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23S rDNA Spacers
System. Appl. Microbiol. 19,61-65 (1996) © Gustav Fischer Verlag· Stuttgart· Jena . New York
Phylogenetic Analysis of the Archaeal Order of Sulfolobales Based on Sequences of 235 rRNA Genes and 165/235 rONA Spacers SIRO I. TREVISANATO l , NIELS LARSEN 2 , ANDREAS H. SEGERER3 , KARL O. STETTER3 , and ROGER A. GARRETT l 1
2 3
Institute of Molecular Biology, Copenhagen University, DK-1307 Copenhagen K, Denmark Department of Microbiology, Michigan State University, East Lansing, MI 48824, USA Lehrstuhl flir Mikrobiologie, Universitat Regensburg, D-93053 Regensburg, Germany Received July 21, 1995
Summary 23S rRNA genes from six members of the order of Sulfolobales, within the crenarchaeotal branch of the archaea, were cloned and sequenced. For each of the organisms Acidianus brierleyi, Acidianus infernus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus shibatae and Stygiolobus azoricus the 23S rRNA gene is linked directly to that of 16S rRNA by a short (- 200 bp) spacer with no intervening tRNA gene and no closely linked 5S rRNA gene which is typical for the Crenarchaeota. A phylogenetic tree, derived from the 23S rRNA gene sequences, shows that the six organisms are closely related, compatible with their belonging to one Order, and cluster in three pairs: the two Acidianus species, S. shibatae and S. solfataricus, and S. acidocaldarius and St. azoricus.
Key words: Phylogeny - 235 rRNA - 5ulfolobales - Acidianus brierleyi - Acidianus infernus - Sulfolobus acidocaldarius - Sulfolobus solfataricus - Sulfolobus shibatae - Stygiolobus azoricus - 165/235 intergenic spacer
Introduction Members of the order 5ulfolobales, and in particular
Sulfolobus, are amongst the best characterized of the ex-
tremely thermophilic archaea. The ease with which the latter can be grown, under aerobic conditions, renders them the organisms of choice for molecular genetic studies on the Crenarchaeota and, currently, different cloning vectors are being developed for Sulfolobus (Aagaard et al., 1994; Schleper et al., 1994). Therefore, it is important to classify these diverse organisms phylogenetically. The archaeal order of 5ulfolobales comprises the genera
Acidianus, Desulfurolobus, Metallosphaera, Stygiolobus
and Sulfolobus (Stetter et al., 1990). 5ulfolobales generally metabolize sulphur by oxidation but some can grow anaerobically and reduce sulphur (Segerer et al., 1986; Grogan, 1989). These assignments were made on the basis of morphology and growth requirements, their low genomic G+C contents (31-45%) (Segerer et al., 1986; 1991; Grogan, 1989) and the subunit composition of their DNA-dependent RNA polymerases (Zillig et al., 1980), all
of which are only approximate criteria for phylogenetic relatedness. The rRNA sequences provide a more exact evolutionary chronometer, and the 235 rRNAs have an advantage over the 165 rRNAs (which are presented in the accompanying paper - Fuchs et al., 1996) because of their larger size and larger number of variable regions. Here, we present 235 rDNA sequences, and a phylogenetic analysis, for six members of the 5ulfolobales, including three species of Sulfolobus. The 165/235 rDNA spacer regions which are devoid of tRNA genes (reviewed by Garrett et al., 1991) and can form archaeal-specific secondary structures (Kjems and Garrett, 1990) are also presented.
Materials and Methods
Growth ofcells. A brierleyi strain SP3a/1, DSM 6334 (Zillig et aI., 1980; Segerer et aI., 1986), A. infernus strain S04a, DSM 3191 (Segerer et al., 1986), S. acidocaldarius, DSM 639 (Grogan,
62
S. I. Trevisanato et al.
1989) and S. solfataricus, DSM 1616 (Zillig et aI., 1980) were grown on Brock's medium (Brock et aI., 1972) at their optimal growth temperatures and St. azoricus strain FC6, DSM 6296 was grown as described earlier (Segerer et aI., 1991). Cells were centrifuged and stored as frozen pellets. S. acidocaldarius and S. solfataricus cells were grown from single colonies. A culture of S. shibatae, DSM 5389 (Grogan et aI., 1990) was obtained from Prof. W. Zillig and grown in Brock's medium (Brock et aI., 1972). Purification of nucleic acids. DNA was extracted from suspended cells using glass beads (Leffers et aI., 1987) and purified by isopycnic ultracentrifugation (Sambrook et aI., 1989). RNA was extracted from cells, suspended in guanidinium-thiocyanate buffer and lysed by vortexing with glass beads (Chomoczynski and Sacchi, 1987). Amplification of 23S rRNA genes. Polymerase chain reaction (PCR) amplification of genomic DNA was performed in 100 IAI under a layer of mineral oil, using a Hybaid Thermocycler (Teddington, UK) (Sambrook et aI., 1989). The following oligonucleotide primers were used to amplify overlapping segments of the 235 rRNA gene; where numbers in brackets correspond to the position within S. acidocaldarius rRNA (Maidak et aI., 1994). 165 rRNA: TCGTAACAAGGTAGCCGT (1451-1468); 235 rRNA: TACCCAGGGGCCGAAGCCTCCCGG (1-24), GAAAGGGGGACAGCCCAAA (242-261), TAACACCACATCCCCACC (300-283), TTCTGACGTGCAATTCGTTC (884-903), GGGAACTAGCTATCACCG (959-942), CCTGACTGTTTAATAAAA (1921-1938), GTACACACCCTTTCGGGCT (1972-1954), AGTACGAGAGGAACAGGG (2787-2804), AGAAGAGGG-
GGTTGATGG (2943-2960), CCATCAACCCCCTCTTCT (2960-2943), CCAGGGGGGAGCCTGATT (3033-3016), 55 rRNA: GGCTTAACTTCCGGGTTC (64-43). Cloning and sequencing of PCR products. PCR products were 5'-phosphorylated and treated with Klenow enzyme to produce blunt ends (Sambrook et aI., 1989) before cloning into polylinker sites in M13mp18 and M13mp19 and transforming into competent Escherichia coli HB101 cells (Sambrook et aI., 1989). The cloned fragments were sequenced using standard protocols for the dideoxy sequencing method (Leffers et aI., 1987, Sambrook et aI., 1989). Phylogenetic analyses. 235 rRNA sequences were aligned using the ae2 editor (Maidak et aI., 1994). Evolutionary distances were estimated by the program DNADIST in PHYLIP Version 3.5c (Felsenstein, 1993) using the Kimura 2-parameter model program that weights transitions and transversions at 2:1 (Kimura, 1980). The tree was computed from the distance matrix using the Fitch-Margoliash least square fit program coupled to global optimization (Fitch and Margoliash, 1967). 165-235 spacer sequences were aligned using the PileUp program (Genetics Computer Group, Inc., 1994) and adjusted on the basis of the localization of common stem-loop structures (Kjems and Garrett, 1990).
Results and Discussion 23S rRNA gene sequences Alignment of the 235 rRNA gene sequences from the six organisms showed an identity level of 82-95% (Fig. 1)
A. A. brierleyi A.brierleyi
A. in/emus
S. acidncaJdarius
S. solfataricus
S. solfataricusMT4
100
86.6
87.5
100
87.5 S. solfataricus
88.5
89.3
100
S. solfataricusMT4
87.0
87.6
87.5
97.4
100
S.shibatae
81.9
86.2
87.0
95.1
95.2
88.8
88.6
89.1
87.3
A. brierleyi
A. in/emus
S. acidncaldarius
S. solfataricus
S. solfata- S. ricusMT4 shibatae
31
31
37.2
St. azoricus
St. awricus
100
A. in/emus 93.6
S. acidncaJdarius
S. shibatae
88.1
100 88.0
B.
35.1
40
34.6
St. azoricus
38
100
·Fig. 1. 235 rDNA sequence comparisons for the six members of 5ulfolobales. A: Nucleotide sequence identities of the 235 rDNAs expressed as a percentage. B: The G+C content of the genomic DNA for the six organisms where the data derive from the following sources: the two Acidianus species (Segerer et aI., 1986), the three Sul(olobus species (Grogan et aI., 1990) and St. azoricus (Segerer et al.,1991).
235 rRNAs of Sulfolobales
and introns that have been detected in other crenarchaeotal rRNA genes (Garrett et a1., 1994) were not detected. The sequence of the 235 rRNA gene of S. acidocaldarius was 99.9% identical to that of "S. solfataricus" in the Ribosomal Database (Maidak et a1., 1994) consistent with the use of a contaminated culture in the earlier work (see Zillig, 1993); it also corresponded to the S. acidocaldarius sequence of Durovic and Dennis (1994). Complete 235 rDNA sequences are presented for A. brierleyi (3046 bp), S. acidocaldarius (3034 bp) and S. solfataricus (3043 bp) and minor omissions, mainly at the downstream end of the gene, are present in the sequences A. infernus (2955 bp), S. shibatae (2972 bp) and St. azoricus (2909 bp). EMBL-Genbank accession numbers for the six 235 rRNA sequences are as follows: A. brierleyi - U32317, A. infernus - U32318, S. acidocaldarius U32320, S. solfataricus - U32322, S. shibatae - U32321 and St. azoricus - U32319.
1
63
16S rRNA ~
A. B. C. D. E. F.
GCGGCUGGAU GCGGCUGGAU GCGGCUGGAU GCGGCUGGAU GCGGCUGGAU GCGGCUGAAU
A. B. C. D. E. F.
GGAUGCUCAC CAGGAGAGCC GUAGGAGGUC GUAGGAAGCC AGGGCUUCAC AGGGCUUCAC
A. B. C. D. E. F.
UCUCA .. AAU ACUC .. UUCG CCUCU . UGGC CCUCU . UGGC CCUCUUGGGC UCCCUUUGGG
A. B. C. D. E. F.
GACAUUACAU . UA . AUA . AUA CAUA CA GGCU UA
A. B. C. D. E. F.
UACGACCAAC AACGACCGGA UGGCCUUAGG UGGCUUAGGG CUAGGUAGCC CCAGGCGACC
51
50
CACCUCACAU CACCUCACAU CACCUCACAG CACCUCAUAG CACCUCAUA. CACCUCACA.
..... AAAGC UCCACAACUU UUACAAACUC UUACAAACUC UAUUUA ... C UAAAUAACUC
processing stem
101
UAUAAUCUCG ACUUAAGCUC CAUAAAACCC CAUAAAACCC UAAAACUCGU UAUAAUCCUC
UCUCCUGUUU UUGGUGCUCC CCCCGUCAAU CCCCGUCAAU UCCCCCGCUA CCCCGCCAAA
~
GUUCCUCUUG UCGGCUUCCC UUGGUUCCCU UUGGUUCCCU AAUCUUCCCU UGGUUCUCCU
CUU.. . . . .. CAUUUACUUA UCUUACAG.. UUUACAG. .. UUUAUAG. .. CUGGCAG. ..
CUAAGUAGCU CUAAGAGCCU AUAAGAGCAA AUAAGAGCAA UGAAGUGCCU UGAAGUGCCU
AAGAGUGCCC A.. GGACUCG A. GGGCCUAU A. GGGCCUAU A. GGGCCCAU A. GAGCCCGU
GA. UAAUCUG GCUUAUUGUG GC AGCUC CA . . . . . . • . . . . CUCCCUA CUC. . . . . •. . UCCCUA AA. UAUGUCU CUGCCAAGUU AA .. GGUCAC CUAUCAAUAU
GGUCUC. AGU GGGUCC.AGU AGGGCU AGGGCU. . .. AGGGCUCAAU UGGGCUCAAU
helix E
~
. UGAGAGGAA .. GAGUGGAG CAGAGGAGAA CAGAGGAGAA CAGAGGGGAA CGGGGAGGAA
AGAGGAGGCA CCUAGAGGGG UUCAGUAGGG UUCCAUAGGG AUUGGGUGGG UUAGGGCGGG
~
~
100
AUGCCGUUCC AUGCGGUUCC AUGCGAUUCU AUGCGGUUCU AUGCAGUUCU AUGCAAUUCC
150 ACAAUAGGUU AGUU . GGGAG ...•. GGGAA ..... UUGGCAGAGA AUCGGUAGGU
Secondary structures 5econdary structural models derived for each of the 235 rRNAs correspond very closely to the structure presented earlier for Desulfurococcus mobilis 235 rRNA (Leffers et a1., 1987) and they revealed only minor differences from one another. The main differences fall within hypervariable sequence regions of domains I (helices 9 and 10) and domain IV (helix 98) (Leffers et a1., 1987). Multiple compensating base changes occur within helices 9 and 10 and small changes in helix length (4-5 bp for helix 9 and 7-10 bp for helix 10) whereas in helix 98, larger changes in length were observed; 11 bp for A. brierleyi and A. infernus and only 3 to 5 bp for the three Sulfolobus and S. solfataricus MT4 (Martayan et a1., 1994); the corresponding sequence for St. azoricus was not determined. Complete secondary structures are available from Gutell et a1., (1993) on request.
Sequences of the 16S123S rRNA intergenic spacer 5equences of the 165/235 rRNA spacer region were determined by binding a primer at the conserved downstream end of the 165 rRNA gene, which invariably exhibits the sequence 5'-CCUCA-3' rather than 5'-CCUCC-3' found in other archaeal165 rRNAs, and at the start of the 235 rRNA gene before amplifying the intervening spacer sequence by PCR. The results revealed a spacer of about 200 bp in each organism and the absence of tRNA genes. The sequences are aligned in Fig. 2 and were submitted to the EMBL-Genbank together with the corresponding 235 rDNA sequences. The conserved sequence motif, 5'-AUGC ... AAG-3', flanking helix E (Fig.2) is common to the crenarchaeotal 165/235 RNA spacer sequences (Kjems and Garrett, 1990; Garrett et a1., 1994) including that of S. solfataricus strain MT4 (Londei P., pers. comm.). Helix E, as for other archaea (Kjems and Garrett, 1990), is conserved in length (10 bp) and exhibits several compensating base changes for the six organisms. In contrast, helix F is less conserved and varies in length from 11-23 bp with a variable terminal loop sequence.
151
201
helix F
processing stem GCCAGACAGC GCUAAACAGC GCGAAAGGCC CCGAAAGGCC ACAUUAUAGC GCCUAAUAAC
~
~
200
GAGGCUAG. U GAGGCUAGCC CGAUGAAGC . CGAUGAAGC GAGGCUAGUA GAGGCUGGUG
236 CACCUAGGGA CACCUAGGGA GCUUAGAAUC GCUUAGAAUC CGUCUAGGAG CGCCUAGGGA
UGGC .. CCGACU GUUAA. GUUAA. UU . AA .
Fig. 2. Alignments of the 165/235 rRNA spacer region for A. brierleyi, B: A. infernus, C: S. solfataricus, D: S. shibatae, E: S. acidocaldarius and F: St. azoricus. The limits of the secondary structural elements are indicated by arrow heads and include the approximate ends of the 165 and 235 rRNA processing stems and helices E and F (Kjems and Garrett, 1990). A conserved sequence flanking helix E is underlined. The 3'-terminal nucleotide of the 165 rRNA (A-17) is conserved as an adenosine amongst the 5ulfolobales whereas other Crenarchaeota exhibit a cytidine (Maidak et aI., 1994).
Absence of linked 5S rRNA genes No PCR product was obtained using primers complementary to the 3'-end of the 235 rRNA and the centre of 55 rRNA of S. acidocaldarius. The latter primer was complementary to a conserved sequence GGCTTAACTTCCGGGTTC (positions 64 to 43) in crenarchaeotal 55 rRNA. A positive control experiment was performed with primers complementary to the 3'-end of 165 rRNA and the 3'-end of 235 rRNA which generated a 3.4 kbp fragment (data not shown). We infer, therefore, that the spacing between the 235 rRNA and 55 rRNA genes was at least several kilo base pairs (Ponce and Micol, 1992) reinforcing the view that the rRNA gene organization of the 5ulfolobales is typical of Crenarchaeota (re-
S. I. Trevisanato et al.
64
viewed in Garrett et aI., 1991), including S. shibatae for which conflicting evidence appeared (Neumann et aI., 1983; Reiter et aI., 1987). Phylogenetic relationships
The relatively high sequence identity (85-95%) of the six 235 rDNA sequences (Fig. 1), the degree of conservation of the inferred rRNA secondary structures, and the similar sequences and secondary structures of the 165/235 rRNA spacers (Fig. 2) all indicate that the six organisms are related and belong to the same phylogenetic lineage. This correlates with both the similar genomic G+C contents of the different organisms (Fig. 1) and the comparative rRNA-DNA hybridization studies for A. brierleyi, S. acidocaldarius, S. shibatae, and S. solfataricus (Zillig et aI., 1987). M. vannielii H. morrhuae S. solfatancus S. MT4
S. shibatae S. acidocaldarius SI. azoncus A. brierleyi
D. mobilis 0.10
Fig. 3. Phylogenetic tree for the Sulfolobales. The tree is derived from aligned 23S rRNA sequences for the six members of Sulfolobales and S. solfataricus strain sp. MT4 (Martayan et aI., 1994). Included in the analysis are 23S rRNA sequences from Methanococcus vannielii Uarsch and Bock, 1985), Halococcus morrhuae, and Desulfurococcus mobilis (Leffers et aI., 1987) from the order Thermoproteales. Programs used in the analyses are described under Materials and Methods.
A phylogenetic tree is presented for the six organisms and S. solfataricus MT4 (Martayan et aI., 1994) in Fig. 3. It shows clusters of three pairs of organisms, A. brierleyi and A. infernus, S. shibatae and S. solfataricus, and S. acidocoldarius and St. azoricus, and it correlates well with the phylogenetic analysis of the 165 rRNA sequences in the accompanying paper (Fuchs et aI., 1996). Acknowledgements. We are grateful to Ilia Leviev and jonathan Trent for their helpful advice. The research was supported by the Danish Natural Science Research Council, the NOVO-Nordisk Fund and the EU Generic Project, Biotechnology of Extremophiles, Contract Bio-CT93-02734.
References
c., Phan, H., Trevisanato, S., Garrett, R. A.: A spontaneous point mutation in the single 23S rRNA gene of the
Aagaard,
thermophilic archaeon Sulfolobus acidocaldarius confers multiple drug resistance. J. Bacteriol. 176, 7744-7747 (1994) Brock, T. D., Brock, K., Belly, R. T, Weiss, R. L.: Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Microbiol. 84, 54-68 (1972) Chomczynski, P., Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162, 156-159 (1987) Durovic, P., Dennis, P. P.: Separate pathways for excision and processing of 16S and 23S rRNA from the primary rRNA operon transcript from the hyperthermophilic archaebacteria Sulfolobus acidocaldarius: similarities to eukaryotic rRNA processing. Mol. Microbiol. 13, 229-242 (1994) Felsenstein, ].: PHYLIP (Phylogeny Inference Package) Version 3.5c. Distributed by the author at Department of Genetics, Un.iversity of Washington, Seattle, USA, 1993 Fitch, W. M., Margoliash, E.: Construction of phylogenetic trees. Science 155, 279-284 (1967) Fuchs, T, Huber, H., Burggraf, S., Stetter, K.O.: 16S rRNAbased phylogeny of the Sulfolobales. System. Appl. Microbiol. in press (1996)
Garrett, R. A., Dalgaard, j., Larsen, N., Kjems,]., Mankin, A. S.:
Archaeal rRNA operons. Trends Biochem. Sci. 16, 22-26 (1991)
Garrett, R. A., Aagaard, c., Andersen, M., Dalgaard, j. Z., Lykke-Andersen, ]., Phan, H. T. N., Trevisanato, S., 0stergaard, L., Larsen, N., Leffers, H.: Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny. System. Appl. Microbiol. 16,680-691 (1994) Genetics Computer Group Inc.: Wisconsin Package, Version 8.0 - open VMS, 575 Science Drive, Madison, Wisconsin (1994) Grogan, D. W.: Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains. J. Bacteriol. 171, 6710-6719 (1989) Grogan, D., Palm, P., Zillig, W.: Isolate B12, which harbours a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov. Arch. Microbiol. 154, 594-599 (1990) Gutell, R. R., Damburger, S. H., Gray, M., Schnare, M. N.: Compendium of large subunit (235 and 23S-like) rRNA structures. Nucl. Acids Res. 21, 3055-3074 (1993) jarsch, M., Bock, A.: Sequence of the 23S rRNA gene from the archaebacterium Methanococcus vannielli: evolutionary and functional implications. Mol. Gen. Genet. 200, 305-312 (1985) Kimura, M.: A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111-120, (1980) Kjems, ]., Garrett, R. A.: Secondary structure elements exclusive to the sequences flanking rRNAs lend support to the monophyletic nature of the archaebacteria. J. Mol. Evol. 31, 25-32 (1990) Leffers, H., Kjems, j., 0stergaard, L., Larsen, N., Garrett, R. A.: A comparative study of 23S rRNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophile methanogen. J. Mol. BioI. 195,43-61 (1987) Maidak, B. L., Larsen, N., McCaughey, M.j., Overbeek, R.,
Macke, T]., Olsen, G.]., Fogel, K., Blandy,]., Woese, C. R.:
The ribosomal database project. Nucleic Acids Res. 22, 3485-3487 (1994) Martayan, A., Caprini, E., Londei, P.: The 23S-rRNA of Sulfolobus solfataricus (strain MT4): sequence, structure and functional homology with other 235-rRNAs of thermophilic, sulfur-dependent archaea. System. Appl. Microbiol. 16, 170-176 (1994) Neumann, H., Gierl, A., Tu,]., Leibrock,]., Staiger, D., Zillig,
23S rRNAs of Sulfolobales
W.: Organization of the genes for rRNA in archaebacteria. Mol. Gen. Genet. 192, 66-72 (1983) Ponce, M. R., Mical, j. 1.: PCR amplification of long fragments. Nucleic Acids Res. 20, 623 (1992) Reiter, W.-D., Palm, P., Voos, W., Kaniecki, j., Grampp, B., Schulz, W., Zillig, W.: Putative promoter elements for the rRNA genes of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12. Nucleic Acids Res. 15, 5581-5595 (1987) Sambrook, W., Fritsch, E. F., Maniatis, T. H.: Molecular cloning, a laboratory manual, 2nd edition. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) Schleper, Roder, R., Singer, T., Zil/ig, W.: An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous ~-galactosidase gene. Mol. Gen. Genet. 243, 91-96 (1994) Segerer, A. H., Neuner, A., Kristjansson, j. K., Stetter, K.O.: Acidianus infernus gen. nov., spec. nov., and Acidianus brierleyi comb. nov.: facultatively aerobic, extremely acidophilic thermophilic sulfur-metabolizing archaebacteria. IntI. ]. System. Bacteriol. 36, 559-564 (1986)
c.,
Segerer, A. H., Trincone, A., Gahrtz, M., Stetter, K.O.: Stygiolobus azoricus gen. nov., sp. nov. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. IntI. ]. System. Bacteriol. 41, 495-501 (1991) Stetter, K.O., Fiala, G., Huber, G., Huber, R. and Segerer, A.: Hyperthermophilic microorganisms. FEMS Microbiol. Revs. 75, 117-124 (1990) Zil/ig, W., Stetter, K. 0., Wunderl, S., Schulz, W., Priess, H., Scholz, T.: The Sulfolobus "Caldariella" group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch. Microbiol. 125, 259-269 (1980) Zil/ig, W., Holz, T., Klenk, H. P., Trent, J., Wunderl, S., Janekovic, D., Imsel, E. and Haas, B.: Pyrococcus woesei sp. nov., an ultra-thermophilic marine archaebacterium, representing a novel order, Thermococcales. System. Appl. MicrobioI. 9, 62-70 (1987) Zillig, W.: Confusion in the assignments of Sulfolobus sequences to Sulfolobus species. Nucleic Acids Res. 21, 5273 (1993)
Prof. Roger Garrett, Institute of Molecular Biology, S0lvgade 83H, DK-1307 Copenhagen K, Denmark
5 System. Appl. Microbiol. Vol. 19/1
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Phylogenetic Analysis of the Archaeal Order of Sulfolobales Based on Sequences of 235 rRNA Genes and 165/235 rONA Spacers SIRO I. TREVISANATO l , NIELS LARSEN 2 , ANDREAS H. SEGERER3 , KARL O. STETTER3 , and ROGER A. GARRETT l 1
2 3
Institute of Molecular Biology, Copenhagen University, DK-1307 Copenhagen K, Denmark Department of Microbiology, Michigan State University, East Lansing, MI 48824, USA Lehrstuhl flir Mikrobiologie, Universitat Regensburg, D-93053 Regensburg, Germany Received July 21, 1995
Summary 23S rRNA genes from six members of the order of Sulfolobales, within the crenarchaeotal branch of the archaea, were cloned and sequenced. For each of the organisms Acidianus brierleyi, Acidianus infernus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus shibatae and Stygiolobus azoricus the 23S rRNA gene is linked directly to that of 16S rRNA by a short (- 200 bp) spacer with no intervening tRNA gene and no closely linked 5S rRNA gene which is typical for the Crenarchaeota. A phylogenetic tree, derived from the 23S rRNA gene sequences, shows that the six organisms are closely related, compatible with their belonging to one Order, and cluster in three pairs: the two Acidianus species, S. shibatae and S. solfataricus, and S. acidocaldarius and St. azoricus.
Key words: Phylogeny - 235 rRNA - 5ulfolobales - Acidianus brierleyi - Acidianus infernus - Sulfolobus acidocaldarius - Sulfolobus solfataricus - Sulfolobus shibatae - Stygiolobus azoricus - 165/235 intergenic spacer
Introduction Members of the order 5ulfolobales, and in particular
Sulfolobus, are amongst the best characterized of the ex-
tremely thermophilic archaea. The ease with which the latter can be grown, under aerobic conditions, renders them the organisms of choice for molecular genetic studies on the Crenarchaeota and, currently, different cloning vectors are being developed for Sulfolobus (Aagaard et al., 1994; Schleper et al., 1994). Therefore, it is important to classify these diverse organisms phylogenetically. The archaeal order of 5ulfolobales comprises the genera
Acidianus, Desulfurolobus, Metallosphaera, Stygiolobus
and Sulfolobus (Stetter et al., 1990). 5ulfolobales generally metabolize sulphur by oxidation but some can grow anaerobically and reduce sulphur (Segerer et al., 1986; Grogan, 1989). These assignments were made on the basis of morphology and growth requirements, their low genomic G+C contents (31-45%) (Segerer et al., 1986; 1991; Grogan, 1989) and the subunit composition of their DNA-dependent RNA polymerases (Zillig et al., 1980), all
of which are only approximate criteria for phylogenetic relatedness. The rRNA sequences provide a more exact evolutionary chronometer, and the 235 rRNAs have an advantage over the 165 rRNAs (which are presented in the accompanying paper - Fuchs et al., 1996) because of their larger size and larger number of variable regions. Here, we present 235 rDNA sequences, and a phylogenetic analysis, for six members of the 5ulfolobales, including three species of Sulfolobus. The 165/235 rDNA spacer regions which are devoid of tRNA genes (reviewed by Garrett et al., 1991) and can form archaeal-specific secondary structures (Kjems and Garrett, 1990) are also presented.
Materials and Methods
Growth ofcells. A brierleyi strain SP3a/1, DSM 6334 (Zillig et aI., 1980; Segerer et aI., 1986), A. infernus strain S04a, DSM 3191 (Segerer et al., 1986), S. acidocaldarius, DSM 639 (Grogan,
62
S. I. Trevisanato et al.
1989) and S. solfataricus, DSM 1616 (Zillig et aI., 1980) were grown on Brock's medium (Brock et aI., 1972) at their optimal growth temperatures and St. azoricus strain FC6, DSM 6296 was grown as described earlier (Segerer et aI., 1991). Cells were centrifuged and stored as frozen pellets. S. acidocaldarius and S. solfataricus cells were grown from single colonies. A culture of S. shibatae, DSM 5389 (Grogan et aI., 1990) was obtained from Prof. W. Zillig and grown in Brock's medium (Brock et aI., 1972). Purification of nucleic acids. DNA was extracted from suspended cells using glass beads (Leffers et aI., 1987) and purified by isopycnic ultracentrifugation (Sambrook et aI., 1989). RNA was extracted from cells, suspended in guanidinium-thiocyanate buffer and lysed by vortexing with glass beads (Chomoczynski and Sacchi, 1987). Amplification of 23S rRNA genes. Polymerase chain reaction (PCR) amplification of genomic DNA was performed in 100 IAI under a layer of mineral oil, using a Hybaid Thermocycler (Teddington, UK) (Sambrook et aI., 1989). The following oligonucleotide primers were used to amplify overlapping segments of the 235 rRNA gene; where numbers in brackets correspond to the position within S. acidocaldarius rRNA (Maidak et aI., 1994). 165 rRNA: TCGTAACAAGGTAGCCGT (1451-1468); 235 rRNA: TACCCAGGGGCCGAAGCCTCCCGG (1-24), GAAAGGGGGACAGCCCAAA (242-261), TAACACCACATCCCCACC (300-283), TTCTGACGTGCAATTCGTTC (884-903), GGGAACTAGCTATCACCG (959-942), CCTGACTGTTTAATAAAA (1921-1938), GTACACACCCTTTCGGGCT (1972-1954), AGTACGAGAGGAACAGGG (2787-2804), AGAAGAGGG-
GGTTGATGG (2943-2960), CCATCAACCCCCTCTTCT (2960-2943), CCAGGGGGGAGCCTGATT (3033-3016), 55 rRNA: GGCTTAACTTCCGGGTTC (64-43). Cloning and sequencing of PCR products. PCR products were 5'-phosphorylated and treated with Klenow enzyme to produce blunt ends (Sambrook et aI., 1989) before cloning into polylinker sites in M13mp18 and M13mp19 and transforming into competent Escherichia coli HB101 cells (Sambrook et aI., 1989). The cloned fragments were sequenced using standard protocols for the dideoxy sequencing method (Leffers et aI., 1987, Sambrook et aI., 1989). Phylogenetic analyses. 235 rRNA sequences were aligned using the ae2 editor (Maidak et aI., 1994). Evolutionary distances were estimated by the program DNADIST in PHYLIP Version 3.5c (Felsenstein, 1993) using the Kimura 2-parameter model program that weights transitions and transversions at 2:1 (Kimura, 1980). The tree was computed from the distance matrix using the Fitch-Margoliash least square fit program coupled to global optimization (Fitch and Margoliash, 1967). 165-235 spacer sequences were aligned using the PileUp program (Genetics Computer Group, Inc., 1994) and adjusted on the basis of the localization of common stem-loop structures (Kjems and Garrett, 1990).
Results and Discussion 23S rRNA gene sequences Alignment of the 235 rRNA gene sequences from the six organisms showed an identity level of 82-95% (Fig. 1)
A. A. brierleyi A.brierleyi
A. in/emus
S. acidncaJdarius
S. solfataricus
S. solfataricusMT4
100
86.6
87.5
100
87.5 S. solfataricus
88.5
89.3
100
S. solfataricusMT4
87.0
87.6
87.5
97.4
100
S.shibatae
81.9
86.2
87.0
95.1
95.2
88.8
88.6
89.1
87.3
A. brierleyi
A. in/emus
S. acidncaldarius
S. solfataricus
S. solfata- S. ricusMT4 shibatae
31
31
37.2
St. azoricus
St. awricus
100
A. in/emus 93.6
S. acidncaJdarius
S. shibatae
88.1
100 88.0
B.
35.1
40
34.6
St. azoricus
38
100
·Fig. 1. 235 rDNA sequence comparisons for the six members of 5ulfolobales. A: Nucleotide sequence identities of the 235 rDNAs expressed as a percentage. B: The G+C content of the genomic DNA for the six organisms where the data derive from the following sources: the two Acidianus species (Segerer et aI., 1986), the three Sul(olobus species (Grogan et aI., 1990) and St. azoricus (Segerer et al.,1991).
235 rRNAs of Sulfolobales
and introns that have been detected in other crenarchaeotal rRNA genes (Garrett et a1., 1994) were not detected. The sequence of the 235 rRNA gene of S. acidocaldarius was 99.9% identical to that of "S. solfataricus" in the Ribosomal Database (Maidak et a1., 1994) consistent with the use of a contaminated culture in the earlier work (see Zillig, 1993); it also corresponded to the S. acidocaldarius sequence of Durovic and Dennis (1994). Complete 235 rDNA sequences are presented for A. brierleyi (3046 bp), S. acidocaldarius (3034 bp) and S. solfataricus (3043 bp) and minor omissions, mainly at the downstream end of the gene, are present in the sequences A. infernus (2955 bp), S. shibatae (2972 bp) and St. azoricus (2909 bp). EMBL-Genbank accession numbers for the six 235 rRNA sequences are as follows: A. brierleyi - U32317, A. infernus - U32318, S. acidocaldarius U32320, S. solfataricus - U32322, S. shibatae - U32321 and St. azoricus - U32319.
1
63
16S rRNA ~
A. B. C. D. E. F.
GCGGCUGGAU GCGGCUGGAU GCGGCUGGAU GCGGCUGGAU GCGGCUGGAU GCGGCUGAAU
A. B. C. D. E. F.
GGAUGCUCAC CAGGAGAGCC GUAGGAGGUC GUAGGAAGCC AGGGCUUCAC AGGGCUUCAC
A. B. C. D. E. F.
UCUCA .. AAU ACUC .. UUCG CCUCU . UGGC CCUCU . UGGC CCUCUUGGGC UCCCUUUGGG
A. B. C. D. E. F.
GACAUUACAU . UA . AUA . AUA CAUA CA GGCU UA
A. B. C. D. E. F.
UACGACCAAC AACGACCGGA UGGCCUUAGG UGGCUUAGGG CUAGGUAGCC CCAGGCGACC
51
50
CACCUCACAU CACCUCACAU CACCUCACAG CACCUCAUAG CACCUCAUA. CACCUCACA.
..... AAAGC UCCACAACUU UUACAAACUC UUACAAACUC UAUUUA ... C UAAAUAACUC
processing stem
101
UAUAAUCUCG ACUUAAGCUC CAUAAAACCC CAUAAAACCC UAAAACUCGU UAUAAUCCUC
UCUCCUGUUU UUGGUGCUCC CCCCGUCAAU CCCCGUCAAU UCCCCCGCUA CCCCGCCAAA
~
GUUCCUCUUG UCGGCUUCCC UUGGUUCCCU UUGGUUCCCU AAUCUUCCCU UGGUUCUCCU
CUU.. . . . .. CAUUUACUUA UCUUACAG.. UUUACAG. .. UUUAUAG. .. CUGGCAG. ..
CUAAGUAGCU CUAAGAGCCU AUAAGAGCAA AUAAGAGCAA UGAAGUGCCU UGAAGUGCCU
AAGAGUGCCC A.. GGACUCG A. GGGCCUAU A. GGGCCUAU A. GGGCCCAU A. GAGCCCGU
GA. UAAUCUG GCUUAUUGUG GC AGCUC CA . . . . . . • . . . . CUCCCUA CUC. . . . . •. . UCCCUA AA. UAUGUCU CUGCCAAGUU AA .. GGUCAC CUAUCAAUAU
GGUCUC. AGU GGGUCC.AGU AGGGCU AGGGCU. . .. AGGGCUCAAU UGGGCUCAAU
helix E
~
. UGAGAGGAA .. GAGUGGAG CAGAGGAGAA CAGAGGAGAA CAGAGGGGAA CGGGGAGGAA
AGAGGAGGCA CCUAGAGGGG UUCAGUAGGG UUCCAUAGGG AUUGGGUGGG UUAGGGCGGG
~
~
100
AUGCCGUUCC AUGCGGUUCC AUGCGAUUCU AUGCGGUUCU AUGCAGUUCU AUGCAAUUCC
150 ACAAUAGGUU AGUU . GGGAG ...•. GGGAA ..... UUGGCAGAGA AUCGGUAGGU
Secondary structures 5econdary structural models derived for each of the 235 rRNAs correspond very closely to the structure presented earlier for Desulfurococcus mobilis 235 rRNA (Leffers et a1., 1987) and they revealed only minor differences from one another. The main differences fall within hypervariable sequence regions of domains I (helices 9 and 10) and domain IV (helix 98) (Leffers et a1., 1987). Multiple compensating base changes occur within helices 9 and 10 and small changes in helix length (4-5 bp for helix 9 and 7-10 bp for helix 10) whereas in helix 98, larger changes in length were observed; 11 bp for A. brierleyi and A. infernus and only 3 to 5 bp for the three Sulfolobus and S. solfataricus MT4 (Martayan et a1., 1994); the corresponding sequence for St. azoricus was not determined. Complete secondary structures are available from Gutell et a1., (1993) on request.
Sequences of the 16S123S rRNA intergenic spacer 5equences of the 165/235 rRNA spacer region were determined by binding a primer at the conserved downstream end of the 165 rRNA gene, which invariably exhibits the sequence 5'-CCUCA-3' rather than 5'-CCUCC-3' found in other archaeal165 rRNAs, and at the start of the 235 rRNA gene before amplifying the intervening spacer sequence by PCR. The results revealed a spacer of about 200 bp in each organism and the absence of tRNA genes. The sequences are aligned in Fig. 2 and were submitted to the EMBL-Genbank together with the corresponding 235 rDNA sequences. The conserved sequence motif, 5'-AUGC ... AAG-3', flanking helix E (Fig.2) is common to the crenarchaeotal 165/235 RNA spacer sequences (Kjems and Garrett, 1990; Garrett et a1., 1994) including that of S. solfataricus strain MT4 (Londei P., pers. comm.). Helix E, as for other archaea (Kjems and Garrett, 1990), is conserved in length (10 bp) and exhibits several compensating base changes for the six organisms. In contrast, helix F is less conserved and varies in length from 11-23 bp with a variable terminal loop sequence.
151
201
helix F
processing stem GCCAGACAGC GCUAAACAGC GCGAAAGGCC CCGAAAGGCC ACAUUAUAGC GCCUAAUAAC
~
~
200
GAGGCUAG. U GAGGCUAGCC CGAUGAAGC . CGAUGAAGC GAGGCUAGUA GAGGCUGGUG
236 CACCUAGGGA CACCUAGGGA GCUUAGAAUC GCUUAGAAUC CGUCUAGGAG CGCCUAGGGA
UGGC .. CCGACU GUUAA. GUUAA. UU . AA .
Fig. 2. Alignments of the 165/235 rRNA spacer region for A. brierleyi, B: A. infernus, C: S. solfataricus, D: S. shibatae, E: S. acidocaldarius and F: St. azoricus. The limits of the secondary structural elements are indicated by arrow heads and include the approximate ends of the 165 and 235 rRNA processing stems and helices E and F (Kjems and Garrett, 1990). A conserved sequence flanking helix E is underlined. The 3'-terminal nucleotide of the 165 rRNA (A-17) is conserved as an adenosine amongst the 5ulfolobales whereas other Crenarchaeota exhibit a cytidine (Maidak et aI., 1994).
Absence of linked 5S rRNA genes No PCR product was obtained using primers complementary to the 3'-end of the 235 rRNA and the centre of 55 rRNA of S. acidocaldarius. The latter primer was complementary to a conserved sequence GGCTTAACTTCCGGGTTC (positions 64 to 43) in crenarchaeotal 55 rRNA. A positive control experiment was performed with primers complementary to the 3'-end of 165 rRNA and the 3'-end of 235 rRNA which generated a 3.4 kbp fragment (data not shown). We infer, therefore, that the spacing between the 235 rRNA and 55 rRNA genes was at least several kilo base pairs (Ponce and Micol, 1992) reinforcing the view that the rRNA gene organization of the 5ulfolobales is typical of Crenarchaeota (re-
S. I. Trevisanato et al.
64
viewed in Garrett et aI., 1991), including S. shibatae for which conflicting evidence appeared (Neumann et aI., 1983; Reiter et aI., 1987). Phylogenetic relationships
The relatively high sequence identity (85-95%) of the six 235 rDNA sequences (Fig. 1), the degree of conservation of the inferred rRNA secondary structures, and the similar sequences and secondary structures of the 165/235 rRNA spacers (Fig. 2) all indicate that the six organisms are related and belong to the same phylogenetic lineage. This correlates with both the similar genomic G+C contents of the different organisms (Fig. 1) and the comparative rRNA-DNA hybridization studies for A. brierleyi, S. acidocaldarius, S. shibatae, and S. solfataricus (Zillig et aI., 1987). M. vannielii H. morrhuae S. solfatancus S. MT4
S. shibatae S. acidocaldarius SI. azoncus A. brierleyi
D. mobilis 0.10
Fig. 3. Phylogenetic tree for the Sulfolobales. The tree is derived from aligned 23S rRNA sequences for the six members of Sulfolobales and S. solfataricus strain sp. MT4 (Martayan et aI., 1994). Included in the analysis are 23S rRNA sequences from Methanococcus vannielii Uarsch and Bock, 1985), Halococcus morrhuae, and Desulfurococcus mobilis (Leffers et aI., 1987) from the order Thermoproteales. Programs used in the analyses are described under Materials and Methods.
A phylogenetic tree is presented for the six organisms and S. solfataricus MT4 (Martayan et aI., 1994) in Fig. 3. It shows clusters of three pairs of organisms, A. brierleyi and A. infernus, S. shibatae and S. solfataricus, and S. acidocoldarius and St. azoricus, and it correlates well with the phylogenetic analysis of the 165 rRNA sequences in the accompanying paper (Fuchs et aI., 1996). Acknowledgements. We are grateful to Ilia Leviev and jonathan Trent for their helpful advice. The research was supported by the Danish Natural Science Research Council, the NOVO-Nordisk Fund and the EU Generic Project, Biotechnology of Extremophiles, Contract Bio-CT93-02734.
References
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W.: Organization of the genes for rRNA in archaebacteria. Mol. Gen. Genet. 192, 66-72 (1983) Ponce, M. R., Mical, j. 1.: PCR amplification of long fragments. Nucleic Acids Res. 20, 623 (1992) Reiter, W.-D., Palm, P., Voos, W., Kaniecki, j., Grampp, B., Schulz, W., Zillig, W.: Putative promoter elements for the rRNA genes of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12. Nucleic Acids Res. 15, 5581-5595 (1987) Sambrook, W., Fritsch, E. F., Maniatis, T. H.: Molecular cloning, a laboratory manual, 2nd edition. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) Schleper, Roder, R., Singer, T., Zil/ig, W.: An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous ~-galactosidase gene. Mol. Gen. Genet. 243, 91-96 (1994) Segerer, A. H., Neuner, A., Kristjansson, j. K., Stetter, K.O.: Acidianus infernus gen. nov., spec. nov., and Acidianus brierleyi comb. nov.: facultatively aerobic, extremely acidophilic thermophilic sulfur-metabolizing archaebacteria. IntI. ]. System. Bacteriol. 36, 559-564 (1986)
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Segerer, A. H., Trincone, A., Gahrtz, M., Stetter, K.O.: Stygiolobus azoricus gen. nov., sp. nov. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. IntI. ]. System. Bacteriol. 41, 495-501 (1991) Stetter, K.O., Fiala, G., Huber, G., Huber, R. and Segerer, A.: Hyperthermophilic microorganisms. FEMS Microbiol. Revs. 75, 117-124 (1990) Zil/ig, W., Stetter, K. 0., Wunderl, S., Schulz, W., Priess, H., Scholz, T.: The Sulfolobus "Caldariella" group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch. Microbiol. 125, 259-269 (1980) Zil/ig, W., Holz, T., Klenk, H. P., Trent, J., Wunderl, S., Janekovic, D., Imsel, E. and Haas, B.: Pyrococcus woesei sp. nov., an ultra-thermophilic marine archaebacterium, representing a novel order, Thermococcales. System. Appl. MicrobioI. 9, 62-70 (1987) Zillig, W.: Confusion in the assignments of Sulfolobus sequences to Sulfolobus species. Nucleic Acids Res. 21, 5273 (1993)
Prof. Roger Garrett, Institute of Molecular Biology, S0lvgade 83H, DK-1307 Copenhagen K, Denmark
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