Saccharolobus caldissimus gen. nov., sp. nov., a facultatively anaerobic iron-reducing hyperthermophilic archaeon isolated from an acidic terrestrial hot spring, and reclassification of Sulfolobus solfataricus as Saccharolobus solfataricus comb. nov. and Sulfolobus shibatae as Saccharolobus shibatae comb. nov.

A novel hyperthermophilic archaeon of strain HS-3T, belonging to the family Sulfolobaceae, was isolated from an acidic terrestrial hot spring in Hakone Ohwaku-dani, Japan. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain HS-3T were, first, Sulfolobus solfataricus (96.4 %) and, second, Sulfolobus shibatae (96.2 %), indicating that the strain belongs to the genus Sulfolobus. However, the sequence similarity to the type species of the genus Sulfolobus (Sulfolobus acidocaldarius) was remarkably low (91.8 %). In order to determine whether strain HS-3T belongs to the genus Sulfolobus, its morphological, biochemical and physiological characteristics were examined in parallel with those of S. solfataricus and S. shibatae. Although there were some differences in chemolithotrophic growth between strain HS-3T, S. solfataricus and S. shibatae, their temperature, pH and facultatively anaerobic characteristics of growth, and their utilization of various sugars were almost identical. In contrast, the utilization of various sugars by S. acidocaldarius was quite different from that of HS-3T, S. solfataricus and S. shibatae. Phylogenetic evidence based on the 16S and the 23S rRNA gene sequences also clearly distinguished the monophyletic clade composed of strain HS-3T, S. solfataricus, and S. shibatae from S. acidocaldarius. Based on these results, we propose a new genus and species, Saccharolobus caldissimus gen. nov., sp. nov., for strain HS-3T, as well as two reclassifications, Saccharolobus solfataricus comb. nov. and Saccharolobus shibatae comb. nov. The type strain of Saccharolobus caldissimus is HS-3T (=JCM 32116T and InaCC Ar80T). The type species of the genus is Saccharolobus solfataricus.

The ATPases CopA and CopB both contribute to copper resistance of the thermoacidophilic archaeon Sulfolobus solfataricus.

Certain heavy metal ions such as copper and zinc serve as essential cofactors of many enzymes, but are toxic at high concentrations. Thus, intracellular levels have to be subtly balanced. P-type ATPases of the P(IB)-subclass play a major role in metal homeostasis. The thermoacidophile Sulfolobus solfataricus possesses two P(IB)-ATPases named CopA and CopB. Both enzymes are present in cells grown in copper-depleted medium and are accumulated upon an increase in the external copper concentration. We studied the physiological roles of both ATPases by disrupting genes copA and copB. Neither of them affected the sensitivity of S. solfataricus to reactive oxygen species, nor were they a strict prerequisite to the biosynthesis of the copper protein cytochrome oxidase. Deletion mutant analysis demonstrated that CopA is an effective copper pump at low and high copper concentrations. CopB appeared to be a low-affinity copper export ATPase, which was only relevant if the media copper concentration was exceedingly high. CopA and CopB thus act as resistance factors to copper ions at overlapping concentrations. Moreover, growth tests on solid media indicated that both ATPases are involved in resistance to silver.

Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences.

The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.

CopR of Sulfolobus solfataricus represents a novel class of archaeal-specific copper-responsive activators of transcription.

In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)(-1). Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.

Comparative analysis of the catechol 2,3-dioxygenase gene locus in thermoacidophilic archaeon Sulfolobus solfataricus strain 98/2.

A catechol 2,3-dioxygenase (C23O) gene was found from Sulfolobus solfataricus strain 98/2. Heterologous thermophilic C23O expressed in Escherichia coli showed the highest activity against catechol and 4-chlorocatechol, and at neutral pH. The C23O gene located with a putative multicomponent monooxygenase (MM) gene cluster that exactly matched with the homologous region of S. solfataricus strain P2. Primary sequence comparison identified an insertion sequence (IS) element inserted into a putative MM protein A N-terminal fragment gene in strain 98/2. Both ends of the transposase gene in the IS element, ISC1234, were flanked by 19 bp inverted repeat and 4 bp direct repeat sequences which are typical features of mobile elements. Our analysis and the two geographically distant origins of strains 98/2 and P2 (USA and Italy, respectively) suggest that the two strains have evolved from a common ancestor.

An improved method for single cell isolation of prokaryotes from meso-, thermo- and hyperthermophilic environments using micromanipulation.

This study presents an improved system that enables isolation of single viable prokaryotic cells from a mixture of cells. The system is based on an inverted microscope, a microinjector and a micromanipulator. The isolated cell is captured in a microcapillary from a volume of 400 mul and transferred to an appropriate growth medium. Validation of the system was performed using two fluorescent strains: Pseudomonas putida expressing red fluorescent protein (DsRed), and Escherichia coli expressing green fluorescent protein (GFP). A mixture (100:1) of the constructed fluorescent strains was subjected to isolation experiments and nine out of ten individually isolated cells yielded axenic cultures of E. coli. Upon construction and validation, the system was used to isolate and subsequently cultivate axenic cultures of the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efficiency of single-cell isolation and cultivation demonstrated over a range of temperatures-90% (30 degrees C), 85% (70 degrees C) and 95% (80 degrees C)-from different environments is probably due to the elimination of osmotic stress and limitation of temperature fluctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.

A xylan-degrading strain of Sulfolobus solfataricus: isolation and characterization of the xylanase activity.

Two strains (O(alpha) and X(2)) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O(alpha) and X(2), grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O(alpha) underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100 degrees C, and exhibited an optimal temperature and pH of 90 degrees C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.