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SummaryArsenic resistance is commonly clustered in ars operons in bacteria; main ars operon components encode an arsenate reductase, a membrane extrusion protein, and an As‐sensitive transcription factor. In the As‐resistant thermophile Thermus thermophilus HB27, genes encoding homologues of these proteins are interspersed in the chromosome. In this article, we show that two adjacent genes, TtsmtB, encoding an ArsR/SmtB transcriptional repressor and, TTC0354, encoding a Zn2+/Cd2+‐dependent membrane ATPase are involved in As resistance; differently from characterized ars operons, the two genes are transcribed from dedicated promoters upstream of their respective genes, whose expression is differentially regulated at transcriptional level. Mutants defective in TtsmtB or TTC0354 are more sensitive to As than the wild type, proving their role in arsenic resistance. Recombinant dimeric TtSmtB binds in vitro to both promoters, but its binding capability decreases upon interaction with arsenate and, less efficiently, with arsenite. In vivo and in vitro experiments also demonstrate that the arsenate reductase (TtArsC) is subjected to regulation by TtSmtB. We propose a model for the regulation of As resistance in T. thermophilus in which TtSmtB is the arsenate sensor responsible for the induction of TtArsC which generates arsenite exported by TTC0354 efflux protein to detoxify cells.

BackgroundThe genus Thermus, which has been considered for a long time as a fruitful source of biotechnological relevant enzymes, has emerged more recently as suitable host to overproduce thermozymes. Among these, α-galactosidases are widely used in several industrial bioprocesses that require high working temperatures and for which thermostable variants offer considerable advantages over their thermolabile counterparts.Results Thermus thermophilus HB27 strain was used for the homologous expression of the TTP0072 gene encoding for an α-galactosidase (TtGalA). Interestingly, a soluble and active histidine-tagged enzyme was produced in larger amounts (5 mg/L) in this thermophilic host than in Escherichia coli (0.5 mg/L). The purified recombinant enzyme showed an optimal activity at 90 °C and retained more than 40% of activity over a broad range of pH (from 5 to 8).Conclusions TtGalA is among the most thermoactive and thermostable α-galactosidases discovered so far, thus pointing to T. thermophilus as cell factory for the recombinant production of biocatalysts active at temperature values over 90 °C.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0638-4) contains supplementary material, which is available to authorized users.

To maintain a proper intracellular redox environment, aerobic microorganisms use redox systems and antioxidants that protect cells from the attack of reactive oxygen species (ROS) such as superoxide anions, H 2 O 2 and hydroxyl radicals. Increased ROS concentration inside a cell results in damage to the main biomolecules and membranes and essential metabolic functions [1]; to maintain a low intracellular ROS level, cells are equipped with an array of antioxidant systems, in the first place superoxide dismutases (SODs), which catalyze the dismutation of superoxide anions into H 2 O 2 and oxygen. H 2 O 2 is reduced by various systems, in the main by catalases and peroxidases. Peroxiredoxins (Prx) are thiol-peroxidases that scavenge peroxides using the enzyme-recycling thioredoxin (Trx) ⁄ thioredoxin reductase (Tr) system as an electron donor [2,3]. Peroxiredoxins 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 H 2 O 2 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.

Disulfide bonds between cysteine pairs are a major structural feature of many proteins. Proteins capable of catalyzing protein disulfide bond formation include a large number of thiol-disulfide oxidoreductases which occur in all living cells, from bacteria to humans. Protein disulfide oxidoreductases fall into several families [thioredoxins (Trxs), glutaredoxins (Grxs), protein disulfide isomerases (PDIs) and disulfide bond-forming (Dsb) proteins] and are characterized by an active site containing a CXXC motif and a thioredoxin fold (five strands of b-sheets enclosed by four a-helices) [1]. Thioredoxins and glutaredoxins are small ubiquitous acidic proteins whose main function is to preserve redox homeostatis in the cytoplasm by acting as disulfide reductase in several biological reactions. In bacteria, correct disulfide bond formation is assisted by proteins of the Dsb family, which are either located in extracytoplasmic compartments or secreted into the medium. In eukaryotic cells, disulfide bond formation and rearrangement are catalyzed both by PDI, a protein localized in the endoplasmic reticulum, and by its homologues [2]. PDI is organized into four domains (a, b, b¢, and a¢) followed by a C-terminal extension referred to as c. The a and a¢ domains contain one catalytically active CGHC motif each, while b and b¢ are redox inactive domains. The threedimensional structure of yeast PDI has recently been solved [3]. The overall structure of PDI has the shape of a twisted 'U' with the a and a¢ domains at the ends of the 'U' and the b and b¢ domains forming the base. A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K M 0.3 lm) and not thioredoxins (TrxA1 and TrxA2). SsPDO ⁄ S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.Abbreviations AaPDO, Aquifex aeolicus protein disulfide oxidoreductase; Dsb, disulfide bond-forming; GSH, glutathione reduced form; GSSG, GSH oxidized form; Nbs 2 , 5,5¢-dithio-bis-(2-nitrobenzoic acid); PDI, protein disulfide isomerase; PDO, protein disulfide oxidoreductase; PfPDO, Pyrococcus furiosus PDO; SsADH, Sulfolobus solfataricus alcohol deydrogenase; SsPDO, Sulfolobus solfataricus PDO; SsTR, Sulfolobus solfataricus thioredoxin reductase; TR, thioredoxin reductase; Trx, th...

Fuselloviruses SSV1 and SSV2 are model systems to investigate virusehost relationships in stably infected cells thanks to their temperate nature. Although they are very similar in morphology, genome organization and gene synteny, their replication is induced by different stimuli, i.e.: by UV-light exposure (for SSV1) and by the growth progression of the host (for SSV2). In this study, we have analysed global gene expression in SSV1-and SSV2-lysogens of Sulfolobus solfataricus P2 in the absence of any stimuli. Additionally, the interplay among SSV1, SSV2 and the host has been investigated in a double-infected strain to explore both virusehost and virusevirus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease of the SSV2 copy number has been observed, which in turn led to provirus-capture into the host chromosome. Results of this study have revealed novel aspects of the hosteviral interaction in the frame of the CRISPRresponse.

The presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated pmB and pmA, were cloned, sequenced and characterized. Biochemical analysis of the pmB-and pmA-encoded enzymes showed that the proline biosynthetic pathway of s 8 thennophilus is similar t o the one previously described in Escherichia coli. The deduced amino acid sequence of a 2408 kb DNA region containing the genes revealed the similarity of the s 8 thennophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N-and C-terminal domains of the bifunctional plant enzyme A1-pyrroline-5-carboxylate synthetase of Wgna aconitifolia. Northern blot analysis showed that the two genes in 5. thennqphilus are organized in a single operon with pmB proximal and pmA distal t o the promoter; primer extension analysis indicated that pmBA transcription is not under repressive control by exogenously supplied proline.