J. Gen. App!. Microbiol., 41, 75-81 (1995) Short Communication CHARACTERIZATION OF THE 16S RIBOSOMAL RNA GENES AND PHYLOGENETIC RELATIONSHIPS OF SULFUR-DEPENDENT THERMOACIDO- PHILIC ARCHAEBACTERIA NORIO KUROSAWA,* KIYOTAKA OHKURA, TADAO HORIUCHI, AND YUKO H. ITOH Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji 192, Japan (Received August 22, 1994; Accepted November 4, 1994) A comparative sequence study of the 16S rRNAs showed that living organisms can be divided into the traditional eubacterial and eukarcyotic kingdoms and the newly recognized archaebacterial kingdom (28). Although archaebacteria exhibit a prokaryotic cell structure and organization, they show distinguishing and unify- ing features (1, 9,12,16,18,22). In sulfur-dependent thermophilic archaebacteria, the following five genera have been assigned to the order Sulfolobales: (i) the genus Sulfolobus, which is the type genus and includes the species S. acidocaldarius (3), S. solfataricus (31), S. shibatae (5) and S. metallicus (8); (ii) the genus Acidianus, which includes the species A. infernus (25) and A, brierleyi (2); (iii) the genus Metallosphaera, with one species, M. sedula (7); (iv) the genus Stygiolobus, with one species, S. azoricus (26); and (v) the genus Desulfurolobus, with one species, D. ambivalens (32). D. ambivalens has been suggested to belong to the genus Acidianus based on DNA-DNA hybridization experiments (26). Only in S. acidocaldarius and S. shibatae, have the complete nucleotide sequences of the 16S rRNA genes have been reported and were shown to have 90.8% homology (5,15). Except for their relationships, a phylogenetic analysis has never been done among the other species in the order Sulfolobales. Although the 16S rRNA sequence of S. solfataricus was reported by Olsen et al. (19), the strain used by Olsen was recognized as S. acidocaldarius (15, 30). We have determined the complete nucleotide sequences of the 16S rRNA genes as well as their flanking regions from the sulfur-dependent thermoacidophilic * Address reprint requests to: Dr . Norio Kurosawa, Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji 192, Japan. 75 76 KUROSAWA et al. VOL. 41 archaebacteria S. solfataricus, A. brierleyi and M sedula in order to establish their intra- or inter-genus phylogenetic relationships and to characterize the structures of the 16S rRNA genes. Sulfolobus acidocaldarius ATCC 33909 was obtained from the American Type Culture Collection (ATCC). Sulfolobus solfataricus IFO 15331 (derived from DSM 1616), Acidianus brierleyi IFO 15269 (derived from DSM 1651) and Metal- losphaera sedula IFO 15509 (derived from DSM 5348) were obtained from the Institute for Fermentation, Osaka (IFO). S. acidocaldarius, S. solfataricus and M. sedula were grown in Brock's medium (3), pH 2.5, supplemented with 1.0g of yeast extract per liter at 80, 85 and 70°C, respectively. A. brierleyi was grown at 69°C in IFO 286 medium ((NH4)2SO4, 3.0 g; K2HPO4, 0.5 g; MgSO4. 7H2O, 0.5 g; KCI, 0.1 g; Ca(N03)2, 0.01 g/l) supplemented with 0.5 g of yeast extract per liter, pH 2.0. All cells were grown aerobically in a long-necked conical flask with an aluminum cap. Cells lysed in 0.5% SDS were extracted with phenol and precipitated from the aqueous phase at 0.3 M sodium acetate with ethanol. Genomic DNAs from S. solfataricus, A. brierleyi and M. sedula were digested with restriction enzymes (Sad, EcoRI, XhoI, and EcoRI XhoI), fractionated on 1.0% agarose gels, transfered onto nylon membranes, and probed with horseradish peroxidase (HRP)-labelled 16S rRNA gene fragment (around position 480-1370) of S. acidocaldarius obtained by PCR. HRP labelling was performed using the ECL direct nucleic acid labelling system (Amersham, U.K.). The sequences of the oligonucleotide primers used in the PCR were 5'-CAGCAGCCGCGGTAATAC and 5'-ACGGGCGGTGTGTGC. These sequences are universally found in the 16S rRNA genes not only in eubacte- ria but also in archaebacteria (6). In S. solfataricus, A. brierleyi and M. sedula, a single band was found for each restriction enzyme and their previously mentioned combinations. These results suggest that the 16S rRNA genes are present in one copy per genome of all the three strains. The same result was reported in S. shibatae by Reiter et al. (20). A 5.6-kbp EcoRI XhoI fragment of S. solfataricus, an 8.1-kbp EcoRI fragment of A. brierleyi and a 2.8-kbp EcoRI XhoI fragment of M sedula were cloned from genomic libraries constructed on t Zap II (Stratagene, La Jolla, U.S.A.) or plasmid Bluescript SK- (Stratagene) using standard methods (24). The DNA probe used in plaque or colony hybridization was prepared by the same method used in southern hybridization. Subclones of smaller fragments in plasmid Bluescript SK - or KS - (Stratagene) were used as sequencing templates. DNA sequence analysis was carried out by the dideoxynucleotide chain termination method with FITC- labelled primers using a HITACHI SQ-3000 DNA sequencer (Hitachi, Tokyo, Japan). Bca-best DNA polymerase (Takara, Ohtsu, Japan) was used and dGTP was replaced with dITP in all four sequencing mixtures to avoid "peak compres- sion." The lengths of 16S rRNA genes of S solfataricus, A. brierleyi and M. sedula were presumed to be 1495, 1492 and 1496 nucleotides, respectively. The 5'- and 3'- 1995 Phylogeny of Thermophilic Archaebacteria 77 ends of the 16S rRNA genes were deduced from the other archaebacterial 16S rRNA sequences reported. The percentage homologies within the 16S rRNA sequences from five strains of the order Sulfolobales and four strains of order Thermoproteales were estimated using the GENITYX-MAC software (Software Development Co., Ltd., Tokyo, Japan) (Table 1). We also present the phylogenetic tree based on their 16S rRNA sequences (Fig. 1). This tree, rooted by assuming four strains of order Thermo- proteales as the outgroup, was constructed by the neighbor joining method (23). Evolutionary distances were estimated by Kimura's two-parameter method (11), and were used for constructing the neighbor joining tree. All sites with gaps in any sequences were deleted for determination of evolutionary distances. Bootstrap probabilities, based on 1,000 resamplings, were calculated for each internal branch Table 1. Percentage homologies between the 16S rRNA sequences from five species of Sulfolobales and four species of Thermoproteales. Fig. 1. Phylogenetic tree based on the 16S rRNA sequences, constructed by the neighbor joining method (see text for details). Branch lengths, given below each branch, are proportional to the estimated number of nucleotide substitutions. Bootstrap probabilities (in percentage) are given above the internal branches. 78 KUROSAWA et al. VOL. 41 of the neighbor-joining tree using the CLUSTAL-V program developed by Dr. D. Higgins of the European Molecular Biology Laboratory. The homologies between pairs of the 16S rRNA sequences in the order of Sulfolobales have been more than 87%. Between S, solfataricus and S. shibatae, the homology of the 16S rRNA sequences was 99.5% (only 7 by among 1,495 by were Fig. 2. DNA sequences of flanking regions of 16S rRNA genes from Sulfolobus acidocaldarius, Sulfolobus solfataricus, Acidianus brierleyi and Metallosphaera sedula. Homologous bases (*), the archaebacterial promoter consensus sequences (boxed) and the inverted repeat sequences surrounding the 16S rRNA genes (underlined) are indicated. Putative 5' and 3' terminal of the 16S rRNA coding sequences are written in lower case letters. The last nucleotides of upstream regions (-1) and the first nucleotides of downstream regions (+ 1) are indicated. 1995 Phylogeny of Thermophilic Archaebacteria 79 altered). This result indicates that S. solfataricus and S. shibatae are closely related phylogenetically. In contrast, S. acidocaldarius showed 90.7 and 90.8% homologies with S. solfataricus and S. shibatae, respectively, and was diverged from the cluster of S. solfataricus and S. shibatae with bootstrap probabilities of 66% on the phylogenetic tree. These results suggest that S. acidocaldarius and S. solfataricus or S. shibatae may belong to different genera, although Sulfolobus strains are grouped based on mainly their aerobic characters and GC contents. The 16S rRNA sequences of A. brierleyi and MMsedula showed about 88% homology with any species of the genus Sulfolobus. A. brierleyi and MMsedula form another cluster against three species of genus Sulfolobus with bootstrap probabilities of 100%. We also sequenced the 5' and 3' flanking regions of the 16S coding regions. These sequences are aligned in Fig. 2 with corresponding sequence of S. acido- caldarius. The archaebacterial promoter consensus sequences, TTTATA (21, 27), was found in 171,170 and 165 nucleotides upstream from the putative 5' end of the 16S rRNA coding regions of S. solfataricus, A. brierleyi and M. sedula, respectively. The 23S rRNA genes begin from about 200 bases downstream of the 3' end of the 16S rRNA genes, and no tRNA genes were found in the spacer regions between the 16S and 23S sequences of any of the three strains. Each of the 16S rRNA genes was surrounded by long, nearly perfect, inverted repeat sequences. These sequences are able to form stem-loop structures which are similar to those shown in E. coli (29), also in archaebacteria; H. cutirubrum (4) and D. mobilis (13) except for the slightly shorter length of the stem. It seems that these regions would be attacked by the RNase III-like endonuclease depending on their locations and secondary structures. Although the 16S rRNA sequences of S. solfataricus and S. shibatae are very similar to each other, they showed only 50% homology in the 570-bp sequences upstream from the archaebacterial promoter consensus sequences. These results are consistent with the previous report presented by Grogan et al. in 1990 (5). In their report, although these two strains represented many similarities, they were classified into separate species because of the low genomic homology and differences in the RNA polymerase component patterns.