INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Voi. 24, No. 1 January 1974, p. 102-1 12 Printed in U.S.A. Copyright 0 1974 International Association of Microbiological Societies Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a Nonsporulating Thermophilic Bacterium from a Japanese Thermal Spa TAIRO OSHIMA' and KAZUTOMO IMAHORI Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Tokyo, Japan The properties of an extremely thermophilic bacterium isolated from water at a Japanese hot spring and previously named Flavobacterium thermophihm are described. The cells are gram-negative, nonsporulating, aerobic rods containing yellow pigment. The optimum temperature for growth is between 65 and 72 C, the maximum being 85 C and the minimum being 47 C. The guanine plus cytosine content of the deoxyribonucleic acid of the thermophile is 69 mol %. This microorganism is sensitive to various antibiotics including those which are known to be rather ineffective against gram-negative bacteria. Spheroplast-like bodies are formed upon treating intact cells with egg-white lysozyme at 60 C. The spheres are osmotically more stable than mesophile protoplasts, and their rupture under hypotonic conditions is not complete unless 0.5% Brij 58 is added to the suspension. Bulk protein extracted from this thermophile is much more stable to heat than mesophile proteins, and only about 10% of the total protein is denatured by heating at 110 C for 5 min. Nevertheless, the amino acid composition of the bulk protein is similar to that of mesophile proteins. As the properties of this organism are similar to those of Thermus aquaticus (Brock and Freeze) and inasmuch as Flavobacterium is a poorly defined genus, this thermophilic microorganism is transferred to the genus Thermus as T. thermophilus (Yoshida and Oshima) comb. nov. The type strain is HB8 (=ATCC 2 7 634). For many years thermophilic organisms have temperature for growth of 79 C. We have been of general biological interest, and thermo- isolated nonsporulating, thermophilic bacteria philic bacteria have been isolated from a wide from hot springs in Japan. Among those variety of sources, such as compost, sewage, isolated, one strain is capable of growing at over soil, and water. The ability of thermophiles to 80 C. Although a variety of bacteria has been grow at temperatures that denature enzymes, found in thermal springs in Japan (7), this nucleic acids, and cellular organelles of meso- isolate has not been reported previously by philes has been intensively studied in the past other authors. In the previous studies involving few years, and much is now known about the this organism (15, 22-25), it was tentatively growth of thermophilic microorganisms and the placed in the genus Flavobacterium. The results biochemical properties of their cellular com- of an investigation to determine the proper ponents. Heretofore, studies on thermophilic taxonomic niche of this organism and to bacteria have mainly been on spore formers of attempt to gain some understanding of the the genus Bacillus, and especially on B. stearo- molecular basis of thermophily are reported thermophilus, which is a moderate thermophile here. having a maximum temperature for growth of 75 to 70 C. Recently, Brock and Freeze (5)isolated from MATERIALS AND METHODS water at a hot spring in the United States an strain. (HB8) extremely thermophilic bacterium which they Bacterial The strain studied here was isolated on 23 September 1968, from a hot spring (80 named Thermus aquaticus. T. aquaticus is a C, pH 6.3) at Mine, Shizuoka Prefecture, Japan. It has nonsporulating bacterium having a maximum been deposited at the Fermentation Research Insti- tute, Agency of Industrial Science and Technology, Present address: Mitsubishi-Kasei Institute of Life Chiba, Japan, under the number 2074 and at the Sciences, Minamiooya, Machida, Tokyo 194, Japan. American Type Culture Collection (ATCC), Rockville, 102 VOL. 24, 1974 T. THERMOPHILUS (YOSHIDA AND OSHIMA) COMB. NOV. 103 Md., under the number 27634. This strain has been Streptomycin and penicillin G were purchased from maintained by the authors in a freeze-dried state for-at Banyu Pharmaceutical Co. Ltd., Tokyo, Japan. Meth- least 30 months at -18 C and under reduced pressure icillin and tetracycline were from the Meiji Seika Co. and on an agar slant for 5 weeks in a refrigerator. Ltd., Yokohama? Japan, and Japan Lederle, Ltd., Culture media. The medium used for the cultivation Saitama, respectively. Ristocetin, ampicillin, D-cyclo- of strain HB8 consisted of 0.8% polypeptone (Daigo- serine, and novobiocin were provided by H. Matsu- eiyo Chemical Co. Ltd., Osaka, Japan), 0.4% yeast zawa in our laboratory, and actinomycin D was kindly extract (Kyokuto Seiyaku Co. Ltd., Tokyo, Japan), given by M. Kageyama, Mitsubishi-Kasei Institute of and 0.3% sodium chloride in distilled water. In some Life Sciences. experiments, polypeptone was replaced by 0.6% The effects of various inhibitors wqe studied by Casamino Acids (Kyokuto Seiyaku Co. Ltd.). The observing growth in liquid media containing inhibitors. medium was adjusted to pH 7.5 (measured at room A drop of inoculum obtained from a culture grown temperature using Toyo pH test paper no. 20) by overnight in the standard polypeptone-yeast extract adding 1.O N sodium hydroxide solution. metemper- medium was transferred into polypeptone-yeast ex- aturedependent coefficient of the medium was tract medium containing a suitable amount of an estimated to be -0.01 pH unit per degree. Unless antibiotic or antibacterial agent. The medium was then otherwise noted, cultures were incubated at 75 C in a incubated in an oven at 75 C for 48 h. dry oven. When necessary, sterile distilled water was Chemical analysis of DNA. Deoxyribonucleic acid added to the medium at suitable intervals to replace (DNA) was extracted by the method of Saito and the water lost by evaporation. Agar plates were Miura (16) and further purified by isopropanol prepared by adding powdered agar (final concentra- fractionation (12). The nucleic acid preparation was tion, 276, Wako Pure Chemical Industries, Ltd., Osaka, hydrolyzed by treatment with 60% perchloric acid in a Japan) to the polypeptone-yeast extract medium. The boiling water bath for 60 min (13). Paper chroma- agar plates were incubated at 60 C. tography of the hydrolysate was carried out by The medium used for the gelatin liquefaction test ascending development on paper strips (2 by 40 cm) was prepared by dissolving 300 g of gelatin (Junsei (Toyo Roshi No51A). The solvent systems used were Pure Chemicals Co. Ltd., Tokyo, Japan) in 100 ml of methanol-1 2 N HCl-water (7 :2 :1 , vol/vol) and iso- the Casamino Acids-yeast extract medium. After propanol (6.5 m1)-12 N HCl (1.67 m1)-water (final inoculation, tubes containing the gelatin medium were volume, 10 ml) (22). The spots were cut out and incubated at 75 C for 2 weeks. Every 2 days, water eluted by soaking in 0.1 N HCl. The nucleotide bases was added to replace that lost during the incubation thus eluted were determined by their ultraviolet period. One tube was not inoculated and served as a absorption spectra using the molar extinction coeffi- control. Liquefaction of gelatin was judged by cients recorded in the literature (22). A Beckman observing solidification of the medium after cooling in DK-IIA ratio recording spectrophotometer (Fullerton, an ice bath for 20 min. Calif.) was used in the spectrophotometric determina- Cell growth. Bacterial growth was followed by tions. measuring the absorption of the culture at 600 nm Amino acid analysis of the bulk protein. The bulk using a spectrophotometer (Model QU-3, Ito Electro protein of the thermophile in the cell-free extract was Medical Instruments Mfg. Co. Ltd., Tokyo, Japan). A S-carboxymethylated as described by Crestfield et al. linear relation between the cell concentration and the (6) but with a slight modification. To 20 ml of the absorbancy was observed in the range of absorbancy extract, 4 ml of 0.1 M sodium ethylenediaminetetra- of 0.1 to 0.6. By counting the cell population with a acetate, 36.1 g of urea, and 1.0 ml of p-mercapto- hematometer, it was estimated that 1.0 unit of ethanol were added. The mixture was diluted to 75 absorbancy at 600 nm corresponded to 2.1 X 10' cells ml, adjusted to pH 7.8, and incubated at 70 C for 1 h. per ml. The results are in fairly good accord with those No nitrogen barrier was used during the incubation. obtained by counting viable cells on agar plates. Carboxymethylation was carried out by adding a CeU-free extract. For large-scale cultivation, cells solution of monoiodoacetic acid (2.7 g, Merck and Co. were grown in polystyrene bottles containing 3 liters Inc., in 10 ml of 1.0 M sodium hydroxide) to the of the polypeptone-yeast extract medium in an oven mixture. After standing for 30 min in the dark, the at 75 C. The medium was aerated during the reaction mixture was dialyzed against distilled water incubation at a rate of 1 liter/min through a glass-filter and then freeze-dried. The Scarboxymethylated pro- ball. Usually it was not necessary to sterilize the tein thus obtained was dissolved in 6 N hydrochloric medium. A few drops of octyl alcohol were added to acid (about 2 mg/ml) and then hydrolyzed by prevent foaming. incubation at 105 C for 24 h. The amino acid content Harvested cells (10 g wet weight) were suspended in of the hydrolysate was analyzed using a Shibata amino 20 ml of 0.05 M tris(hydroxymethy1)aminomethane acid analyzer, model AA-600 (Shiba ta Seisakusho, (Tris)hydrochloride buffer, pH 7.5 , and subjected to Tokyo, Japan).