- Email: [email protected]
Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12
GENE AN t N T E R N A T I O N A L J O U R N A L G E N E S AND G E N O M E 5
ELSEVIER
ON
Gene 180 (1996) 183-187
Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12 Miho Aoshima a, Yoshihisa Nishibe b, Masami Hasegawa c, Akihiko Yamagishi a, Tairo Oshima a,, a Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-I, Hachioji-shi, Tokyo 192-03, Japan b Institute for Bio-Medical Research, Teijin Limited, Hino-shi, Tokyo 191, Japan c The Institute of Statistical Mathematics, Minato-ku, Tokyo 106, Japan Received 19 February 1996; revised 16 May 1996; accepted 16 May 1996
Abstract A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus. Keywords: Archaea; Phylogeny; Evolution; rRNA; Acidophile; Thermophile
1. Introduction Until recently, all life on earth was divided into two kingdoms (eukaryotes and prokaryotes) from the viewpoint of whether or not they possess nucleus. However, 16S rRNA gene sequence analyses revealed that prokaryotes can be further divided into two groups, eubacteria and archaea (Woese and Fox, 1977). Nowadays, a proposal of Woese et al. (1990) to divide organisms into three domains, i.e., archaea (Archaea), eubacteria (Bacteria), and eukaryotes (Eucarya) has been widely accepted. Archaea grow in extreme environments such as halophilic, anaerobic, and thermophilic habitats and have many distinctive features from eubacteria and eukaryotes. The phylogenetic tree for archaea based upon 16S rRNA sequence comparisons suggests that archaea can be subdivided into two branches, a cluster of sulfur-dependent extreme thermophiles (Crenarchaeota) on one hand, and a cluster of methanogens and extreme halophiles (Euryarchaeota) on the other (Woese, 1987). * Corresponding author. Tel. +81 426 767134; Fax +81 426 767145; e-mail: [email protected] Abbreviations: PCR, polymerase chain reaction; rRNA, ribosomal RNA. 0378-1119/96/$15.00© 1996 ElsevierScienceB.V. All rights reserved PH S0378-1119(96)00451-9
In this study, we report a hyperthermophile NC12 newly isolated from Noboribetsu hot spring. Furthermore, we report a complete sequence of 16S rRNA gene and a phylogenetic position of this organism.
2. Experimental and discussion 2.1. Isolation and characterization of NC12 In a glass vial, 10 ml of autoclaved base medium (0.01% NaC1, 0.01% CaC12, 0.005% F e S O 4 7 H 2 0 , 0.01% Bacto yeast extract, 0.01% casamino acid, and 0.08% peptone, adjusted to pH 2.7 with sulfuric acid), 10 ~tl of vitamin solution (0.002% biotin, 0.002% folic acid, 0.01% pyridoxine hydrochloride, 0.005% thiamine hydrochloride, 0.005% riboflavin, 0.005% nicotinic acid, 0.005% calcium pantothenate, 0.0001% cyanocobalamin, 0.005% p-aminobenzoic acid, and 0.005% lipoic acid), 10 ktl of trace mineral solution (0.5% MnSO42H20, 0.1% CoSO4, 0.1% ZnSO4, 0.01% C u S O 4 5 H 2 0 , 0.01% H3BO3, and 0.01% NazMoO42H20), 10~tl of 0.1% resazurin solution, and 0.02 g of sulfur were added. The vial was sealed by a buthylgum cap and the air in the vial was removed by a vacuum pump. After generation of air bubbles had stopped, 100 ~tl of 5% NazS9H20
M. Aoshima et al./Gene 180 (1996) 183-187
184
4
3
2
1
M
m
m
m
(kb)
(kb) 23 9.4 6.6 4.4
23 4.4
0.56
Fig. 1. Electrophoresis pattern (0.8% agarose gel) of the DNA obtained by PCR. Final concentrations of MgC12 added in the reaction mixture were 0raM (lane 1), 0.5 mM (lane 2), 1.0mM (lane 3), 1.5 mM (lane 4), respectively. Lambda DNA digested with HindIII was used as a size marker (lane M). Methods: NC12 was cultured anaerobically at 96°C to about 108 per ml. Cultured NC12 (500 ml) was harvested, suspended in 10 mM Tris-HC1 (pH 8.0) containing 10 mM EDTA. SDS (final 0.5%), Proteinase K, and RNase A were added to the suspension and incubated at 60°C for 2 h. After extraction with saturated phenol and phenol-chloroform, genomic DNA was concentrated by ethanol precipitation. From the available sequences in a database (EMBL), specific and conserved sequences for the archaebacterial 16S rRNA were searched and picked out as candidates for the regions to design the PCR primer. From the selected regions, one set of primers were designed as follows: 5'-primer, 5'-TAAAGGAATTGGCGGGGGAGCACC-3'; T-primer, 5'-GAGGTGATCCAGCCGCAGGTTC-3'. Positions of the primers correspond to nucleotide numbers 876-899 and 1494-1473 in 16S rRNA sequences from Desulfurococcus mobilis (Kjems et al., 1987), respectively. PCR was performed in a solution containing 400 ng of NC12 genomic DNA as a template, 20 pmol each of the primers, 0.2 mM each of dNTPs, 2 units of Taq polymerase (TOYOBO), and a reaction buffer appendant to the enzyme to be, finally, a 1 x concentration. MgC12 was added to the solution to be, finally, 0, 0.5, 1.0, and L5 mM, respectively. The solutions were subjected to 40 cycles of PCR (denaturation, 30 s at 94°C; annealing, 30 s at 58°C; and extension, 1 min at 72°C).
solution was added in the medium and further vacuuming was continued until color (derived from resazurin) of the medium had disappeared. Then, a gas mixture (2 atm) composed of hydrogen and carbon dioxide (8:2) was introduced into the vial. In this Vial, water from Noboribetsu hot spring (Hokkaido, Japan) (1 ml) was added by using a syringe and incubated at 90°C. A strain isolated from the culture by limiting dilution method was named NC12. Electron microscopic analysis showed that NC12 is a coccus with a flagellum and that its diameter ranges from 1 to 3 ~tm. Cell growth was observed between 70 and 96°C, and maximum growth rate was obtained at about 92°C. NC12 could grow under acidic conditions (pH range from 1.5 to 4.0), and the optimum pH for cell growth was about 3.0. NC12 did not grow auto-
2.3 2.0
0.56
Fig. 2. Autoradiogram of the Southern hybridization analysis using PCR product (#PCRsmall) as a probe. Methods: Genomic DNA of NC12 was digested with four restriction enzymes (HindIII, KpnI, PstI, and Sinai), respectively. The digested DNA fragments were separated on a 0.8% agarose gel, blotted onto a nylon membrane (Hybond-N, Amersham), and fixed by UV irradiation. PCR product (#PCRsmall) was isolated from an agarose gel and 32p-labelled using an oligolabelling kit (Pharmacia). Hybridization was carried out at room temperature. The membrane was then washed with 0.1 x SSC, 0.1% SDS at 60°C and exposed on an X-ray film.
trophically. Although sulfur was not essential, the growth rate became extremely low when it was excluded from the medium.
Table 1 Similarities of 16S rRNA between that from NC12 Organisma
Identityb (%)
Pyrodictium occultum Desulfurococcus mobilis Thermofilum pendens Thermoproteus tenax Sulfolobus shibatae Sulfolobus acidoealdarius Thermococcus celer Methanothermus fervidus
91.2 90.0 86.8 85.7 85.6 85.0 81.4 79.6
aSequences of 16S rRNA were obtained from EMBL database. Percentages were calculated using GENETYX (SDC) software.
M. Aoshima et al./Gene 180 (1996) 183-187
185
(a) Hin dill
Hin dill Sma I
T'I
Sma
I
Sma
T'I
I 5'primer
---,,-,- : 200 bp
I
3'primer PCR product
I
16SrRNAgene
>
(b) ACTCCGGTTG CGCGCCCAGC GACGGGGATA TCCCTCGCCG CGGCCCATCA TGGGAGCGGG GGCGCGAAAC GGAGCTTTTC CGCCGCGGTA AGCCGGCCCC CTGCGGGGCT A.ATCCCTGGG GCGAAAGCCG GGGCTAGGTG CCGCCTGGGG GGGGTGGAGC GATGACGGCC AGCTCGTGCT GCGACCCAGC AGGAGGGGGC TGGCGGGGAC GTCGGGATTG AACATCGCGC AGAAGGAGGG GAGAAGTCGT
ATCCTGCCGG ACCCGACTGC TATCGGGGTG AGGCTAAGCC ATGGGAGTCG CGCCGCTGGG CGGCGCACGG CTGAGTAACA CGTAGCTAAC CTACCCTCGG ACCCCGGGAA ACTGGGGCTA ATCCCCGATA GGCGAGGGGG CCTGGAACGG AAAGGGACCC TGGGGGGTTA TCGCCTGGGG TCCGCCCGAG GATGGGGCTG TGGTTGTTGG CGGGGTAATG GCCCGCCAAG CCGACGACGG GTAGGGGCCG AGCCCCAGAT GGGCCCTGAG ACAAGGGCCC AGGCCCTACG GGGCGCACCA CTCCGCAATG CGGGAAACCG TGACGGGGTC ACCCCGAGTG CTCCCGTAAG CCCGCTGCAA GGAGGCGGGG GAATAAGCGG GGGGCAAGTC TGGTGTCAGC ATACCAGCCC CGCGAGTGGT CGGGACGTTT ACTGGGCCTA AAGCGCCCGT GTAAGTCCCT CCTGAAAGCC CTGGGCTCAA CCCAGGGAGT GGGGGGGATA AGGGGGCGGG AGAGGCCGGG GGTACCCCAG GGGTAGGGGC GAAATCCGAT GGACCACCAG TGGCGAAAGC GCCCGGCTGG AACGCGCCCG ACGGTGAGGG GGGGAGCGAA CCGGATTAGA TACCCGGGTA GTCCCGGCTG TAAACGATGC TCGGGCGGGC GTTAGAGCCC GCCGGTGCCG CAGGGAAGCC GTTAAGCCCG AGTACGGCCG CAAGGCTGAA ACTTTAAGGA ATTGGCGGGG GGGCACACAA CTGCGGCTCA ATTGGAGTCA ACGCCGGGAA CCTCACCGGG GGCGACACAG AGGCTAACGA CCTTGCCCGA CGCGCTGAGG GGAGGTGCAT GGCCGTCGCC GTGAAGTGTC CTGTTAAGTC AGGCAACGAG CGAGACCCCC GCCCCTAGTT GGGCGACCGC TGGGGCACAC TAGGGGGACT GCCGCCGCTA AGGCGGAGGA CACGGCAGGT CAGCATGCCC CTAAACCCCC GGGCTGCACG CGGGCTACAA AGCGGGATCC GACCCCGAAA GGGGGAGGCA ATCCCTCAAA CCCCGCCGTA GGGGCTGCAA CTCGCCCCCA TGAACCTGGA ATCCCTAGTA ACCGCGCGTC GGTGAATACG TCCCTGCCCC TTGTACACAC TGCCCGTCGC TCCACCTGAG GTGAGGCTTC CTCCTTCGGG AGGGAGTCGA ACCCCTCCTT CTCGAGGGGG AACAAGGTAG CCGTAGGGGA ACCTGCGGCT GGATCACCTC CC
Fig. 3. (a) Restriction map of the HindlII fragment which contains the whole coding region of 16S rRNA gene. Positions of 16S rRNA gene, PCR product used as a probe, and primers used for the PCR are also indicated. (b) Complete nucleotide sequence of the 16S rRNA gene. The nucleotide sequence data will be appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number D85038. Methods: The genomic DNA of NC12 was digested with HindIII and the resultant fragments were separated on a 0.8% agarose gel. The fragments, ranging from 1 to 3 kb, were collected and ligated to HindflI-digested pBluescript II SK(+) (Stratagene). Competent cells of Escherichia coli HB101 were transformed with the ligated DNA, and more than I0 000 independent recombinant colonies were used as a library for the screening. Using PCR product (#PCRsmall) as a probe, a HindlII (1-3 kb) library was screened under the same condition as the Southern hybridization. The physical map of the inserted DNA of the positive clone was analyzed by treating the DNA with several restriction enzymes, and the whole coding region of the gene was sequenced from the both strands by a dideoxy chain termination method using delta-Tth sequencing kit (Toyobo).
2.2. Genomic D N A f r o m N C I 2 T h e y i e l d o f g e n o m i c D N A p r e p a r e d f r o m 500 m l c u l t u r e o f N C 1 2 w a s a b o u t 2 0 0 ~tg. T h e D N A w a s n o t
well d i g e s t e d b y E c o R I a n d B a m H I ( d a t a n o t s h o w n ) . T h e r e a s o n f o r t h e r e s i s t a n c e is u n c l e a r , b u t s i n c e it is u n l i k e l y t h a t t h e r e a r e few E c o R I a n d B a m H I sites i n the genomic DNA, the existence of some kinds of
M. Aoshima et aL/Gene 180 (1996) 183-187
186
NC12
Pyrodictiurn occultum Desulfurococcus mobilis
1001
1O0
761
Sulfolobus shibatae Sulfolobu$ aciclocaldarius
Thermofilum pendens Thermoproteus tenax
Thermococcus celer Methanothermu$ fervidus
0.1 substitutions/site Fig. 4. The 16S rRNA tree inferred by using NucML ver. 2.3 of program package MOLPHY (Adachi and Hasegawa, 1996). Thermococcus celer and Methanothermus fervidus were assumed to be the outgroup to the other species. Positions with gaps and regions where the alignment was ambiguous were excluded from the analysis, and 1351 sites were used for the phylogenetic analysis. The HKY model for the nucleotide substitution (Hasegawa et al., 1985) was used and the local rearrangement topology search option was applied. The transition/transversion ratio was estimated to be 2.57 so as to maximize the likelihood. The horizontal length of each branch is proportional to the estimated number of substitutions. Numerals attached to internal branches refer to local bootstrap probabilities estimated by the RELL method (Kishino et al., 1990; Hasegawa and Kishino, 1994) with 1000 replications.
restriction systems which protect the DNA from EcoRI and BamHI digestion is suggested. For the Southern analyses in this study, HindIII, KpnI, PstI, and Sinai were selected and used since well-digested electrophoresis patterns were obtained by treating the DNA with these enzymes.
2.3. PCR for cloning 16S rRNA gene As a result of PCR using the primers specific to archaeal 16S rRNA gene, two very closely migrating but distinguishable bands (about 600-800 bp) were obtained when 1.0 mM MgC12 was added in the reaction solution (Fig. 1, lane 3). No obvious band was obtained when the concentration of MgC12 was lower (lanes 1 and 2), and the yield of the two bands decreased when the concentration of MgC12 was higher (lane 4). Sequence analysis showed that the larger fragment (#PCRlarge) was amplified from the both directions with the same primer (5'-primer), and has no homology, not only with 16S rRNA genes but also with any other genes reported to date (data not shown). On the other hand, the smaller fragment (#PCRsmall) showed a characteristic 16S rRNA sequence and thus was identified as a fragment containing 16S rRNA gene of NC12. Since #PCRsmall contains less than one-half of the whole coding region of the gene, a further cloning procedure was needed for the full length cloning of the gene. To estimate the fragment size to be cloned, Southern analysis was performed using #PCRsmall as a probe (Fig. 2). As a result of the analysis, a 2.5-kb HindlII band and a 5-kb PstI band (arrows in the figure) seemed to be good candidates,
and a 9-kb KpnI band seemed to be too large for the cloning target. Sinai is not a suitable enzyme to be used since several SmaI sites exist in #PCRsmall.
2.4. 16S rRNA genefrom NC12 genomic library From the HindlII (1-3 kb) NC12 genomic library, a 2.5-kb HindlII fragment which contains the entire 16S rRNA gene was obtained as a fragment which hybridized to the probe (#PCRsmall). Fig. 3(a) shows a physical map of the cloned fragment and the position of the 16S rRNA gene in the fragment. Positions of the PCR product used as a probe (#PCRsmall) and the primers for the PCR are also indicated in the figure. Fig. 3(b) shows a complete nucleotide sequence of the gene determined from the both strands. The gene was comprised of 1492 bp, which both 5'- and 3'-termini were deduced from the reported sequence (Kjems et al., 1987). The sequence obtained had three mismatches with the 5'-primer sequence while it completely matched to the 3'-primer sequence used for the PCR. Especially, there was a mismatch at the 3'-end of the 5'-primer, which seemed to be due to the requirement of the delicate condition and the byproduct in the PCR as mentioned above. Although the desired fragment was obtained with these primers by good fortune in this work, it is necessary to improve the primers for future use.
2.5. Sequence homology of l6S rRNA sequence The 16S rRNA sequence obtained was subjected to homology search using a software GENETYX (SDC)
M. Aoshima et al./Gene 180 (1996) 183-187
with all of the available sequences in the database (EMBL), and sources of 16S rRNA sequences which show high sequence similarities (Leinfelder et al., 1985; Kjems et al., 1987; Achenbach-Richter et al., 1988; Kaine et al., 1989; Grogan et al., 1990; Haas et al., 1990; Kjems et al., 1990; Kurosawa and Itoh, 1993) are listed in order in Table 1. As shown in the table, the sequence showed high homology with archaeal 16S rRNA sequences, indicating that NC12 belongs to the archaeal domain in the phylogenetic tree. The highest identity score was found between the Pyrodictium occultum sequence (91.2%) and the Desulfurococcus mobilis sequence (90.0%), suggesting that NC12 belongs to a group of sulfur-dependent extreme thermophiles (Crenarchaeota) in the archaeal domain. Though NC12 is most closely related to P. occultum from the viewpoint of 16S rRNA similarity, the identity score was only 91.2%. Thus, it is shown that N C12 is a novel organism which has not been reported so far. Furthermore, there is a possibility that NC12 is not only a new species but may constitute a new genus. To estimate evolutional distances between other related organisms listed in the table, maximum likelihood analysis was carried out. Fig. 4 shows the best tree of the 16S rRNA sequences obtained from the analysis. Since the bootstrap probabilities of the common ancestral lineages of D. mobilis and Sulfolobus and of these species and P. occultum are not high (60 and 54%, respectively), the branching order among NC12, P. occulturn, D. mobilis and Sulfolobus cannot be determined with confidence by this analysis. Nevertheless, it seems highly likely that these species form a monophyletic clade excluding T. pendens and T. tenax as outgroup, since the local bootstrap probability of this clade is as high as 97%. Here, we propose to designate the newly isolated hyperthermophilic archaebacterium NC12 as Caldococcus noboribetus. For detailed discussion about the evolutional meaning of the organism, further characterization is needed.
3. Conclusions (1) A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring and was characterized. (2) A gene coding for 16S rRNA from NC12 was cloned and sequenced. (3) Genomic DNA prepared from NC12 was found to be resistant to EcoRI or BamHI treatment, suggesting the existence of some kinds of restriction systems in NC12.
187
(4) Sequence comparisons of 16S rRNA showed that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain of the phylogenetic tree. (5) Sequence comparisons of 16S rRNA showed that NC12 is a novel hyperthermophile which may constitute a new genus.
References Achenbach-Richter, L., Gupta, R., Zillig, W. and Woese, C.R. (1988) Rooting the archaebacterial tree: The pivotal role of Thermococcus celer in archaebacterial evolution. Syst. Appl. Microbiol. 10, 231 240. Adachi, J. and Hasegawa, M. (1996) MOLPHY: Programs for Molecular Phylogeneties ver. 2.3, Institute of Statistical Mathematics, Tokyo. Grogan, D., Palm, P. and Zillig, W. (1990) 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. Haas, E.S., Brown, J.W., Daniels, C.J. and Reeve, J.N. (1990) Genes encoding the 7S RNA and a Ser-tRNA are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermusfervidus. Gene 90, 51-59. Hasegawa, M. and Kishino, H. (1994) Accuracies of the simple methods for estimating the bootstrap probability of a maximum likelihood tree. Mol. Biol. Evo[. 11, 142-145. Hasegawa, M., Kishino, H. and Yano, T. (1985) Dating of the humanape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160-174. Kaine, B.P., Schurke, C. and Stetter, K.O. (1989) Genes for the 16S and 5S ribosomal RNAs and the 7S RNA of Pyrodictium occultum. Syst. Appl. Microbiol. 12, 8-14. Kishino H., Miyata, T and Hasegawa, M. (1990) Maximum likelihood inference of protein phylogeny, and the origin of chloroplasts. J. Mol. Evol. 30, 151 160. Kjems, J., Garrett, R.A. and Ansorge, W. (1987) The sequence of the 16S RNA gene and its flanking region from the archaebacterium Desulfurococcus mobilis. Syst. Appl. Microbiol. 9, 22 28. Kjems, J., Leffers, H., Olesen, T., Ingelore, H. and Garrett, R.A. (1990) Sequence, organization and transcription of the ribosomal RNA operon and the downstream tRNA and protein genes in the archaebacterium Thermofilum pendens. Syst. Appl. Microbiol. 13, 117-127. Kurosawa, N. and Itoh, Y.H. (1993) Nucleotide sequence of the 16S rRNA gene from thermoacidophilic archaea Sulfolobus acidocaldarius ATCC33909. Nucleic Acids Res. 21, 357. Leinfelder, W., Jarsch, M. and Back, A. (1985) The phylogenetic position of the sulfur-dependent archaebacterium Thermoproteus tenax. Syst. Appl. Microbiol. 6, 164-170. Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51,221-271. Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Natl. Acad. Sci. USA 74, 5088 5090. Woese, C.R., Kandler, O. and Wheelis, M.L. (1990) Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576 4579.
ELSEVIER
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Gene 180 (1996) 183-187
Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12 Miho Aoshima a, Yoshihisa Nishibe b, Masami Hasegawa c, Akihiko Yamagishi a, Tairo Oshima a,, a Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-I, Hachioji-shi, Tokyo 192-03, Japan b Institute for Bio-Medical Research, Teijin Limited, Hino-shi, Tokyo 191, Japan c The Institute of Statistical Mathematics, Minato-ku, Tokyo 106, Japan Received 19 February 1996; revised 16 May 1996; accepted 16 May 1996
Abstract A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus. Keywords: Archaea; Phylogeny; Evolution; rRNA; Acidophile; Thermophile
1. Introduction Until recently, all life on earth was divided into two kingdoms (eukaryotes and prokaryotes) from the viewpoint of whether or not they possess nucleus. However, 16S rRNA gene sequence analyses revealed that prokaryotes can be further divided into two groups, eubacteria and archaea (Woese and Fox, 1977). Nowadays, a proposal of Woese et al. (1990) to divide organisms into three domains, i.e., archaea (Archaea), eubacteria (Bacteria), and eukaryotes (Eucarya) has been widely accepted. Archaea grow in extreme environments such as halophilic, anaerobic, and thermophilic habitats and have many distinctive features from eubacteria and eukaryotes. The phylogenetic tree for archaea based upon 16S rRNA sequence comparisons suggests that archaea can be subdivided into two branches, a cluster of sulfur-dependent extreme thermophiles (Crenarchaeota) on one hand, and a cluster of methanogens and extreme halophiles (Euryarchaeota) on the other (Woese, 1987). * Corresponding author. Tel. +81 426 767134; Fax +81 426 767145; e-mail: [email protected] Abbreviations: PCR, polymerase chain reaction; rRNA, ribosomal RNA. 0378-1119/96/$15.00© 1996 ElsevierScienceB.V. All rights reserved PH S0378-1119(96)00451-9
In this study, we report a hyperthermophile NC12 newly isolated from Noboribetsu hot spring. Furthermore, we report a complete sequence of 16S rRNA gene and a phylogenetic position of this organism.
2. Experimental and discussion 2.1. Isolation and characterization of NC12 In a glass vial, 10 ml of autoclaved base medium (0.01% NaC1, 0.01% CaC12, 0.005% F e S O 4 7 H 2 0 , 0.01% Bacto yeast extract, 0.01% casamino acid, and 0.08% peptone, adjusted to pH 2.7 with sulfuric acid), 10 ~tl of vitamin solution (0.002% biotin, 0.002% folic acid, 0.01% pyridoxine hydrochloride, 0.005% thiamine hydrochloride, 0.005% riboflavin, 0.005% nicotinic acid, 0.005% calcium pantothenate, 0.0001% cyanocobalamin, 0.005% p-aminobenzoic acid, and 0.005% lipoic acid), 10 ktl of trace mineral solution (0.5% MnSO42H20, 0.1% CoSO4, 0.1% ZnSO4, 0.01% C u S O 4 5 H 2 0 , 0.01% H3BO3, and 0.01% NazMoO42H20), 10~tl of 0.1% resazurin solution, and 0.02 g of sulfur were added. The vial was sealed by a buthylgum cap and the air in the vial was removed by a vacuum pump. After generation of air bubbles had stopped, 100 ~tl of 5% NazS9H20
M. Aoshima et al./Gene 180 (1996) 183-187
184
4
3
2
1
M
m
m
m
(kb)
(kb) 23 9.4 6.6 4.4
23 4.4
0.56
Fig. 1. Electrophoresis pattern (0.8% agarose gel) of the DNA obtained by PCR. Final concentrations of MgC12 added in the reaction mixture were 0raM (lane 1), 0.5 mM (lane 2), 1.0mM (lane 3), 1.5 mM (lane 4), respectively. Lambda DNA digested with HindIII was used as a size marker (lane M). Methods: NC12 was cultured anaerobically at 96°C to about 108 per ml. Cultured NC12 (500 ml) was harvested, suspended in 10 mM Tris-HC1 (pH 8.0) containing 10 mM EDTA. SDS (final 0.5%), Proteinase K, and RNase A were added to the suspension and incubated at 60°C for 2 h. After extraction with saturated phenol and phenol-chloroform, genomic DNA was concentrated by ethanol precipitation. From the available sequences in a database (EMBL), specific and conserved sequences for the archaebacterial 16S rRNA were searched and picked out as candidates for the regions to design the PCR primer. From the selected regions, one set of primers were designed as follows: 5'-primer, 5'-TAAAGGAATTGGCGGGGGAGCACC-3'; T-primer, 5'-GAGGTGATCCAGCCGCAGGTTC-3'. Positions of the primers correspond to nucleotide numbers 876-899 and 1494-1473 in 16S rRNA sequences from Desulfurococcus mobilis (Kjems et al., 1987), respectively. PCR was performed in a solution containing 400 ng of NC12 genomic DNA as a template, 20 pmol each of the primers, 0.2 mM each of dNTPs, 2 units of Taq polymerase (TOYOBO), and a reaction buffer appendant to the enzyme to be, finally, a 1 x concentration. MgC12 was added to the solution to be, finally, 0, 0.5, 1.0, and L5 mM, respectively. The solutions were subjected to 40 cycles of PCR (denaturation, 30 s at 94°C; annealing, 30 s at 58°C; and extension, 1 min at 72°C).
solution was added in the medium and further vacuuming was continued until color (derived from resazurin) of the medium had disappeared. Then, a gas mixture (2 atm) composed of hydrogen and carbon dioxide (8:2) was introduced into the vial. In this Vial, water from Noboribetsu hot spring (Hokkaido, Japan) (1 ml) was added by using a syringe and incubated at 90°C. A strain isolated from the culture by limiting dilution method was named NC12. Electron microscopic analysis showed that NC12 is a coccus with a flagellum and that its diameter ranges from 1 to 3 ~tm. Cell growth was observed between 70 and 96°C, and maximum growth rate was obtained at about 92°C. NC12 could grow under acidic conditions (pH range from 1.5 to 4.0), and the optimum pH for cell growth was about 3.0. NC12 did not grow auto-
2.3 2.0
0.56
Fig. 2. Autoradiogram of the Southern hybridization analysis using PCR product (#PCRsmall) as a probe. Methods: Genomic DNA of NC12 was digested with four restriction enzymes (HindIII, KpnI, PstI, and Sinai), respectively. The digested DNA fragments were separated on a 0.8% agarose gel, blotted onto a nylon membrane (Hybond-N, Amersham), and fixed by UV irradiation. PCR product (#PCRsmall) was isolated from an agarose gel and 32p-labelled using an oligolabelling kit (Pharmacia). Hybridization was carried out at room temperature. The membrane was then washed with 0.1 x SSC, 0.1% SDS at 60°C and exposed on an X-ray film.
trophically. Although sulfur was not essential, the growth rate became extremely low when it was excluded from the medium.
Table 1 Similarities of 16S rRNA between that from NC12 Organisma
Identityb (%)
Pyrodictium occultum Desulfurococcus mobilis Thermofilum pendens Thermoproteus tenax Sulfolobus shibatae Sulfolobus acidoealdarius Thermococcus celer Methanothermus fervidus
91.2 90.0 86.8 85.7 85.6 85.0 81.4 79.6
aSequences of 16S rRNA were obtained from EMBL database. Percentages were calculated using GENETYX (SDC) software.
M. Aoshima et al./Gene 180 (1996) 183-187
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(a) Hin dill
Hin dill Sma I
T'I
Sma
I
Sma
T'I
I 5'primer
---,,-,- : 200 bp
I
3'primer PCR product
I
16SrRNAgene
>
(b) ACTCCGGTTG CGCGCCCAGC GACGGGGATA TCCCTCGCCG CGGCCCATCA TGGGAGCGGG GGCGCGAAAC GGAGCTTTTC CGCCGCGGTA AGCCGGCCCC CTGCGGGGCT A.ATCCCTGGG GCGAAAGCCG GGGCTAGGTG CCGCCTGGGG GGGGTGGAGC GATGACGGCC AGCTCGTGCT GCGACCCAGC AGGAGGGGGC TGGCGGGGAC GTCGGGATTG AACATCGCGC AGAAGGAGGG GAGAAGTCGT
ATCCTGCCGG ACCCGACTGC TATCGGGGTG AGGCTAAGCC ATGGGAGTCG CGCCGCTGGG CGGCGCACGG CTGAGTAACA CGTAGCTAAC CTACCCTCGG ACCCCGGGAA ACTGGGGCTA ATCCCCGATA GGCGAGGGGG CCTGGAACGG AAAGGGACCC TGGGGGGTTA TCGCCTGGGG TCCGCCCGAG GATGGGGCTG TGGTTGTTGG CGGGGTAATG GCCCGCCAAG CCGACGACGG GTAGGGGCCG AGCCCCAGAT GGGCCCTGAG ACAAGGGCCC AGGCCCTACG GGGCGCACCA CTCCGCAATG CGGGAAACCG TGACGGGGTC ACCCCGAGTG CTCCCGTAAG CCCGCTGCAA GGAGGCGGGG GAATAAGCGG GGGGCAAGTC TGGTGTCAGC ATACCAGCCC CGCGAGTGGT CGGGACGTTT ACTGGGCCTA AAGCGCCCGT GTAAGTCCCT CCTGAAAGCC CTGGGCTCAA CCCAGGGAGT GGGGGGGATA AGGGGGCGGG AGAGGCCGGG GGTACCCCAG GGGTAGGGGC GAAATCCGAT GGACCACCAG TGGCGAAAGC GCCCGGCTGG AACGCGCCCG ACGGTGAGGG GGGGAGCGAA CCGGATTAGA TACCCGGGTA GTCCCGGCTG TAAACGATGC TCGGGCGGGC GTTAGAGCCC GCCGGTGCCG CAGGGAAGCC GTTAAGCCCG AGTACGGCCG CAAGGCTGAA ACTTTAAGGA ATTGGCGGGG GGGCACACAA CTGCGGCTCA ATTGGAGTCA ACGCCGGGAA CCTCACCGGG GGCGACACAG AGGCTAACGA CCTTGCCCGA CGCGCTGAGG GGAGGTGCAT GGCCGTCGCC GTGAAGTGTC CTGTTAAGTC AGGCAACGAG CGAGACCCCC GCCCCTAGTT GGGCGACCGC TGGGGCACAC TAGGGGGACT GCCGCCGCTA AGGCGGAGGA CACGGCAGGT CAGCATGCCC CTAAACCCCC GGGCTGCACG CGGGCTACAA AGCGGGATCC GACCCCGAAA GGGGGAGGCA ATCCCTCAAA CCCCGCCGTA GGGGCTGCAA CTCGCCCCCA TGAACCTGGA ATCCCTAGTA ACCGCGCGTC GGTGAATACG TCCCTGCCCC TTGTACACAC TGCCCGTCGC TCCACCTGAG GTGAGGCTTC CTCCTTCGGG AGGGAGTCGA ACCCCTCCTT CTCGAGGGGG AACAAGGTAG CCGTAGGGGA ACCTGCGGCT GGATCACCTC CC
Fig. 3. (a) Restriction map of the HindlII fragment which contains the whole coding region of 16S rRNA gene. Positions of 16S rRNA gene, PCR product used as a probe, and primers used for the PCR are also indicated. (b) Complete nucleotide sequence of the 16S rRNA gene. The nucleotide sequence data will be appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number D85038. Methods: The genomic DNA of NC12 was digested with HindIII and the resultant fragments were separated on a 0.8% agarose gel. The fragments, ranging from 1 to 3 kb, were collected and ligated to HindflI-digested pBluescript II SK(+) (Stratagene). Competent cells of Escherichia coli HB101 were transformed with the ligated DNA, and more than I0 000 independent recombinant colonies were used as a library for the screening. Using PCR product (#PCRsmall) as a probe, a HindlII (1-3 kb) library was screened under the same condition as the Southern hybridization. The physical map of the inserted DNA of the positive clone was analyzed by treating the DNA with several restriction enzymes, and the whole coding region of the gene was sequenced from the both strands by a dideoxy chain termination method using delta-Tth sequencing kit (Toyobo).
2.2. Genomic D N A f r o m N C I 2 T h e y i e l d o f g e n o m i c D N A p r e p a r e d f r o m 500 m l c u l t u r e o f N C 1 2 w a s a b o u t 2 0 0 ~tg. T h e D N A w a s n o t
well d i g e s t e d b y E c o R I a n d B a m H I ( d a t a n o t s h o w n ) . T h e r e a s o n f o r t h e r e s i s t a n c e is u n c l e a r , b u t s i n c e it is u n l i k e l y t h a t t h e r e a r e few E c o R I a n d B a m H I sites i n the genomic DNA, the existence of some kinds of
M. Aoshima et aL/Gene 180 (1996) 183-187
186
NC12
Pyrodictiurn occultum Desulfurococcus mobilis
1001
1O0
761
Sulfolobus shibatae Sulfolobu$ aciclocaldarius
Thermofilum pendens Thermoproteus tenax
Thermococcus celer Methanothermu$ fervidus
0.1 substitutions/site Fig. 4. The 16S rRNA tree inferred by using NucML ver. 2.3 of program package MOLPHY (Adachi and Hasegawa, 1996). Thermococcus celer and Methanothermus fervidus were assumed to be the outgroup to the other species. Positions with gaps and regions where the alignment was ambiguous were excluded from the analysis, and 1351 sites were used for the phylogenetic analysis. The HKY model for the nucleotide substitution (Hasegawa et al., 1985) was used and the local rearrangement topology search option was applied. The transition/transversion ratio was estimated to be 2.57 so as to maximize the likelihood. The horizontal length of each branch is proportional to the estimated number of substitutions. Numerals attached to internal branches refer to local bootstrap probabilities estimated by the RELL method (Kishino et al., 1990; Hasegawa and Kishino, 1994) with 1000 replications.
restriction systems which protect the DNA from EcoRI and BamHI digestion is suggested. For the Southern analyses in this study, HindIII, KpnI, PstI, and Sinai were selected and used since well-digested electrophoresis patterns were obtained by treating the DNA with these enzymes.
2.3. PCR for cloning 16S rRNA gene As a result of PCR using the primers specific to archaeal 16S rRNA gene, two very closely migrating but distinguishable bands (about 600-800 bp) were obtained when 1.0 mM MgC12 was added in the reaction solution (Fig. 1, lane 3). No obvious band was obtained when the concentration of MgC12 was lower (lanes 1 and 2), and the yield of the two bands decreased when the concentration of MgC12 was higher (lane 4). Sequence analysis showed that the larger fragment (#PCRlarge) was amplified from the both directions with the same primer (5'-primer), and has no homology, not only with 16S rRNA genes but also with any other genes reported to date (data not shown). On the other hand, the smaller fragment (#PCRsmall) showed a characteristic 16S rRNA sequence and thus was identified as a fragment containing 16S rRNA gene of NC12. Since #PCRsmall contains less than one-half of the whole coding region of the gene, a further cloning procedure was needed for the full length cloning of the gene. To estimate the fragment size to be cloned, Southern analysis was performed using #PCRsmall as a probe (Fig. 2). As a result of the analysis, a 2.5-kb HindlII band and a 5-kb PstI band (arrows in the figure) seemed to be good candidates,
and a 9-kb KpnI band seemed to be too large for the cloning target. Sinai is not a suitable enzyme to be used since several SmaI sites exist in #PCRsmall.
2.4. 16S rRNA genefrom NC12 genomic library From the HindlII (1-3 kb) NC12 genomic library, a 2.5-kb HindlII fragment which contains the entire 16S rRNA gene was obtained as a fragment which hybridized to the probe (#PCRsmall). Fig. 3(a) shows a physical map of the cloned fragment and the position of the 16S rRNA gene in the fragment. Positions of the PCR product used as a probe (#PCRsmall) and the primers for the PCR are also indicated in the figure. Fig. 3(b) shows a complete nucleotide sequence of the gene determined from the both strands. The gene was comprised of 1492 bp, which both 5'- and 3'-termini were deduced from the reported sequence (Kjems et al., 1987). The sequence obtained had three mismatches with the 5'-primer sequence while it completely matched to the 3'-primer sequence used for the PCR. Especially, there was a mismatch at the 3'-end of the 5'-primer, which seemed to be due to the requirement of the delicate condition and the byproduct in the PCR as mentioned above. Although the desired fragment was obtained with these primers by good fortune in this work, it is necessary to improve the primers for future use.
2.5. Sequence homology of l6S rRNA sequence The 16S rRNA sequence obtained was subjected to homology search using a software GENETYX (SDC)
M. Aoshima et al./Gene 180 (1996) 183-187
with all of the available sequences in the database (EMBL), and sources of 16S rRNA sequences which show high sequence similarities (Leinfelder et al., 1985; Kjems et al., 1987; Achenbach-Richter et al., 1988; Kaine et al., 1989; Grogan et al., 1990; Haas et al., 1990; Kjems et al., 1990; Kurosawa and Itoh, 1993) are listed in order in Table 1. As shown in the table, the sequence showed high homology with archaeal 16S rRNA sequences, indicating that NC12 belongs to the archaeal domain in the phylogenetic tree. The highest identity score was found between the Pyrodictium occultum sequence (91.2%) and the Desulfurococcus mobilis sequence (90.0%), suggesting that NC12 belongs to a group of sulfur-dependent extreme thermophiles (Crenarchaeota) in the archaeal domain. Though NC12 is most closely related to P. occultum from the viewpoint of 16S rRNA similarity, the identity score was only 91.2%. Thus, it is shown that N C12 is a novel organism which has not been reported so far. Furthermore, there is a possibility that NC12 is not only a new species but may constitute a new genus. To estimate evolutional distances between other related organisms listed in the table, maximum likelihood analysis was carried out. Fig. 4 shows the best tree of the 16S rRNA sequences obtained from the analysis. Since the bootstrap probabilities of the common ancestral lineages of D. mobilis and Sulfolobus and of these species and P. occultum are not high (60 and 54%, respectively), the branching order among NC12, P. occulturn, D. mobilis and Sulfolobus cannot be determined with confidence by this analysis. Nevertheless, it seems highly likely that these species form a monophyletic clade excluding T. pendens and T. tenax as outgroup, since the local bootstrap probability of this clade is as high as 97%. Here, we propose to designate the newly isolated hyperthermophilic archaebacterium NC12 as Caldococcus noboribetus. For detailed discussion about the evolutional meaning of the organism, further characterization is needed.
3. Conclusions (1) A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring and was characterized. (2) A gene coding for 16S rRNA from NC12 was cloned and sequenced. (3) Genomic DNA prepared from NC12 was found to be resistant to EcoRI or BamHI treatment, suggesting the existence of some kinds of restriction systems in NC12.
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(4) Sequence comparisons of 16S rRNA showed that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain of the phylogenetic tree. (5) Sequence comparisons of 16S rRNA showed that NC12 is a novel hyperthermophile which may constitute a new genus.
References Achenbach-Richter, L., Gupta, R., Zillig, W. and Woese, C.R. (1988) Rooting the archaebacterial tree: The pivotal role of Thermococcus celer in archaebacterial evolution. Syst. Appl. Microbiol. 10, 231 240. Adachi, J. and Hasegawa, M. (1996) MOLPHY: Programs for Molecular Phylogeneties ver. 2.3, Institute of Statistical Mathematics, Tokyo. Grogan, D., Palm, P. and Zillig, W. (1990) 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. Haas, E.S., Brown, J.W., Daniels, C.J. and Reeve, J.N. (1990) Genes encoding the 7S RNA and a Ser-tRNA are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermusfervidus. Gene 90, 51-59. Hasegawa, M. and Kishino, H. (1994) Accuracies of the simple methods for estimating the bootstrap probability of a maximum likelihood tree. Mol. Biol. Evo[. 11, 142-145. Hasegawa, M., Kishino, H. and Yano, T. (1985) Dating of the humanape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160-174. Kaine, B.P., Schurke, C. and Stetter, K.O. (1989) Genes for the 16S and 5S ribosomal RNAs and the 7S RNA of Pyrodictium occultum. Syst. Appl. Microbiol. 12, 8-14. Kishino H., Miyata, T and Hasegawa, M. (1990) Maximum likelihood inference of protein phylogeny, and the origin of chloroplasts. J. Mol. Evol. 30, 151 160. Kjems, J., Garrett, R.A. and Ansorge, W. (1987) The sequence of the 16S RNA gene and its flanking region from the archaebacterium Desulfurococcus mobilis. Syst. Appl. Microbiol. 9, 22 28. Kjems, J., Leffers, H., Olesen, T., Ingelore, H. and Garrett, R.A. (1990) Sequence, organization and transcription of the ribosomal RNA operon and the downstream tRNA and protein genes in the archaebacterium Thermofilum pendens. Syst. Appl. Microbiol. 13, 117-127. Kurosawa, N. and Itoh, Y.H. (1993) Nucleotide sequence of the 16S rRNA gene from thermoacidophilic archaea Sulfolobus acidocaldarius ATCC33909. Nucleic Acids Res. 21, 357. Leinfelder, W., Jarsch, M. and Back, A. (1985) The phylogenetic position of the sulfur-dependent archaebacterium Thermoproteus tenax. Syst. Appl. Microbiol. 6, 164-170. Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51,221-271. Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Natl. Acad. Sci. USA 74, 5088 5090. Woese, C.R., Kandler, O. and Wheelis, M.L. (1990) Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576 4579.