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The peroxiredoxins as molecular guardians in the oxidative stress in Sulfolobus solfataricus.
No full textThe machinery for oxidative protein folding in thermophiles.
No full textDisulfide bonds are required for the stability and function of many proteins. A large number of thiol-disulfide oxidoreductases, belonging to the thioredoxin superfamily, catalyze protein disulfide bond formation in all living cells, from bacteria to humans. The protein disulfide isomerase (PDI) is the eukaryotic factor that catalyzes oxidative protein folding in the endoplasmic reticulum; by contrast, in prokaryotes, a family of disulfide bond (Dsb) proteins have an equivalent outcome in the bacterial periplasm. Recently the results from genome analysis suggested an important role for disulfide bonds in the structural stabilization of intracellular proteins from thermophiles. A specific protein disulfide oxidoreductase (PDO) has a key role in intracellular disulfide shuffling in thermophiles. Here we focus on the structural and functional characterization of PDO correlated with the multifunctional eukaryotic PDI. In addition, we highlight the chimeric nature of the machinery for oxidative protein folding in thermophiles in comparison with the mesophilic bacterial and eukaryal counterparts
How Sulfolobus responds to enviromental stress
No full textBacteria and Archaea occupy a considerable
diversity of niches that vary with respect to the physical
conditions. Survival and colonisation requires the
capacity to sense, and adapt to environmental change.
In this chapter we consider some basic mechanisms
adopted by prokaryotes to answer to high temperature,
oxidative stress and toxic substances. We lay particular
attention on the molecular strategies of Sulfolobus sp.
to survive at high temperature and to respond to
thermal stress, focusing on the peculiar composition of
the membrane and on the involvement of chaperonins.
This chapter also analyses biological defense
mechanisms and genetic responses to oxidative stress
and chemical damage with particular attention to
detoxification from metals and drugs
Antioxidant system of peroxiredoxins in Sulfolobus solfataricus.
No full textThe maintenance of the proper intracellular redox environment in aerobic microorganisms is guaranteed by redox systems and antioxidants that safeguard cells from the attack of reactive oxgen species (ROS) such as superoxide anions (O∙-), hydrogen peroxide (H2O2), hydroxyl radicals (OH∙). The increase of ROS concentration inside the cell damage biomolecules, membranes and essential metabolic functions. To kept low the intracellular level of ROS the cells are equipped of an array of antioxidant systems that have in the first line the superoxide dismutase (SODs) that catalyze the dismutation of O∙- to form H2O2 and oxygen. The H2O2 is reduced by different systems represented mainly by catalases and peroxidases.
The peroxiredoxins (Prx) are thiol-peroxidase that can scavange the peroxides utilizing generally Thioredoxin Reductase (TR) / Thioredoxin (Trx) system as electron donors that allow the recycling of the enzymes (1). Prxs are ubiquitous enzymes that share the same basic catalytic mechanisms, in which an activated cysteine (the peroxidative cysteine) is oxidized to sulphenic acid by peroxide substrate. Reductive regeneration of the oxidized catalytic thiol generally depends on glutathione, thioredoxin, glutaredoxin, cyclophilin and tryparedoxin.
The aim of this project is to expand the knowledge on antioxidant system in Sulfolobus solfataricus. For this purpose we have expressed in E. coli and characterized the four Prxs, Bcp1, Bcp2 (2), Bcp3, Bcp4. The purified recombinant proteins are able to remove both H2O2 and tert-butyl hydroperoxide and show high thermostability. Furthermore Bcp1, Bcp3 and Bcp4 can use as electron donor in the reycling of enzymes, the redox system Protein Disulfide Oxidoreductase (SsPDO) / TR previously characterized in S.solfataricus (3) while Bcp2 can use only DTT.
Comparative functional and transcriptional analysis suggest different role of Bcps during oxidative stress: Bcp2 and Bcp3 could act as inducible enzyme when the cell are attacked by exogenous peroxides, while Bcp1 and Bcp4 could support a protective role against endogenous peroxide formed during metabolism
Thermophilic arsenic binding proteins: characterization and exploitation as biosensors.
No full textArsenic is an ubiquitous toxic metalloid naturally present in the soil, water and air that adversely affects human health. The abundance of arsenic in the environment has guided the evolution of multiple defence strategies in almost all microorganisms which must therefore sense the metalloid and regulate the transcription of genes coding for resistance proteins. In this sense microorganisms participate to the geochemical cycling of arsenic in their living environments, promoting or inhibiting arsenic release from sediment material (1).
The thermophilic gram negative bacterium Thermus thermophilus HB27 is able to grow in the presence of both arsenate and arsenite in a range of concentrations which are lethal for other microorganisms. The putative resistance genes have not been found in a single resistance operon but associated to chromosomal genes apparently not functionally related. One of them codes for a thioredoxin-coupled arsenate reductase (TtArsC) which catalyzes the reduction of pentavalent arsenate to trivalent arsenite (2); the second codes for a transcriptional repressor (TtSmtB), sensitive to arsenic, belonging to the ArsR/SmtB family of transcriptional regulators.
Here we present studies addressed to the elucidation of the role of TtarsC and TtsmtB in the arsenic resistance mechanism, among which the characterization of the recombinant TtArsC and TtSmtB proteins. The results obtained represent the starting point for the development of stable whole-cell or protein- based arsenic biosensors (3).
THIS WORK IS SUPPORTED BY GRANTS FROM THE REGIONE CAMPANIA, LEGGE 5 (ITALY, CUP NUMBER E69D15000210002), AND FROM THE PROJECT BIO2013-4496R.
REFERENCES:
1 ROSEN BP . “BIOCHEMISTRY OF ARSENIC DETOXIFICATION”. FEBS LETTERS 2002; 529:86-92.
2. DEL GIUDICE I, ET AL. “A NOVEL ARSENATE REDUCTASE FROM THE BACTERIUM THERMUS THERMOPHILUS HB27: ITS ROLE IN ARSENIC DETOXIFICATION”. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2013; 1834:2071-2079.
3. POLITI J ET AL. “INTERACTION OF THERMUS THERMOPHILUS ARSC ENZYME AND GOLD NANOPARTICLES NAKED-EYE ASSAYS SPECIATION BETWEEN AS(III) AND AS(V). NANOTECHNOLOGY. 2015;26(43):435703
Identification and molecular characterisation of thermophilic endoglucanase from Sulfolobus solfataricus.
No full textINTRODUCTION: Microbial degradation of cellulose has enormous economic potential for the conversion of plant biomass into fuel and chemicals. Cellulose is a linear polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. Its enzymatic hydrolysis requires the action of both endoglucanases (1,4--D-glucan glucanohydrolase [EC 3.2.1.4]) and exoglucanases (1,4--D-glucan cellobiohydrolase [EC 3.2.1.91]). A synergic interaction of these enzymes is necessary for the complete hydrolysis of crystalline cellulose. Thermophilic microrganisms have received considerable attention as source of highly active and thermostable cellulolytic enzymes; genes encoding endoglucanases are widely distributed among fungi and Bacteria; recently one was identified in the Archaeon Pyrococcus furiosus suggesting the occurrence of polysaccharides in hydrothermal vent environments (1).
Our results report the identification of cellulasic activity in the Archaeon Sulfolobus solfataricus and molecular characterisation of celS, encoding a putative cellulase homologous to thermophilic endoglucanases.
MATERIALS AND METHODS: S. solfataricus MT4 strain, kindly provided by Prof. Mario De Rosa, was grown at 82°C in a rotary shaker.
A gene bank of S solfataricus MT4 strain was constructed as described previously (2). pGEM1.1 was the recombinant plasmid screened with adh gene as probe and containing celS . It was sequenced using primers constructed ad hoc. Analysis of the putative open reading frames (ORFs), was performed using Blast program.
Total RNA was extracted in the late exponentially growth phase and primer extension was performed (3).
Detectection of cellulasic activity was determined on CMC-cellulose plates and after SDS-PAGE (4).
RESULTS: Cellulase activitiy was detected in the supernatant of S. solfataricus MT4 cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 49 kDa and 40 kDa. At the same time we have cloned a DNA fragment from S. solfataricus MT4 containing an ORF (CelS) of 322 aminoacids (molecular weight 36703 Da) with significant homology to the P. furiosus (1), Thermotoga maritima (4) and T. neapolitana (5) endo-1,4--glucanases. The gene was demonstrated to be transcribed in vivo.
In order to optimise the expression of celS we tried to grow MT4 on different -glucans, namely in minimal media containing Avicel, lichenan, carboxylmethylcellulose, but they were unable to support growth. Therefore enzymatic and transcriptional analysis were performed on cells cultured in more unspecific minimal and rich media. The results obtained suggested a catabolite repression by glucose and in general a down regulation by complex nutrients.
The transcription start site was unambiguously identified by primer extension and revealed coincident with translational initiation.
In order to demonstrate the relationship between structure and function of celS, the gene was fused with gst (glutathione-S- tranferase) in the expression vector pGEX-2tk and expressed in E.coli. The purification and the characterisation of the recombinant protein are underway
Involvement of peroxiredoxin bcp2 in oxidative stress in Sulfolobus solfataricus.
No full textReactive oxygen species (ROS) such as superoxide and hydroxyl radicals, hydrogen peroxide, are potent oxidant capable of damaging all cellular components including DNA, proteins and membrane lipids. In order to protect against the toxicity of ROS, organisms are equipped with an array of defence mechanisms. Among these, peroxiredoxins (Prxs) (1), a family of thiol-specific antioxidant proteins, have received significant attention in the last years. Prxs exert their protective antioxidant role in cells through their peroxidase activity, whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides are reduced and detoxified.
These enzymes have been identified in each domain of life: Eucarya (2), Bacteria (3)and Archaea (4). Prxs use a redox-active cysteine to reduce peroxides and can be divided into two groups, the 1-Cys and 2-Cys Prxs, based on the cysteinyl residues involved in the catalysis.
[i]S.[/i] [i]solfataricus[/i], is an aerobic hyperthermophilic Archaea whose genome has been sequenced. The genome analysis shows the presence of four putative Prxs (Bcp1, Bcp2, Bcp3, Bcp4) whose physiological roles in the oxidative stress are underway.
We report the involvement of [i]bcp2[/i] gene in oxidative stress, the characterization of the recombinant protein rSsBcp2, its capability to scavenge H2O2 and to protect DNA against the cleavage caused from thiol-mixed –function oxidation (MFO) system
Peroxiredoxins as cellular guardians in Sulfolobus solfataricus characterization of Bcp1, Bcp3 and Bcp4.
No full textPeroxiredoxins are ubiquitous enzymes that are part of the oxidative stress defense system. In the present study, we identified three peroxiredoxins [bacterioferritin comigratory protein (Bcp)1, Bcp3 and Bcp4] in the genome of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Based on the cysteine residues conserved in the deduced aminoacidic sequence, Bcp1 and Bcp4 can be classified as 2-Cys peroxiredoxins and Bcp3 as a 1-Cys peroxiredoxin. A comparative study of the recombinant Bcps produced in Escherichia coli showed that these enzymes protect DNA plasmid from oxidative damage and remove both H2O2 and tert-butyl hydroperoxide, although at different efficiencies. We observed that all of them were particularly thermostable and that peak enzymatic activity fell within the range of the growth temperature of S. solfataricus. Furthermore, we discovered an alternative Bcp reduction system whose composition differs from that of the peroxiredoxin reduction system previously characterized in the aerobic hyperthermophilic archaeon Aeropyrum pernix. Whereas the latter uses the thioredoxin/thioredoxin reductase/NADPH system, this alternative Bcp system is formed of the protein disulfide oxidoreducatase, SSO0192, the thioredoxin reductase, SSO2416, and NADPH. The role of Bcps in oxidative stress was investigated using transcriptional analysis. Different northern blot analysis responses suggested that the Bcp antioxidant system of S. solfataricus can both operate at the constitutive level, with Bcp1 and Bcp4 preventing endogenous peroxide formation, and at the inducible level, with Bcp3 and the already characterized Bcp2 protecting cells from the attack of external peroxides
Comparative activity of Ceftizoxime and four other Cephalosporins against Gram negative bacteria and their sensitivity to Beta Lactamases. Microbiologica:
No full textCharacterization of 1-cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress.
No full textIntroduction
To protect against toxic Reactive oxygen species (ROS), aerobic organisms are equipped with a full array of defence mechanisms. In recent years, much attention has been given to peroxiredoxins (Prxs) [1], a new family of thiol-specific antioxidant proteins. Prxs use a redox-active cysteine to reduce peroxides. In the aerobic hyperthermophilic archaeon Sulfolobus solfataricus the investigation of the mechanisms governing ROS protection is at initial stage. Recent analyses of the genomic sequence of S. solfataricus [2] have revealed the presence of four putative Prxs (Bcp1, Bcp2, Bcp3, Bcp4).
We report the involvement of the Bcp2 in oxidative stress in S. solfataricus and the characterization of the recombinant protein (rBcp2) in order to shed light on its role in the detoxification process and on its catalytic mechanism.
Material and Methods
Purification of rBcp2 by affinity chromatography (HisTrap HP Amersham) and size-exclusion chromatography (HiLoad Superdex 75; Amersham).
DNA cleavage assay by the metal ion catalyzed oxidation (MCO) system and assay of peroxidase activity were according to Lim et al. [3].
The mutant rBcp2 C49S was obtained by QuickChange II site directed mutagenesis kit (Stratagene).
Results
To understand the function of Bcp2 in oxidative stress, we provided evidence of its involvement in response to various oxidant agents. The transcriptional and the western blot analysis have showed the increased level of specific mRNA and Bcp2 respectively in response to Paraquat, tert-butyl hydroperoxide and H2O2.
rBcp2, expressed in E.coli, was purified and functionally characterized: rBcp2 removes the H2O2 in a DTT-dependent manner and protects plasmidic DNA against the MCO system (DTT/ Fe3+/O2). Lastly the loss of activity in the Bcp2 mutant (C49S) obtained shows that Cys49 must necessarily be involved in the catalysis
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