The genus Sulfolobus was established in 1972 and is the type genus of the order Sulfolobales. Members of the Sulfolobus were first isolated from acid thermal soils and acid hot springs in Yellowstone National Park (United States), El Salvador, Dominica, and Italy (Brock et al., 1972). Eight species of Sulfolobus have been characterized, described, and validly named under the International Code of Nomenclature of Prokaryotes (ICNP)¹; however, six of them were later reassigned to other genera. The type species is Sulfol. acidocaldarius, which was isolated from Locomotive Spring in Yellowstone National Park. Sulfol. yangmingensis and Sulfol. tengchongensis (which is not validly named) were isolated from a geothermal vent in Yang-Ming National Park in northern Taiwan, and an acidic hot spring in Tengchong, Yunnan, China, respectively (Jan et al., 1999; Xiang et al., 2003). Two species, Sulfol. islandicus isolated from Icelandic solfataras (Zillig et al., 1993; Reno et al., 2009) and Sulfol. sp. A20 isolated from a hot spring in Costa Rica (Dai et al., 2016), were also described and sequenced, although they were not validly named under the ICNP.

Cells of Sulfolobus are irregular cocci with frequent lobes, with diameter 0.8–1.5μm. Cells grow in the temperature range 55–95°C (optimal 65–85°C) and pH range 1.0–6.5 (optimal 2.0–4.0). Aerobic and facultatively chemolithoautotrophic growth occurs on S⁰ or a variety of complex organic compounds and sugars. Anaerobic growth of this genus has not been detected (Jan et al., 1999). The type strain Sulfol. acidocaldarius cannot oxidize elemental sulfur autotrophically in aerobic conditions (Brock et al., 1972; Huber et al., 1989; Huber and Prangishvili, 2006; Sakai and Kurosawa, 2018). Corresponding to this, genes encoding sulfur oxygenase for sulfur oxidation were not found in its genome (Sakai and Kurosawa, 2018).

Metallosphaera contains five members with valid name. The type species M. sedula was isolated from a continental solfataric field in Italy (Huber et al., 1989). Metallosphaera prunae was isolated from a smoldering slag heap of a uranium mine in Thuringen (Germany; Fuchs et al., 1995), and M. hakonensis was isolated from an acidic hot spring at a geothermal area in Hakone (Japan; Takayanagi et al., 1996; Kurosawa, 2003). Metallosphaera cuprina (Liu et al., 2011a) and M. tengchongensis (Peng et al., 2015) were both isolated from sulfuric hot springs in Tengchong (Yunnan, China). Metallosphaera hakonensis originally belonged to Sulfolobus, but it was reclassified to Metallosphaera in 2003 by Kurosawa (2003) based on phylogenetic evidence, DNA G+C content, and phenotypic properties (Table 2; Takayanagi et al., 1996; Kurosawa, 2003). Metallosphaera yellowstonensis was isolated from Yellowstone National Park and introduced in 2011 (Kozubal et al., 2011), which has not been validly named under the ICNP.

Cells of Metallosphaera are cocci or irregular cocci with diameter 0.8–1.2μm. Growth happens at 50–80°C (optimal 65–75°C), and pH 1.0–6.5 (optimal 2.5–3.5). Metallosphaera are aerobic and facultatively chemolithoautotrophic (Table 1). They can extract metal ions from several kinds of sulfidic ore, such as pyrite, chalcopyrite, and sphalerite. They can also oxidize S⁰ to sulfate but cannot reduce S⁰ (with or without the presence of H₂). Heterotrophic growth occurs on complex organic compounds, such as beef extract, casamino acids, peptone, tryptone, and yeast extract. Metallosphaera cannot use sugars and amino acids (with the exception of M. cuprina, which can use a few types of sugar and amino acid, such as D-glucose, D-xylose, L-arabinose, and L-tryptophan; Table 2; Huber et al., 1989; Fuchs et al., 1995; Takayanagi et al., 1996; Liu et al., 2011a; Peng et al., 2015).

The genus Sulfuracidifex was proposed by Itoh in 2020. The type species is Sulfura. tepidarius, which was isolated from a solfataric field at Hakone, Japan (Itoh et al., 2020). Another member is Sulfura. metallicus, isolated from continental solfataric fields in Iceland (Huber and Stetter, 1991). Sulfura. metallicus was reclassified from Sulfolobus because its phenotypic properties and 16S rRNA gene sequences are closer to those of Sulfura. tepidarius than to other members of the order Sulfolobales (Itoh et al., 2020).

Cells of Sulfuracidifex are irregular cocci with diameter 0.8–1.2μm. Growth occurs at 45–75°C (optimal around 65°C), and at pH 0.4–5.5 (optimal 2.5–3.5). Sulfuracidifex is obligate aerobes. Cells grow autotrophically on S⁰, reduced sulfur compounds, or sulfide ores. When grown mixotrophically, Sulfura. tepidarius uses several complex organics and sugars, whereas Sulfura. metallicus uses only yeast extract or glycogen as carbon sources (Tables 1 and 2; Huber and Stetter, 1991; Itoh et al., 2020).

The type species of genus Sulfodiicoccus is Sulfod. acidiphilus, isolated from the Hakone Ohwaku-dani hot spring in Japan in 2017 (Sakai and Kurosawa, 2017). At present, it is the only member of the Sulfodiicoccus. Growth of the species is significantly inhibited in the presence of S⁰. The DNA G+C content is 52.0mol%, which is remarkably higher than that of the other known species of the Sulfolobales (30.6–46.2mol%; Sakai and Kurosawa, 2017).

Cells of Sulfodiicoccus are cocci to irregular cocci with diameter 0.8–1.5μm. Cells grow at 50–70°C (optimal 65–70°C), pH 1.4–5.5 (optimal 3.0–3.5), and 0–2.5% (w/v) NaCl. Sulfod. acidiphilus is strictly aerobic and heterotrophic. Growth occurs on various complex substrates or sugars as carbon sources. Chemolithoautotrophic growth does not occur by oxidation of S⁰, pyrite, K₂S₄O₆, Na₂S₂O₃, or FeSO₄·7H₂O, or on H₂ (Sakai and Kurosawa, 2017).

There are six species described in the genus Acidianus: A. ambivalens (Zillig et al., 1986; Fuchs et al., 1996), A. brierleyi (Segerer et al., 1986), A. infernus (Segerer et al., 1986), A. manzaensis (Yoshida et al., 2006), A. sulfidivorans (Plumb et al., 2007), and A. tengchongenses, which is not validly named (He et al., 2004). Among them, A. infernus is the type species of Acidianus (Segerer et al., 1986). Acidianus ambivalens was previously named Desulfurolobus ambivalens (Zillig et al., 1986), but, as it is very similar to A. infernus in physiological and biochemical features, it was reassigned to the genus Acidianus (Fuchs et al., 1996). Members of the Acidianus occur in acidic solfataras and marine hydrothermal systems. Cells of Acidianus are irregular cocci with diameter 0.5–2.0μm. Cells grow at 45–96°C (optimal 70–90°C), pH 1.0–6.0 (optimal 0.8–2.5), and 0.1–4% (w/v) NaCl. Chemolithoautotrophic growth occurs aerobically by means of S⁰ oxidation or anaerobically via S⁰ reduction with H₂ as electron donor (Tables 1 and 2). Acidianus infernus (Segerer et al., 1986), A. ambivalens (Zillig et al., 1986), and A. tengchongenses (He et al., 2004) are obligately chemolithotrophic. A. brierleyi (Segerer et al., 1986), A. manzaensis (Yoshida et al., 2006), and A. sulfidivorans (Plumb et al., 2007) are facultatively autotrophic and can grow heterotrophically on yeast extract in the absence of S⁰ in aerobic conditions.

The genus Sulfurisphaera contains three species at present, Sulfuri. javensis (Tsuboi et al., 2018), Sulfuri. ohwakuensis (Kurosawa et al., 1998), and Sulfuri. tokodaii (Suzuki et al., 2002), which were all isolated from acidic hot springs (Kurosawa et al., 1998; Suzuki et al., 2002; Tsuboi et al., 2018). Sulfuri. tokodaii formerly belonged to Sulfolobus but was reclassified to Sulfurisphaera by Tsuboi et al. (2018) based on the latest phylogenetic data (Suzuki et al., 2002; Tsuboi et al., 2018). The type species of this genus is Sulfuri. ohwakuensis (Kurosawa et al., 1998).

Cells of Sulfurisphaera are irregular cocci with diameter approximately 1μm and grow at 60–96°C (optimal 80–84°C), pH 1.5–6.0 (optimal 2.0–4.0), and 0–1.5% (w/v) NaCl. Cells are facultatively anaerobic. Anaerobic growth occurs on FeCl₃ in the presence of yeast extract. Chemolithoautotrophic growth occurs on S⁰, S₄O₆²⁻, and pyrite in aerobic conditions. The G+C content is in the range 30.6–33.7mol% (Tsuboi et al., 2018).

The type species of the genus is Sa. solfataricus, which was first described by Zillig et al. (1980). The other two species are Sa. shibatae and Sa. caldissimus (Sakai and Kurosawa, 2018). Sa. solfataricus and Sa. shibatae were originally classified into the genus Sulfolobus (Zillig et al., 1980; Grogan et al., 1990). However, later study demonstrated that their abilities to use various sugars were quite different from that of Sulfol. acidocaldarius, the type species of Sulfolobus. The growth temperature and pH, and facultatively anaerobic characteristics, of Sa. solfataricus and Sa. shibatae are almost identical to those of Sa. caldissimus. Phylogenetic evidence based on 16S rRNA and 23S rRNA gene sequences also helped distinguish Sa. solfataricus, Sa. shibatae, and Sa. caldissimus from Sulfol. acidocaldarius. Therefore, Sa. solfataricus and Sa. shibatae were reclassified as Saccharolobus (Sakai and Kurosawa, 2018).

Cells of Saccharolobus are irregular cocci. The temperature and pH ranges for growth are 50–93°C and pH 1.5–6.0 (optima 80–85°C and 3.0–4.5), respectively. Cells are facultatively anaerobic, using FeCl₃ as an electron acceptor and yeast extract as an electron donor. Heterotrophic growth occurs on complex substrates, such as yeast extract and various kinds of sugar. Chemolithoautotrophic growth occurs on pyrite or, poorly, by oxidation of H₂. S⁰ and K₂S₄O₆ cannot be used as electron donors. The G+C content of this genus is in the range 31.7–35.8mol% (Sakai and Kurosawa, 2018).

Stygiolobus isolates were obtained from solfataric fields in the Azores and described by Segerer et al. (1991). The type species is S. azoricus, the only member of Stygiolobus, which is an obligate anaerobe. Cells are irregular cocci or lobed and are approximately 0.5–1.8μm wide in exponential growth phase, and frequently surrounded by pilus- or fimbria-like appendages. The growth temperature and pH ranges are 57–89°C and 1.0–5.5 (optimum around 80°C and 2.5–3.0), respectively. Stygiolobus azoricus is obligately chemolithotroph and grow by means of H₂–S⁰ lithotrophy. Growth was stimulated by a trace amount of yeast extract (0.005–0.02%; Segerer et al., 1991).

The genus Sulfurococcus contains two species: Sulfuro. mirabilis and Sulfuro. yellowsonensis. The original characterization manuscripts are both in Russian. However, the abstract (written in English) states that Sulfuro. yellowsonensis was isolated from the hydrotherm of Yellowstone National Park (United States) and is a spherical, sulfur-oxidizing thermoacidophile. It grows at 40–80°C. Sulfuro. yellowsonensis is a facultative autotroph that grows autotrophically by oxidizing elemental sulfur, ferrous sulfate, and sulfide minerals, and heterotrophically on organic compounds. The DNA G+C content is 44.6mol% (Karavaĭko et al., 1994).

As shown in Table 2, the cells of all Sulfolobales are cocci or irregular cocci, 0.5–2.2μm in diameter. However, they have diverse O₂ requirements and nutrition types. In general, Sulfolobus, Metallosphaera, Sulfuracidifex, and Sulfodiicoccus are obligate aerobes. Acidianus, Sulfurisphaera, and Saccharolobus are facultative anaerobes. Stygiolobus is the only obligate anaerobic genus within the Sulfolobales. Most members within Sulfolobales are facultatively chemolithoautotrophic, but Sulfodiicoccus species are heterotrophs (Sakai and Kurosawa, 2017). Stygiolobus and some species of Acidianus are obligately chemolithoautotrophic.

Compared with members of Sulfolobus, most Metallosphaera have greater ability to oxidize RISCs, such as S⁰, S₄O₆²⁻, and sulfidic ores, but lesser ability to use sugars. The members of Sulfolobus can use different types of sugar. However, compared with Sa. solfataricus, which shows high metabolic versatility and is able to use a broad spectrum of substrates, including mono-, di-, oligo-, and polysaccharides, Sulfol. acidocaldarius has a much narrower substrate spectrum. This could be attributed to its relatively small genome, which lacks numerous transport systems for substrate uptake (Lewis et al., 2021).

Both Sulfurisphaera and Saccharolobus are facultatively anaerobic and facultatively chemolithoautotrophic. They have similar abilities in using FeCl₃ as an electron acceptor in anaerobic conditions, while their abilities to use RISCs vary. In the case of Sulfurisphaera, chemolithoautotrophic growth occurs on various kinds of RISC in aerobic conditions. However, Saccharolobus can only oxidize pyrite poorly (Kurosawa et al., 1998; Sakai and Kurosawa, 2018). Although Zillig et al. (1980) described that Sa. solfataricus can use S⁰ as an energy source, the data of Sakai and Kurosawa (2018) indicate that this species cannot use pyrite or S⁰ at all (Zillig et al., 1980; Sakai and Kurosawa, 2018).

With the increasing number of isolates and phylogenetic data, several species of Sulfolobales have been taxonomically reclassified. Sulfolobus was the first described genus of the Sulfolobales. Six species that were originally classified as Sulfolobus species – A. brierleyi (Zillig et al., 1980), M. hakonensis (Takayanagi et al., 1996), Sa. solfataricus (Zillig et al., 1980), Sa. shibatae (Grogan et al., 1990), Sulfura. metallicus (Huber and Stetter, 1991), and Sulfuri. tokodaii (Suzuki et al., 2002) – have been reclassified into new genera according to later physiological and phylogenetic evidences (Segerer et al., 1986; Kurosawa, 2003; Sakai and Kurosawa, 2018; Tsuboi et al., 2018; Itoh et al., 2020; Table 2; Figures 2, 3).

Although features, such as morphology, temperature and pH for growth, O₂ requirements, and nutrition types of Sulfurisphaera, resemble those of the Acidianus, phylogenetic analyses including 16S rRNA gene similarities and DNA–DNA hybridization data distinguish it from the other genera of the Sulfolobales (Segerer et al., 1986; Kurosawa et al., 1998; Tsuboi et al., 2018).

As the phylogenetic tree in Figures 2, 3 show, members of the other genera of Sulfolobales each cluster together or closely, with the exception of the members of Sulfolobus, which are dispersed in different clusters. Notably, the 16S rRNA gene sequence of Sulfol. yangmingensis is more similar to that of Sulfuri. tokodaii and Sulfuri. ohwakuensis, and these three species form a clade in the phylogenetic tree (Figure 2). Furthermore, the use of organic compounds and RISCs by Sulfol. yangmingensis is also similar to that by Sulfurisphaera, although the G+C content of Sulfol. yangmingensis (42%) is much higher than that of Sulfurisphaera spp. (30–33%; Kurosawa et al., 1998; Jan et al., 1999; Tsuboi et al., 2018). Sulfol. islandicus, Sulfol. tengchongensis, and Sulfol. sp. A20, have been charactered or sequenced but not validly named, are all far from the type strain Sulfol. acidocaldarius but related to the clade containing Saccharolobus, according to phylogenetic analysis based on 16S rRNA gene sequences and whole genome sequences (Figures 2, 3). Average amino acid identity (AAI) and conserved multi-locus sequence alignment (MLSA) also indicate that Sulfol. acidocaldarius is distinct from the other species of Sulfolobus (Counts et al., 2021).

In addition, AAI and MLSA data for Sulfolobales suggest that Sulfod. acidophilus should be classified into a new family (Counts et al., 2021). All these observations indicate that the phylogenetic positions of the members of Sulfolobales need to be reconsidered.

Reduction of elemental sulfur is universal among hyperthermophilic archaea. Three genera of Sulfolobales – Acidianus, Sulfurisphaera, and Stygiolobus – reduce S⁰ to H₂S with H₂ as the electron donor (Table 2). Two membrane-bound, multisubunit enzymes are involved in S⁰ reduction in Acidianus: sulfur reductase (SR) and NiFe hydrogenase. SR is composed of five subunits encoded by the sreABCDE gene cluster: a large subunit (SreA), an Fe–S-cluster-containing subunit (SreB), a membrane-anchor subunit (SreC), and SreD and SreE, whose functions are unknown. Both SreA and SreB share sequence similarity with molybdopterin oxidoreductases belonging to the dimethylsulfoxide reductase family. sreABC gene clusters are found in Sulfol. islandicus, A. ambivalens, A. brierleyi, A. manzaensis, A. sulfidivorans, and Sa. solfataricus. The NiFe hydrogenase is encoded by an operon with 12 open reading frames, hynS–isp1–isp2–hynL–hynYZ–hypDCE–hypYZ–hoxM. HynS, HynL, and Isp1 are the small subunit and large subunit of the hydrogenase and the membrane-anchor protein, respectively. HynS and HynL contain [NiFe] and Fe–S clusters, respectively. HypDCE and HoxM are proteins required for hydrogenase maturation. Isp2, HynYZ, and HypYZ are proteins with unknown functions. Electron transfer between NiFe hydrogenase and SR is probably mediated by quinones in Acidianus (Laska, 2003).

Tetrathionate, the product of TQO, is further hydrolyzed by tetrathionate hydrolase (TetH), a pseudoperiplasmic protein attached to the S-layer, with an overall β-propeller structure. In A. ambivalens, TetH was found only in cells grown on tetrathionate; the gene is poorly expressed in cells grown on sulfur (Protze et al., 2011). TetH secreted by A. hospitalis YS8 forms zipper-like particles (ZLPs). The amounts of ZLPs that increased after cells were treated by mitomycin C, UV light, or by freezing in liquid nitrogen and rapid thawing, and they decreased to nondetectable levels after cells adapted to their growth conditions. TetH from A. hospitalis YS8 has 99% identity with that from A. ambivalens; both are stimulated by general stress (Krupovic et al., 2012).

TetH-coding genes exist in strictly or facultatively chemolithoautotrophic members of the Sulfolobales, which can grow in tetrathionate (Figure 3). Two copies of TetH-encoding genes (tetH1 and tetH2) are found in Acidianus, Sulfuracidifex, and Sulfurisphaera species. TetH1 and TetH2, the function of which is unknown, cluster in distinct clades in a dendrogram (Protze et al., 2011).

Heterodisulfide reductase (Hdr) is an iron–sulfur protein first discovered in methanogenic archaea that catalyzes reversible reduction of the heterodisulfide (CoM–S–S–CoB) of the thiol-coenzymes M (CoM–SH) and B (CoB–SH), coupled with energy conservation. Hdr is composed of three subunits, HdrA, HdrB, and HdrC. HdrA contains a typical FAD-binding motif and four [4Fe–4S] cluster-binding motifs. HdrB harbors two similar non-cubane [4Fe–4S] clusters and each cluster consist of fused [3Fe–4S]-[2Fe–2S] subcluster sharing one iron and one sulfur. The ferredoxin-like HdrC contains two [4Fe–4S] cluster-binding motifs (Hedderich et al., 2005; Wagner et al., 2017). Hdr complex-like proteins in sulfur-oxidizing bacteria and archaea are encoded by the gene cluster hdrC1B1A-hyp-hdrC2B2 (Liu et al., 2014). The Hdr complex in the thermophilic bacterium Aquifex aeolicus is a membrane-bound protein composed of at least five subunits: HdrA, HdrB1, HdrB2, HdrC1, and HdrC2 (Boughanemi et al., 2016). The Hdr complex is supposed to oxidize disulfide intermediates to sulfite and deliver the collected electrons to the membrane quinol pool. Furthermore, sulfur trafficking proteins, such as TusA and DsrE, are involved in transferring the sulfur groups to Hdr (Quatrini et al., 2009). Recent evidence showed that the Hdr complex oxidized thiosulfate to sulfite in Hyphomicrobium denitrificans, and the electrons produced may be transferred via a lipoate-binding protein (LbpA) to generate NADH (Cao et al., 2018; Koch and Dahl, 2018). The expression of Hdr subunits in M. cuprina Ar-4 increases when cells are grown in autotrophic conditions compared with heterotrophic conditions, indicating the participation of the Hdr-like complex in sulfur oxidation in M. cuprina (Jiang et al., 2014).

Sulfur trafficking is normally required for delivery of sulfur-containing groups as protein-bound forms to the sulfur-catalyzing enzymes. During this process, the unstable sulfur groups can be protected. The active site of TQO is suggested to face the cytoplasm; the tetrathionate produced by thiosulfate oxidation is thus released to the cytoplasm. However, tetrathionate is unstable at the near-neutral pH in the cytoplasm (Protze et al., 2011). Whereas the (rhd–)tusA–dsrE2 gene cluster is widely distributed in phototrophic and chemotrophic sulfur-oxidizing bacteria for transfer of sulfane sulfur, the dsrE3A–tusA–hdr gene cluster is ubiquitous in Sulfolobales (Figure 3). TusA appears to be a central and common protein for sulfur trafficking in sulfur-oxidizing pathways (Dahl, 2015). It has been proven in M. cuprina that the dsrE3A–tusA–hdr gene cluster is important in trafficking the sulfane sulfur of tetrathionate to prevent its biological toxicity. As shown in Figure 1, DsrE3A and TusA can both react with tetrathionate to form protein–Cys–S–thiosulfonate, which is stable in the cytoplasm. Then, DsrE3A–Cys–S–thiosulfonate transfers one thiosulfonate to TusA, forming TusA–Cys–S–thiosulfonate, and releases another thiosulfonate to TQO. The reverse transfer reaction does not happen. Next, the thiosulfonate combined with TusA serves as the substrate of the Hdr-like complex to produce sulfite for SAOR/SoeABC. The sulfane group remaining on TusA is then oxidized and released (Liu et al., 2014; Dahl, 2015).