recent studies indicate that, despite the classification into a single family, all MTs do not have a single unifying function, and while some MTs help metal homeostasis others play a role in detoxification of heavy metals (e.g. Cd and As). We identified an mt gene in Cd resistant durum wheat coding for a type 1 MT (dMT) and the recombinant protein (dMT) was over-expressed in E. coli as GST fusion (GSTdMT). For detailed structural and functional investigations GST-fusion constructs of βhinge-, hinge- and the hinge-domains of dMT were over-expressed in E. coli. Proteins were purified and results of characterization by size exclusion chromatography, SDS- and native-PAGE, UV-VIS absorption spectroscopy, atomic absorption spectroscopy, dynamic light scattering, and small-angle solution X-ray scattering will be presented. Studies on the isolated domains indicate distinct metal-binding properties and structural features for these regions. GST fusion of the hinge appears to be stable reflecting an intrinsic structural organization for this domain. These findings will be discussed in terms of relevance for the native structure of dMT

Revisiting the biodesulfurization capability of hyperthermophilic archaeon Sulfolobus solfataricus P2 revealed DBT consumption by the organism in an oil/water two-phase liquid system at high temperatures

The ability of the hyperthermophilic archaeon Sulfolobus solfataricas P2 to grow on organic and inorganic sulfur sources was investigated. A sulfur-free mineral medium was employed with different sources of carbon. The results showed that inorganic sulfur sources display growth curve patterns significantly different from the curves obtained with organic sulfur sources. Solfataricas has the ability to utilize DBT and its derivatives, but it lacks BT utilization. Solfataricas utilizes DBT at a rate of 1.23 mu mol 2-HBP h(-1) g DCW-1 even at 78 degrees C, at which DBT is known to be unstable. After enabling DBT stabilization using a two-phase culture system, stable microbial growth was achieved showing a desulfurization rate of 0.34 mu M DBT g DCW-1 h(-1). Solfataricas offers beneficial properties compared to the other desulfurizing mesophilic/moderate thermophilic bacteria due to its capacity to utilize DBT and its derivatives under hyperthermophilic conditions

Identifying, cloning and structural analysis of differentially expressed genes upon Puccinia infection of Festuca rubra var. rubra

Differentially expressed genes in response to rust infection (Puccinia sp.) in creeping red fescue (Festuca rubra var. rubra) were identified and quantified using the mRNA differential display technique. The differentially induced genes were identified as homologs of mitogen-activated protein kinase (MAPK) 3 of Arabidopsis thaliana, stem rust resistance protein Rpg1 of barley and Hsp70 of Spinacia oleracea. The change in the steady state expression levels of these genes in response to rust infection was tested by Northern blot analysis and further quantified by real-time PCR. A steady accumulation of transcripts in the course of rust infection was observed. Full-length transcript of a fescue MPK-3 was obtained by RACE PCR. Its corresponding cDNA encodes a protein with a predicted MW of 42.5 kDa which was mapped onto the structural model of homologs MAPK to illustrate the corresponding MAPK signature motifs. This study, for the first time, presents evidence on the rust infection dependent metabolic pathways in creeping red fescue

Recombinant Expression and Characterization of A.thaliana Heterotrimeric G protein alpha subunit; GPA1

Current research on plant heterotrimeric G proteins is mainly focused on mutant / overexpression studies performed in model organisms, i.e. A. thaliana and O. sativa. These studies provide information about the pathways involving these proteins; however, the mechanism of G protein activation via signaling molecules remains unknown. Similarly, information on biochemical and biophysical characteristics of the plant heterotrimeric G-proteins is lacking, whereas for the mammalian counterparts, such studies have contributed unique insights into understanding their functional roles. Following this, A.thaliana heterotrimeric G protein  subunit (GPA1) is cloned into P.pastoris expression system and the recombinant protein is purified. Initial analyses reveal that GPA1 is membrane bound and N-terminally blocked indicating the presence of lipid modifications. The purified protein is GDP bound and can bind to GTP. Circular dichroism polarimetry analyses show that the dominant structure is helical and activation by receptor mimetics leads to a decrease in helical content; similar to mammalian counterparts. It appears that, contrary to mammalian G-alphas, the intrinsic fluorescence of GPA1 decreases in the presence of GTP and increases in the presence of GDP. Full characterization of GAPA1, including its nucleotide binding kinetics and dynamics, will help to establish a better understanding of the functional mechanisms of heterotrimerc G-protein activation in plants and will shed light on the level of similarity of plant and mammalian hetetrotrimeric G protein signaling pathways

Functional and structural investigations of a metallothionein from durum wheat, dMT

Plant metallothioneins (MTs) are low molecular weight (7- 8 kDa), sulphydryl-rich, aromatic residue lacking metalbinding proteins which have two terminal metal-binding clusters separated by a conserved distinctively long hinge region of about 50 amino acids. The length and conserved sequence of the hinge region distinguishes plant MTs from their mammalian counterparts, suggesting additional roles other than metal binding and detoxification. Homology modeling indicates mammalian-like folds for the metalbinding domains of dMT, and ab initio calculations yield a DNA-binding like structural motif for the hinge region1. Here, we study the structural features of full-length dMT Abstracts 263 and its constructs in unstructured, metal-free (apo) and metal-bound (holo) states using various biophysical and biochemical characterization techniques and we investigate putative DNA-binding interactions of the hinge region using functional assays (e.g. whole-genome PCR technique) for the apo- and holo-protein. The Cd-binding dMT is expressed in Escherichia coli as a Glutathione-STransferase fusion protein and cleaved from this tag for further analyses. The exact Cd-bound state of dMT and oxidation of thiols are kept under control through preparation of apo-protein from purified Cd-dMT prior to reconstitution by titration of equivalent amounts of Cd solution under anaerobic conditions. 4 mole equivalence of Cd could saturate dMT as followed by spectral changes of circular dichroism and UV-Vis absorption spectroscopy. This finding is further confirmed by parallel inductively coupled plasma optical emission spectroscopy measurements. Small angle X-ray solution scattering measurements and gel-filtration chromatography revealed an oligomeric form for the Cd-bound dMT whereas apo dMT is monomeric as also shown by native state electrospray ionization mass spectrometry experiments. Taken together, our results suggest that structural rearrangements driven by metal-binding result in oligomerization/complex formation that may lead to functional variations. Acknowledgements This work is supported by a joint TUBITAK and JULICH grant, TBAG-U-157 105T535. 1. Bilecen et al., 2005, J. Biol. Chem. 280,13701-1