The kinetics and mechanism of CeIV oxidation of the water oxidation catalyst [(bpy)2(H2O)RuIIIORuIII(OH2)(bpy)2]4+ (1, RuIIIORuIII) have been investigated by UV−visible measurements with application of global analysis. The reaction proceeds by stepwise oxidation of RuIIIORuIII to RuVORuV with oxidation of RuIVORuIII the slow step. RuVORuV has been identified as an intermediate by the appearance of its ClO4 - salt as a black suspension in concentrated solutions at 5 °C. It is the key intermediate in water oxidation. The mechanism may involve a bimolecular step and formation of a peroxo-bridged intermediate. Catalytic water oxidation is greatly retarded after just a few catalytic cycles because of anation induced by O2 evolution.

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Ce(IV) oxidation of the ruthenium blue dimer leads to catalytic oxidation of water. Kinetic and spectroscopic studies have revealed evidence for RuVORuV as the active catalyst. Redox cycling occurs between RuIIIORuIII and RuVORuV via a complex sequence of coupled steps involving oxidations by Ce(IV) and second-order cross reactions of the dimer. The oxygen-evolving step is not rate determining in the catalytic cycle.

The archaea Sulfolobus utilizes the ESCRT-III-based machinery for cell division. This machinery comprises three proteins: CdvA, Eukaryotic-like ESCRT-III and Vps4. In addition to ESCRT-III, Sulfolobus cells also encode three other ESCRT-III homologs termed ESCRT-III-1, -2 and -3. Herein, we show that ESCRT-III-1 and -2 in S. islandicus REY15A are localized at midcell between segregating chromosomes, indicating that both are involved in cell division. Genetic analysis reveals that escrt-III-2 is indispensable for cell viability and cells with reduced overall level of ESCRT-III-1 exhibit growth retardation and cytokinesis defect with chain-like cell morphology. In contrast, escrt-III-3 is dispensable for cell division. We show that S. islandicus REY15A cells generate buds when infected with S. tengchongensis spindle shaped-virus 2 (STSV2) or when ESCRT-III-3 is over-expressed. Interestingly, Δescrt-III-3 cells infected with STSV2 do not produce buds. These results suggest that ESCRT-III-3 plays an important role in budding. In addition, cells over-expressing the C-terminal truncated mutants of ESCRT-III, ESCRT-III-1 and ESCRT-III-2 are maintained predominantly at the early, late, and membrane abscission stages of cell division respectively, suggesting a crucial role of the ESCRTs at different stages of membrane ingression. Intriguingly, intercellular bridge and midbody-like structures are observed in cells over-expressing MIM2-truncated mutant of ESCRT-III-2.

We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P sac7d -hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P sac7d -hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His 6 -tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (⌬herA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

Hjm and Hel308a are novel, RecQ-like DNA helicases recently identified in the euryarchaeotes Pyrococcus furiosus and Methanothermobacter thermautotrophicus, respectively. In this study, an Hjm/Hel308 homologue (designated StoHjm) from Sulfolobus tokodaii, a hyperthermophilic archaeon belonging to the Crenarchaeota subdomain of archaea, was cloned, purified, and characterized. Unlike Hjm and Hel308a, which unwind DNA in a 3-to-5 direction, StoHjm unwound DNA in both 3-to-5 and 5-to-3 directions. Remarkably, StoHjm exhibited structure-specific single-stranded-DNA-annealing and fork regression activities in vitro. In addition, gel filtration, affinity pulldown, and yeast two-hybrid analyses revealed that StoHjm physically interacted with StoHjc, the Holliday junction-specific endonuclease from S. tokodaii. This interaction may have functional significance, because the unwinding activity of StoHjm was inhibited by StoHjc in vitro. These results may suggest that the Hjm/Hel308 family helicases, in association with Hjc endonucleases, are involved in processing of stalled replication forks.

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