Desulfurococcus kamchatkensis sp. nov., a novel hyperthermophilic protein-degrading archaeon isolated from a Kamchatka hot spring

Proteinaceous substrates appear to be the most common energy sources used by anaerobic hyperthermophilic archaea (Vieille & Zeikus, 2001); however, the majority of these organisms utilize hydrolysed proteins, such as peptone, tryptone or yeast extract. Only a few organisms have been shown to be able to use non-hydrolysed proteins as substrates for growth: Pyrococcus horikoshii (Gonzalez et al., 1998), Palaeococcus ferrophilus (Takai et al., 2000) and Palaeococcus helgesonii (Amend et al., 2003).

Keratins, structural proteins of animals, are known to be very resistant to hydrolysis by chemical or biological agents because of their compact structures, high amounts of cross-linkages via disulfide bonds and high amounts of hydrogen bonds (Arai et al., 1996). In 2002, Friedrich and Antranikian examined several strains of thermophilic micro-organisms for their ability to grow on keratin, and only one of 19 was found to possess this capacity. Fervidobacterium pennivorans DSM 7003 represented one of the few thermophilic bacteria that can grow on keratins; however, at the time of writing, keratins have not been found to be utilized by representatives of the domain Archaea. Here we describe a novel species, Desulfurococcus kamchatkensis sp. nov., which is able to use proteins, including native α-keratin, as a sole energy and carbon source.

For enrichment of hyperthermophilic proteolytic micro-organisms, the following basal medium was used: (l⁻¹): NH₄Cl, 0.33 g; KCl, 0.33 g; KH₂PO₄, 0.33 g; CaCl₂.2H₂O, 0.33 g; MgCl₂.6H₂O, 0.33 g; yeast extract, 0.1 g; trace element solution (Kevbrin & Zavarzin, 1992), 10 ml; and vitamin solution (V1 vitamins kit; Sigma), 10 ml. α-Keratin (porcine hair obtained from SIFDDA Co.) was added as a substrate up to a final concentration of 2 g l⁻¹. Elemental sulfur (10 g l⁻¹) was added as an electron acceptor. The medium was prepared anaerobically, reduced by adding Na₂S.9H₂O (600 mg l⁻¹) and dispensed in 10 ml portions in 18 ml Hungate tubes; N₂ was used as the gas phase. The pH of the medium, measured at 20 °C using a pH meter calibrated at 20 °C, was adjusted using anoxic 3 N HCl. The medium was sterilized by autoclaving at 106 °C for 40 min. From a mixed water and sediment sample of Izvilistyi Spring, Central Field, Uzon Caldera, Kamchatka (5 ° 50′ 06.5″ N 16 ° 00′ 57.1″ E; pH 5.9, temperature 81 °C) an enrichment culture was obtained that grew at 80 °C and pH^2 °C 6.0. The dominant micro-organism was isolated by using serial ten-fold dilutions of the primary enrichment. The isolated strain was designated 1221n^T and characterized.

Cells of strain 1221n^T were regular cocci, with diameters varying from 0.6 to 1 μm (Fig. 1⇓). The cells were non-motile, and flagella were not observed on electron micrographs of negatively stained whole cells (data not shown). The cell wall was represented by a mono S-layer consisting of globular particles ∼150 Å in diameter. Growth of the novel isolate was observed at 65–87 °C (optimum, 85 °C) and pH 5.5–7.5 (optimum, pH 6.5). At optimal conditions, the doubling time of strain 1221n^T was 90 min. Strain 1221n^T did not grow at 60 or 90 °C, or at pH 5.0 or 8.0. Therefore, the novel strain was a neutrophilic, hyperthermophilic micro-organism. Strain 1221n^T was an obligate anaerobe that grew only in pre-reduced medium.

Utilization of organic substrates as energy and carbon sources was studied using threefold transfers on the same media. Strain 1221n^T was found to be an organotroph, able to utilize the following substrates (2 g l⁻¹): biotrypticase, tryptone, peptone, beef extract, yeast extract, α-keratin (porcine hair), albumin, gelatin, sucrose, glucose, and (weakly) dextrin and dextran. Growth was not observed when methanol, ethanol, arabinose, fructose, lactose, galactose, maltose, cellobiose, succinate, fumarate, pyruvate, acetate, starch, xylan, microcrystalline cellulose, carboxymethylcellulose, pectin, Casamino acids, β-keratin (ground feathers), chitin, casein or casein hydrolysate were added as substrates. The products of glucose fermentation determined according to Miroshnichenko et al. (2008) were CO₂ (3.5 mmol ml⁻¹), H₂ (2 mmol ml⁻¹), acetate (1 mmol ml⁻¹) and minor amounts of propionate and isovalerate. The presence of elemental sulfur in the medium significantly stimulated the growth of strain 1221n^T (the final cell concentrations after 28 h incubation were 9.75×10⁷ and 2.72×10⁷ ml⁻¹ with and without  °), and sulfide formation was detected (Trüper & Schlegel, 1964), indicating that sulfur was reduced during the fermentation process. In the absence of elemental sulfur, molecular hydrogen produced an inhibitory effect on the growth of strain 1221n^T – 0.67 of regular cell yield at atmosphere, containing 33 % H₂, and absolute inhibition at 100 % H₂. The same effect was shown for all representatives of the genus Desulfurococcus (Slobodkin & Bonch-Osmolovskaya, 1994) except D. fermentans (Perevalova et al., 2005). Possible electron acceptors were added to the sulfur-free medium as 50 mM sodium salts. Sulfate did not influence growth, whereas sulfite, thiosulfate and nitrate inhibited growth.

A number of proteases produced by hyperthermophilic archaea of the genera Desulfurococcus (Cowan et al., 1987), Sulfolobus (Fusek et al., 1990; Lin & Tang, 1990), Pyrococcus (Eggen et al., 1990; Dib et al., 1998), Thermococcus (Klingeberg et al., 1995), Pyrobaculum (Völkl et al., 1994) and others have been described during past decades. Using the zymogram method (Tsiroulnikov et al., 2004), an ∼120 kDa thermostable proteinase, active at 85 °C and pH 6.6 and 9.0 (data not shown), was found in the cell envelope fraction of strain 1221n^T, grown on α-keratin. An additional ∼40 kDa active band appeared when strain 1221n^T grew on albumin and peptone (data not shown). These results indicated that strain 1221n^T possessed a set of substrate-dependent proteinases, including one with keratinolytic ability.

Peptides are the usual substrates of hyperthermophilic organotrophic archaea including the members of the genus Desulfurococcus (Perevalova et al., 2005). We found that, apart from peptides, strain 1221n^T was able to grow on globular (albumin) and fibrillar (keratin) proteins as substrates. We verified the growth of the type strains of various Desulfurococcus species on non-hydrolysed proteins as sole energy and carbon sources, and found that all the organisms tested were able to grow on proteins, showing substrate specificity (Table 1⇓). This is the first evidence that hyperthermophilic archaea are able to grow on keratins, a capacity found in several thermophilic bacteria (Friedrich & Antranikian, 1996; Riessen & Antranikian, 2001). The only evidence of hyperthermophilic micro-organisms growing on keratin (Tsiroulnikov et al., 2004) did not include the phylogenetic characterization of the isolate.

DNA of strain 1221n^T was isolated according to Park (2007). The G+C content of the DNA was measured by using thermal denaturation of DNA (Marmur & Doty, 1962) and was calculated to be 44.4 mol%. An almost complete 16S rRNA gene sequence of strain 1221n^T (1496 nucleotides) was determined as described previously (Sokolova et al., 2002). Comparison of this 16S rRNA gene sequence against the existing database using blast (Altschul et al., 1997) showed that strain 1221n^T is a member of the phylum Crenarchaeota, class Thermoprotei, order Desulfurococcales and family Desulfurococcaceae. The phylogenetic position of strain 1221n^T was revealed by constructing a phylogenetic tree based on 16S rRNA gene sequences of members of the family Desulfurococcaceae (Fig. 2⇓), using mega software (Tamura et al., 2007). The analysis showed that the level of 16S rRNA gene sequence similarity between strain 1221n^T and other Desulfurococcus species was 96.7–98.1 %, and Desulfurococcus amylolyticus (Bonch-Osmolovskaya et al., 1988) was found to be the most closely related micro-organism with 98.1 % 16S rRNA gene sequence similarity. However, strain 1221n^T differed from D. amylolyticus in temperature and pH for growth, use of a different set of carbohydrates and the capacity for growth on α-keratin. Thus, strain 1221n^T should be classified as representing a novel species, for which the name Desulfurococcus kamchatkensis sp. nov. is proposed.