Sulfolobus tokodaii

From MicrobeWiki, the student-edited microbiology resource
Revision as of 15:22, 8 April 2008 by Buschurk (talk | contribs)

A Microbial Biorealm page on the genus Sulfolobus tokodaii


Higher order taxa:

Kingdom- Archaea, Phylum-Crenarchaeota, Class-Thermoprotei, Order-Sulfolobales, Family-Sulfolobaceae, Genus-Sulfolobus


Sulfolobus tokodaii

Description and significance

Sulfolobus tokodaii was originally discovered in an acidic spa in Beppu Hot Springs of Kyushu Island, Japan, during the early 1980s. It is one of the four main species of the genus Sulfolobus. Publications and research projects in recent years mainly focused on a specific strain of this specie, S. tokodaii sp strain7, because it is the most abundant and can be easily isolated. It favors an environment with very low pH and high temperature. This species shows optimal growth through aerobic respiration. (1) Its cells are irregular cocci with diameter varies between 0.5 – 2.0 micrometer. Colonies of the cells are arranged in single unit, pale tan, translucent, smooth, and convex while the cells, itself, have flattened and uneven surfaces. (3)

Through the method of shotgun sequencing, S. tokodaii strain 7 is determined to carry no extra-chromosomal genetic unit and has the ability to directly convert hydrogen sulfide to sulfate; this feature has been widely used in the treatment of industrial wastewater. Moreover, search of its genome sequence shows that it contains 14 ORFs similar to the gene families of those in eukaryotes. Identification of these eukaryote-specific genes suggests that this strain is closer to eukaryotes than any other known archaea strains. (3) Further, recent studies have found that S. tokodaii strain7, similar to its sister Sulfolobus metallicus, contain upregulated genes that are responsible for the oxidation of ferrous iron. This trait is only portrayed in these two species of the whole Sulfolobus genus. (2)

Genome structure

The circular genomic DNA of Sulfolobus tokodaii sp strain7 contains 2,694,756 base pair long with G+C content of 32.8%. RNA-coding genes found consists of a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes of which were 24 intron-containing tRNA genes. The repetitive sequences identified were SR-type, long dispersed-type and Tn-like repetitive elements. The genome of this species is composed of 2826 potential protein-coding regions, also known as the open reading frames – ORFs. Of these, 32.2% are related to functional assigned genes, 32.6% are conserved ORFs of unknown function, 5.1% are of some motifs while 30.0% did not show any important registered sequences. (3)

The functional genes are involved in sulfide metabolism, TCA cycle and respiratory chain. Studies have shown that the large genomic size of this specie, 2.7 million base pair, is due to the integration of plasmid, rearrangement of genomic structure and duplication of genomic regions. Moreover, its genome contains eukaryote-type structure of tRNA genes and the presence of tRNA nucleotidyltrasferase which catalyzed the addition of the CCA sequence to tRNA transcripts. Likewise, this strain contains 37 archaea-specific ORFS and 53 ORFs that exist in both archaea and eukaryotes. (3)

Cell structure and metabolism

The cell structure of S. tokodaii strain7 includes a cell envelope that is formed through a hexagonal S-layer lattice. The cells also contain membrane-bound cytochromes that are used in the specie aerobic respiratory terminal oxidase supercomplex. In addition, a soluble zinc-containing ferrodoxin attached on the membrane also serves as a major cytosolic electron transport protein. (1) Based on its genome structure, Sulfolobus tokodaii sp strain7 has a complete TCA cycle system, similar to that in the mitochondria of eukaryotes, with genes coding for citrate synthase and two subunits of 2-oxoacidferredoxin oxidoreductase. Nonetheless, genes needed in the respiratory chain to produce ATP are not present and cytochrome c – important in electron transfer to oxygen in eukaryotes- is also missing. These features suggest that this microbe may use a different molecule or a different pathway for this function; this information has yet to be discovered. Though, iron-sulfur protein and sulfocyanin are known to be used in place of cytochrome c in Sulfolobus species. (3)

A significant trait of S. tokodaii strain7 is that it is capable of oxidizing hydrogen sulfide to sulfate intracellularly. More specifically, eight ORFs have been found to code for enzymes that oxidizes sulfur from hydrogen sulfide to sulfate. (3) In addition, a gene encoding sulfur oxygenase-reductase is present as the main transcription factor in sulfur-grown cells of the Sulfolobus species. (2)

S. tokodaii strain7 contains a cluster of open reading frames that code for genes encoding proteins similar to that of cytochrome c oxidase subunits and CbsA-like cytochrome. These genes are identified as the fox genes and are predicted to code for membrane proteins. The presence of these genes allowed the growth of this specie and that of Sulfolobus metalicus on ferrous iron. (2)


Sulfolobus tokodaii strain7, like other specie in its genus, is a thermoacidophilic. Optimal growth occurs at pH 2.5 – 3 with temperature of 80 degrees Celsius. Best growth appears under chemoheterotrophic conditions through aerobic respiration. This specie is particularly specific in its environment in that it will show poor or no growth under facultatively chemolithotrophic conditions in the presence of elemental sulfur. Likewise, there is no sign of growth at temperature of 65 and 90 degrees Celsius or at pH 1 and pH6. (1)

The species of Sulfolobus, as they are acidophiles and thermophiles, are commonly found in volcanic springs or in continental areas of acidic geothermal activity. For instance, Sulfolobus solfataricus was located in the Solfatara – volcanic mud pots, as Sulfolobus tokodaii strain7 was isolated from a hot springs. (1) More specificly, many genes from this strain are contributed to sulfide metabolism; therefore, a sulfur rich environment induces its growth.


While Sulfolobus tokodaii strain7 has not been identified as a pathogen, its close relative, Sulfolobus tengchongensis, was found as a host for a virus. The virus infecting the hyperthermophilic archeon is known as Sulfolobus tengchongensis Spindle-Shaped Virus – STSV1, the largest of the known spindle-shaped viruses up to date. This crenarchaeotal virus was isolated from S. tengchongensis in an acidic hot springs in Tengchong of southern China. Virus-like particles of STSV1 have also been located in hot springs in Yellowstone National Park. (7)

Although the function of this virus in human disease is still under observation, STSV1 is noted to slow the growth of the host cells but does not cause lysing. The rapid replication of the virus in the host, by the 10^10 folds, retards the growth of the host. The virus does not integrate into the host genome but infected host cells contain the virus even after repeated transfers; this is a clear indication that STSV1 exists as a carrier trait in S. tengchongensis. (7)

Sulfolobus species are popular viral hosts because they provide protection for viruses in acidic and high temperatures conditions. In addition to the ability to adapt in extreme environments, archaeal genomes possess many opportunities to enter, form long-term colonization and can coexist with endogenous flora in the human host cells. They also have unique flagella and contain Tad-like proteins that are used for tight adherence to surfaces. These proteins enhance fibril formation where elements are structured to protrude out of the cells and “grab-on” to another constituent in the environment. Together, these traits provide the ideal home for viruses to grow. (6)

Application to Biotechnology

The creation of SO2 and other sulfur compounds disturbs the sulfur cycle and results in pollution, toxicity, acidification of rain and freshwater, increase COD- Chemical Oxygen Demand is the amount of organic pollutants in water- and maximize the greenhouse effect. The safest way to break the sulfur cycle is to form elemental sulfur, SO42- → S2- → S0, an insoluble product that can easily be recovered. In areas that contain sulfuric and highly acidic water bodies due to the sulfuric acid product of oxidizied sulfur compounds, such as the hot, acid springs of volcanic areas, Sulfolobus tokodaii strain7, along with other Sulfolobus species, are used as sulfur oxidizers. (4)

S. tokodaii strain7'’s ability to oxidize ferrous iron at high temperatures is efficient in the extraction of copper. The microbe starts off by converting the ferrous iron to ferric iron through oxidation and the electron transport system. Ferric iron is the mineral sulfide-oxidizing agent or the mineral sulfide chalcopyrite, CuFeS2. Presently, this is one of the most common forms of copper production. (4)

Current Research

1) A study done by the Department of Chemistry, Advanced Research Center for Science and Engineering at Waseda University of Tokyo Japan in early 2007 reported that the fen-1 gene from S. tokodaii strain7 is not located at the originally assigned AUG- the bases that code for the starting codon, but is stationed at, approximately, 100 bases upstream of it. Flap endonuclease-1, fen-1, is an endonuclease that is structure-specific and is important in DNA replication and repair processes. In the experiment, AUG was used as the translation start codon for one recombinant of the specie, referred to as StoS-FEN-1. In another recombinant, AUG was inserted upstream of the pre-ATG region, StoL-FEN-1. The results include that StoS-FEN-1 lacks FEN-1 functions and thermostability. On the contrary, StoL-FEN-1 shows FEN-1 activity and thermostability. Moreover, it was discovered that archaeal fen-1 proteins contain N-terminal region that resembles the pre-ATG region. These findings propose that the amino acids from the pre-ATG region, the N-terminal, are crucial in providing fen-1 activity & thermostability to S. tokodaii strain7 and that the translation start codon for fen-1 is upstream of the formerly assumed AUG codon. Nonetheless, the exact location and the bases that code for this codon are still under observations. (5)

2) Archaeal integrases are enzymes that carry out DNA recombination between the attachment sites of a genetic element and the host chromosome. The two integrated element that have been found to be associated within archaeal chromosomes are the SSV, contains a smaller N-terminal and larger C-terminal integrase gene fragment, and pNOB8 type, an intact integrase gene. Studies in recent years revealed that Sulfolobus tokodaii’'s genome contains four integrase gene fragments of pNOB8 type. Of this, an ORF region of 74 amino acids shows 89% identity of N-terminal region of the pNOB8 integrase. More importantly, this ORF segment is also the least conserved part of the tyrosine integrase; thus, this evidence suggests that there must be mechanisms available to maintain an integrated element within the S.tokodaii chromosomes. More importantly, the species of Sulfolobus have continued to use integrative genetic elements to acquire new constituents in their genomes. Nonetheless, the methods of selecting which elements to maintain during genome evolution are still under construction. Because these integrases are one of the largest protein classes known in microbial genomes, knowing how they function and their common traits will allow insight into microbial horizontal gene transfer and their genomes evolution. (8)

3) The use of toxic heavy metals contributes to the high arsenic levels in drinking water in areas such as East & West Bengal, American Midwest, and the Canadian Maritime Provinces. The two common forms of arsenic that humans are exposed to are arsenate or arsenite. Although arsenate, As(V) is highly soluble, but in the presence of calcium or insoluble iron compounds, it precipitates and can easily be removed. Arsenite in water exists as an inorganic equivalent that can cross cell membranes from bacterial to human cells through glyceroporin membrane channel proteins. For this reason, Bacteria and Archaea which contain the enzyme for the respiratory oxidation of arsenite, As(III) to arsenate, As(V), have been examined and considered as potential solutions for removing toxic arsenic-impacted environment. Recent studies proposed that the genome of Sulfolobus tokodaii contains gene pairs that are in the same order as asoA and asoB which code for the enzyme of arsenite oxidase. It has been suggested that this method of microbial metabolism and the precipitation of mineral deposits can be used in practical bioremediation of drinking water arsenic. Nonetheless, further research is required in this prospect because the mechanism for this oxidation process is uncleared. (9)


1. Suzuki, T., Iwasaki, T., Uzawa, T., Hara, K., Nemoto, N., Kon, T., Ueki, T., Yamagishi, A., and Oshima, T. “Sulfolobus tokodaii sp. Nov. (f. Sulfolobus sp. Strain 7), a new member of the genus Sulfolobus isolated from Beppu Hot Springs, Japan.” Extremophiles. 2002. Volume 6. pp 39-44.

2. Bathe, S., Norris, P. “Ferrous Iron-and Sulfur-Induced Genes in Sulfolobus metallicus.” Applied Environmental Microbiology. 2007. April Issue. pp2491-2497.

3. Kawarabayasi, Y., Hino, Y., Horikawa, H., Jin-No, K., Takahashi, M., Sekine, M., Baba, S., Ankai, A., Kosugi, H., Hosoyama, A., Fukui, S., Nagai, Y., Nishijima, K., Otsuka, R., Nakazawa, H., Takamiya, M., Kato, Y., Yoshizawa, T., Tanaka, T., Kudoh, Y., Yamazaki, J., Kushida, N., Oguchi, A., Aoki, K., Masuda, S., Yanagii, M., Nishimura, M., Yamagishi, A., Oshima, T., Kikuchi, H. “Complete Genome Sequence of an Aerobic Thermoacidophilic Crenarchaeon, Sulfolobus tokodaii strain7.” Oxford Journals – DNA Research. 2001 . Volume 8 Number 4 , pp 123 – 140

4. Kuenen, J.G., Robertson, L.A. “The Use of Natural Bacterial Populations for the Treatment of Sulfur-Containing Wastewater.” Biodegradation. 1992. Volume 3 Number 2-3. pp 239-254

5. Horie, M., Fukui, K., Xie, M., Kageyama, Y., Hamada, K., Sakihama, Y., Sugimori, K., Matsumoto, K. The N-Terminal Region Is Important for the Nuclease Activity and Thermostability of the Flap Endonuclease-1 from Sulfolobus tokodaii.” Bioscience, Biotechnology and Biochemistry. 2007. Volume 1 Number 4 . pp 855-865

6. Eckburg, P.B., Lepp, P.W., Relman, D.A. “Archaea and Their Potential Role in Human Disease.” Infection and Immunity. 2003. Volume 71 Number 2 . pp 591-596

7. Xiang, X., Chen, L., Huang, X., Luo, Y., She, Q., Huang, L. “Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features.” Virology. 2005. Volume 79 Number 14. pp 8677 – 8686

8. She, Q., Shen, B., Chen, L. “Archaeal Integrases and Mechanisms of Gene Capture.” Biochemical Society Transactions. 2004. Volume 32 Part 2 . pp 222- 226

9. Silver, S., Phung, L.T. “Genes and Enzymes Involved in Bacterial Oxidation and Reduction of Inorganic Arsenic.” Applied and Environmental Microbiology. 2005. Volume 71 Number 2 . pp 599 – 608.

Kendrix D. Nguyen University of California San Diego, Dr. Larsen - BIMM120