Cellulose Degradation by Sulfolobus solfataricus Requires a Cell-Anchored Endo-β-1-4-Glucanase

ABSTRACTA sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence ofSulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences inEscherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS ofS. solfataricusthat are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system forSulfolobusand on expression control by the promoter of theS. solfataricusgene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed inS. solfataricuswas stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.</jats:p

Characterization and Functional Complementation of a Nonlethal Deletion in the Chromosome of a β-Glycosidase Mutant of Sulfolobus solfataricus

LacS(−) mutants of Sulfolobus solfataricus defective in β-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the β-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of ∼13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(−) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5′ and 3′ flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the β-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside)

Synthesis, Magnetic, Spectroscopic and Electrochemical Studies of Mixed Pyrimidine-2-thiolate/triphenylphosphine Rhenium(V) and Rhenium (III) Complexes.

Equimolar amounts of trans-[ReOX2(OEt)(PPh3)2] (XCl, Br, I) precursors and potentially bidentate N,S-donor pyrimidine-2(1H)-thione (pymSH) react in refluxing acetone to give mononuclear octahedral paramagnetic trans-[ReIIIX2(pymS)(PPh3)2](XCl, Br, I) species. Starting from a metal–ligand molar ratio of 1:3, in the presence of N(C2H5)3 as deprotonating agent inrefluxing ethanol, the same reaction proceeds stepwise, affording octahedral [ReO(pymS)3] or [ReO(pymS)3] and pentagonalbipyramidal[Re(pymS)3PPh3] complexes as a function of the reaction time. The compounds were characterized by elementalanalysis, magnetic susceptibility, UV–Vis–NIR, IR and 1H NMR spectroscopy and by cyclovoltammetric measurements.Reaction pathways and physico-chemical properties of the complexes are discussed

An Autonomously Replicating Transforming Vector for Sulfolobus solfataricus

A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C