The sequence of the 16S RNA gene and its flanking region from the archaebacterium Desulfurococcus mobilis

System. Appl. Microbiol. 9, 22-28 (1987)

The Sequence of the 16S RNA Gene and its Flanking Region from the Archaebacterium Desulfurococcus mobilis J0RGEN KJEMS, ROGER A. GARRETT l , and WILHELM ANSORGE Biostructural Chemistry, Kemisk Institut, Aarhus University, 8000 Aarhus C, Denmark and EMBL Laboratory, Postfach 1022.40, 6900 Heidelberg, Federal Republic of Germany Received April 16, 1986

Summary The sequence of the 165 RNA gene from the archaebacterium Desulfurococcus mobilis and its flanking regions were determined. The mature RNA transcript is 1,495 nucleotides long. The putative secondary structure exhibits typical archaebacterial features and is closely related to those of the other extreme thermophile RNAs. This structure exhibits a very high level of G-C base pairing (74%) and it is possible for this and other thermophile structures to extend the regular base pairing into the internal loops which may reflect adaptation to high temperature. The 165 RNA structure is enclosed by a long processing stem containing a few irregularities that is characteristic of prokaryotes. The structure of 165 RNA from D. mobilis was aligned with all of the sequenced archaebacterial165 RNAs and E. coli 165 RNA, and homology values were determined. A phylogenetictree was derived from these values which showed that D. mobilis, Sulfolobus solfataricus and Thermoproteus tenax constitute a separate phylogeneticgroup. Of these D. mobilis exhibits the lowest genetic drift while T. tenax shows the deepest branching.

Key words: Archaebacteria - Extreme thermophile - 16S RNA secondary structure - RNA processing Phylogenetic sequence analysis

Introduction A program is currently underway to use the 16S ribosomal RNA sequences as a measure of evolutionary distance between organisms. This is appropriate first because the 16S-like RNAs are universal amongst living organisms and, second, because their functional role in protein biosynthesis has remained basically unchanged during evolution. Fox, Woese and colleagues first used this approach by comparing sequence catalogues of oligonucleotides deriving from ribonuclease T 1 digests of the 16S-like RNAs (Woese et al., 1978; Magnum et al., 1978). This study provided the first evidence that the archaebacteria which consist of the extreme halophiles, the methanogens and the extreme thermophiles, are distantly related to both eubacteria and eukaryotes (Fox et al., 1980). Several archaebacterial 16S RNAs are currently being sequenced in order to deduce the evolutionary relationships between these organisms. To date, sequences have 1

To whom editorial correspondence should be sent.

been determined for four extreme halophiles, Halobacterium volcanii (Gupta et al., 1983), Halococcus morrhuae (Leffers and Garrett, 1984), Halobacterium halobium (Mankin et al., 1985) and Halobacterium cutirubrum (Hui and Dennis, 1985), two methanogens Methanococcus vannielii (jarsch and Bock, 1985) and Methanobacterium formicicum (Lechner et al., 1985) and two extreme thermophiles, Thermoproteus tenax (Leinfelder et al., 1985) and Sulfolobus solfataricus (Olsen et al., 1985). Here we present an additional sequence for the extreme thermophile Desulfurococcus mobilis that was isolated from Icelandic springs at temperatures up to 97°C (Zillig et al., 1982). The ribosomal RNA operon of D. mobilis exhibits some unusual properties. There is only one operon (Neumann et al., 1983; our unpublished results); the 5 RNA gene is not coupled to those of 16S RNA and 23S RNA; moreover, the 23S RNA gene contains the only intervening sequence so far detected in a prokaryotic rRNA gene (Kjems and Garrett, 1985; Larsen et al., 1986).

16S RNA Gene of Desulfurococcus mobilis

23

of the 5'-terminal nucleotide was ascertained by comparing the size of the run-off eDNA transcript with the corresponding DNA sequence that was obtained using the same primer on single stranded DNA covering this 5'-region. The 3'-terminus was determined as follows: 10 fAg 16S RNA was partially digested with 0.1 units RNAse T I in 40 ul TMK buffer (30 mM Tris-HCl, pH 7.5,20 mM MgCI 2, 300 mM KCI). The resulting fragments were 3'-end labelled with [32pjpCp. Only 3'-terminal fragments should label since RNAse T I produces 3'-phosphates on the internal fragments. After purifying on a 10% polyacrylamide gel the labelled bands were sequenced using base-specific ribonucleases

Materials and Methods D. mobilis cells and preparation of DNA and rRNA Cells from Desulfurococcus mobilis were kindly provided by W. Zillig. The source and the growth conditions have been described by Zillig et al. (1982). DNA was prepared by lysing cells in 5 vol. of 0.01 M Tris-HCl, pH 8.0, 0.001 M EDTA, 0.1 M NaCI by adjusting to 1.5% Na dodecylsulphate followed by 3 phenol and 2 chloroform extractions, before banding in a caesium chloride gradient in the presence of 5 ug/ml ethidium bromide. Ribosomes were extracted by grinding cells with alumina and the rRNAs were fractionated in a sucrose gradient containing Li dodecylsulphate (Fellner, 1969).

(Donis-Keller, 1979).

Phylogenetic analysis

Cloning and sequencing strategy

The D. mobilis sequence was aligned with those of the archaebacteria T. tenax (Leinfelder et aI., 1985), S. solfataricus (Olsen et aI., 1985), H. volcanii (Gupta et aI., 1983), He. morrhuae (Leffers and Garrett, 1984), H. cutirubrum (Hui and Dennis, 1985), Me. vannielii lJarsch and Bock, 1985) and M. formicicum (Lechner et aI., 1985) and with that of the eubacterium E. coli (Brosius et al., 1978) according to the conserved secondary structural elements. Homology values were calculated for pairs of sequences as follows: homology = match positions/ [match positions + mismatch positions] (McCarroll et aI., 1983). No account was taken of gaps in the archaebacterial sequences since they are minimal. Knuc values were calculated according to Hori and Osawa (1979) and the phylogenetic tree was estimated as described by McCarroll et al. (1983). Conserved regions of the 16S-like RNAs were also aligned for 5 eubacterial, 4 chloroplast, 9 mitochondrial and 7 eukaryotic sequences. All except one of the literature sources (Green et aI., 1985) are listed in Gutell et al. (1985).

The 16S rRNA gene of D. mobilis was isolated on an 8 Kb EcoR1 fragment from a digest of chromosomal DNA and was cloned into A-phage L47.1 essentially as described by Maniatis et al. (1982). Phages containing rRNA genes were selected by plaque hybridization using rRNA fragments prepared by RNAse S1treatment that had been 3'-end labelled using [32pjpCp and RNA ligase (Bruce and Uhlenbeck, 1977). DNA subfragments in the size range 1-4 Kb were prepared and digested with restriction enzymes specific for 4 base pairs. After purifying on polyacrylamide gels each fragment was cloned in both directions into phage M 13 mp 18 or mp 19 vectors. The direction of cloning was determined by 'figure 8' mapping (Maniatis et aI., 1982). Single stranded DNA was prepared and used as a template for DNA sequencing by the dideoxynucleotide method (Sanger et aI., 1977) using [a- 35SjATP (1,000 Ci mmol"; Amersham); the DNA was run on sequencing gels containing 4-6% polyacrylamide and wedge shaped gradients (Ansorge and Labeit, 1984). The DNA was sequenced in both directions and overlapping sequences were read at each restriction site.

Results and Discussion

Determination of 16S RNA gene limits

Primary sequence

The 5'-terminus of the 16S RNA molecule was determined by hybridizing 5 pmol of a 50 bp Dde1 DNA fragment that was positioned close to the 5'-end of the 16S RNA gene to 10 pmol 16S RNA in 10 mM Tris-HCI, pH 7.5, 40 mM KCl by heating at 95°C for 2 min and then at 60 °C for 60 min before cooling quickly. Primer extension was performed in 10 !AI buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCI 2, 2 mM DDT containing 200 !AM dGTP, dTTP and dCTP, 10 fACi [a-uS]ATP (1,000 Ci mmol"; Amersham), 5 pmol primer template complex and 10 units AMV reverse transcriptase (Life Sciences, Florida). After 15 min, the reaction was chased with cold dATP. The exact position

The sequencing strategy for the 165 RNA gene is illustrated in Fig. 1 and the sequence of the RNA gene is shown in Fig. 2. The extremities of the 165 RNA were determined experimentally as described in Materials and Methods, and they align with the terminal sequences of other reported thermophiles that were determined by oligonucleotide sequence analysis (Woese et a!., 1984). They consist of the sequence 5'-A-C-U-3' at the 5'-terminus and the putative 5hine-Dalgarno sequence 5'-C-C-U-C-C-3' at the

1 kb

1-

B B B

E

B

::;:=:::r---

~":!!""":!~~--""!!'!~!'!I!I~-------+----'i t:t:::238~

-. -1-k---b 168 RNA 0

Fig. 1. Restriction endonuclease map of the D.

mobilis operon showing the sequencing strategy

Alu I

for the 16S RNA gene. The upper part shows the 8.0 kb fragment cloned into phage A vectors. Restriction sites are abbreviated as EcoRI (E) and BamHI (B). The lower part shows an enlarged restriction map of the 16S RNA gene. The sequenced fragments and the direction of sequencing are indicated by arrows.

Ava I Ode I Hae III Rsa I Taq I

I I I

II I

I

I

I

I I'

II'

I II

'1 1111'1'

II

I

I

II

E I

J. Kjems,

24

R. A. Garrett , and \VI. Anso rge

CCGACGAGGG GGAGGGAGCC ACTTA AGCCG AGCGCTCCAG AAACCCCGGC CCCAACCCCA AGCGGCCGTA

-7 0

- 60

~16S

- 50

RNA

- 30

-~ o

- 20

- 10

ACTCCGGTTG ATCCTGCCGG TCCCGACCGC TATCGGGGTG GGGCTAAGCC ATGGGAGTCG CACGCTCCGC

10

20

30

~o

50

60

70

CGCTGCGGGG CGTGGCGGAC GGCTGAGTAA CACGTGGCTA AC CTA CCCTC GGGAGGGGGA TAACACCGGG

80

90

100

110

120

130

1~0

AAACTGGTGC TAATCCCCCA TAGGGGAGGA GGCCTGGAAG GGTTCCTCCC CGAAAGGGTG TGGCAGGGGT

150

160

170

180

190

200

210

TAACGCTGCT ACACCGCCCG AGGATGGGGC TACGGCCCAT TAGGTTGTTG GCGGGGTAAC GGCCCGCCA A

220

230

2~0

250

260

270

280

GCCGATAATG GGTAGGGGCC GTGAGAGCGG GAGCCCCCAG ATGGGCACTG AGACAAGGGC CCAGGCCCTA

290

300

310

320

330

3~ 0

350

CGGGGCGCAC CAGGCGCGAA ACCTCCGCAA TGCGGGAAAC CGTGACGGGG CCACCCCGAG TGCCCCCTTA

360

370

380

390

~OO

410

420

CGGGGGCTTT TCCCCGCTGT AGGAAGGCGG GGGAATAAGC GGGGGGCAAG TCTGGTGTCA GCCGCCGCGG

430

440

450

460

470

480

490

TAATACCAGC CCCGCGAGTG GTCGGGACGA TTATTGGGCC TAAAGCGCCC GTAGCCGGCC CGGCAAGTCC

50 0

510

520

530

5~ 0

550

560

CCTCCTAAAT TCCCGGGCTC AACCCGGGGA CTGGAGGGGA TACTGCCGGG CTAGGGGGTG GGAGAGGCCG

570

580

590

600

610

620

630

AG GGTACTCC CGGGGTAGGG GCGAAAT CCT ATAATCC CGG GAGGACCACC AGTGGCGAAG GCGCTCGGCT 6 ~0

650

660

670

680

690

700

GGAACACGCC CGACGGTGAG GGGCGAAAGC CGGGGGAGCG AACCGGATTA GATACCCGGG TAGTCCCGGC

7 10

720

730

7~0

750

760

770

TGTAAACGAT GCGGGCTAGG TGTTGGGTGG GCTTAGAGCC CACCCAGTGC CGCAGGGAAG CCGTTAAGCC

780

790

800

810

820

830

8~0

COCCGCCTGG GGAGTACGGC CGCAAGGCTG AAACTCAAAG GAATTGGCGG GGGAGCACCA CAAGGGGTGG

850

860

870

880

890

900

9 10

AGCCTOCGOT TCAATTGGAG TCAACGCCGG GAATCTCACC GGGGGAGACA GCAGGATGAC GGCCAGGTTA

920

930

9~ 0

950

960

970

980

AAGGCCTTGC CTGACGCGCT GAGAGGAGGT GCATGGCCGT CGCCAGCTCG TGCTGTGAAG TGTCCGGTTA

990

1000

1010

1020

1030

1 0~ 0

1050

AGTCCGGAAA CGAG CGAGAC CCCCACCCCT AGTTGCTACC CGGGGCTACG GCTCCGGGGC ACACTAGGGG

1060

1070

1080

1090

l1 GO

1110

1120

GACTGCCGCC GTTTAAGGCG GAGGAAGGAG GGGGCCACGG CAGGTCAGCA TGCCCCGAAC CCCCCGGGCT

1130

1150

11~ 0

1160

1170

1180

1190

ACACGCGGGC TACA ATGGCG GGGACAGCGG GATCCGACCC CGAAAGGGGG AGGCAATCCC TCAAACCCCG

1200

12 10

1220

1230

1 2~ 0

1250

1260

CCGTGGTTGG GATCGAGGGC TGCAACTCGC CCTCGTOAA C OAOGAATCCC TAGTAACCGC GCGTCUCAT

1270

1280

1290

1300

1310

1320

1330

CGCGCGGTGA ATACGTCCCT GCTCCTTGCA CACACCGCCC GTCGCTCCAC CCGAGGGGAG GGGGAGTGAG 1 3~ 0

1350

1360

1370

1380

1390

1400

GCCCGGCCCC TTGGGTCGGG TCGAACTCCC CCTCCCTGAG GGGGGAGAAG TCGTAACAAG GTAGCCGTAC 1~1 0

1430

1~20

14~0

1~ 50

1460

1~70

~

CGGAAGGTGC GGCTGGATCA CCTCCTGCCT CAGGCCGGGG CTGGGGCCGG GGCTGGAATG CGCTAAGGCT 1~ 80

1490

1

15

25

35

~5

TAAGTGGCTC CCGCCTCCTC TATTCATTGA TGCAACACGG

55

65

75

85

Fig. 2. The nucleot ide sequence of the 165 RNA gene and its flankin g regions from D. mobilis. Th e gene is numbered from 1 to 1495.

3'-terminus. The mature RNA is 1,495 nucleotides long and similar in size to the extreme thermophile RNAs of T. tenax (1,504 n) (Leinfelder et al. , 19 85) and S. solfataricus (1,493 n) (Olsen et al., 1985 ). The se are all slightly longer than the extreme halophile and methanogen RNAs but smaller than those of the eubacteria. The other extreme th ermophile RNAs are subject to extensive modification after transcription (Woese et al., 1984) and, although we ha ve not investigated such modifications in D. mobilis, the strong termination band observed at G 35 , when using reverse transcriptase (data not sho wn), suggests that C 34 is modified.

Secondary structure of 165 RNA The secondary structure for 16S-like RNAs that is based on ph ylogenetic sequence comparison s is drawn for the D . mobilis RNA in Fig. 3. lt exhibits both primary and secondary stru ctur al features that are characteristic of the extr eme therrnophiles, in particular, an d the archaebacteria in general (Woese et aI., 198 3 ; Gutel/ et aI., 1985 ). These features are summarized in Table 1. The D. mobilis RNA structure exhibits a G-C base pair content of 74% which is similar to that of T. tenax il.einfelder et aI., 1985 ) and higher than the 70% value for S. solfataricus (Olsen et al., 1985 ). These high levels of G-C pairing almost certainly reflect ad aptation to high temperature. This view is reinforced by the occurrence of extensive runs of guanosines, and cytidines, in the extreme thermophiles (Woese et al., 1984) which tend to produce greater th ermal stability within double helices than alternating G-C sequences. There are th irty four sequences of four, or more, consecutive guanosines and fifteen of four, or mo re, co nsecutive cytidines in th e D. mobilis RNA , whereas there ar e onl y twel ve and two, respecti vely, in the E. coli 16S RNA (Brosius et aI., 1978 ). A further indication of the adaptation to high temperature is the po ssibility of extending regular base pairing into so me internal loops of th e extreme thermophile (and extreme halophile) RNA st ructure s. Some weak helices do not increase in stability in th e thermophiles and these ma y con stitute protein binding or functional sites. Examples are: helix 2, the purine juxtap ositions in helix 23 and helix 47, G-U base pa irs in helices 28, 37 and 39 , and the C- U juxtaposition in helix 36 (Fig. 3). Var ious nucleotides that can determine antibiotic sensitivity or resistance within the rib osome are indicated on th e secondary structure by a rro ws. C 1 l 5S confers sensitivity to spectinomycin which perturbs translocation, while a C-U transition produces resistance (Sigmund et al., 1984). The stable base pair C13WG1447 at the end of helix 47 (Fig. 3) confers sensitivity to paromomycin which affects translational fidelity; its disruption causes resistance (Li et al. , 1982). C876 is associated with streptomycin-sensitivity and is con sidered in detail below.

Features specific to D. mobilis A few positions a t which the D. mobilis sequence diverges from otherwise conserved ar chaebacterial or extrem e thermophile sequ ences ar c listed in Table 2. Especially interesting is po sition C 876 • Thi s nucleotide is a uridine in all other archaebacteri a and eukaryotes, and a cytidine in eubacteria and chloropl asts. Mutants of Euglena gracilis which contain strepto mycin-resistant chloroplasts wer e shown to undergo a single base change C to T at this position in their single 16S RNA gene (Montandon et al. , 1985). It wa s suggested th at this base change pr oduce s resistance to streptomycin, which affects translation al accuracy. This infer ence is strongly supported by the present data since the D. mobilis contains the onl y archaebacterial RNA that has a C at this position and it is the o nly archaebacterium kn own to be streptomycin-sensitive (R. Amils, personal comm).

165 RNA Gen e of Desulfurococcus mobilis

25

Fig. 3. Putat ive secondary str ucture of the 165 RN A from O. mobili s using th e genera l form at of Gutell er al. (1985 ). Th e dou ble helical segmems are nu mbered from 1 to 48. Dashed lines drawn across internal loo ps indicate th at coordin ated ba se changes support th e existence of these base pa irs. Arrows indicate nucleo tides that dete rmin e ant ibiotic sensit ivity/res istance in ribosomes (see tex t for details).

Structure of the flanking region of 165 RN A The sequence flanking the 5'-end of the 165 RNA can base pair with the sequence flanking the 3'-end to form a long imperfect helix containing 54 base pairs as shown in Fig. 4. The base pair content of 74% G-C corresponds to that of the 165 RNA. The centre of the helix also exhibits two bulged loops, each containing 2-3 nucleotides, at staggered positions on opposite strands. They correspond to the initial processing sites for RNAse III-like enzymes in

the precursor RNAs of H. cutirubrum (Chant and Dennis, 1986). This feature occurs in all the processing stems of archaebacterial 165 RNAs so far investigated for H. halobium (Mankin et al., 1984), I-f. cutirubrum (Hui and Dennis, 1985; Chant and Dennis, 1986); M. [ormicicum (Lechner et al., 1985); Me. vannielii (jarseh and Bock, 1985) and T. tenax (Leinfelder et al., 1985). The sequ-

J. Kjems,

26

Helix C28 G 43 C 248-G ZYO G29j-CJI7 GG 461 Um-A 498 U478 G519 C652 G904-C1J10 G930 G103rU1041 C 1161 G 1169 C n oo C UI7 C 1374

3

10/11 12 13 19 19 19

32 39 37

46

R. A. Garrett, and W. Ansorge Thermophiles

Halophiles/ methanogens

Eubacteria/ chloroplasts

Eukaryo-

C G long/long CoG G-C GG U-A U G C G-C G G-U C G C C G

U A long/short G-C CoG CU CoG C U U C-G Y A-C U C G U A

C

U A

short/variable G-C I

-

G-C YC I

C-G I C A U G-C UI G-U Y C G U A

tes

Table 1. Primary and secondary structural features specific to the extreme thermophile 165 RNAs

C-G CoG GA U-A C A U G-C C G-U C G C G G

Structural characteristics of 16S RNAs from thermophiles are compared with those of other archaebacteria (5), eubacteria/chloroplasts (8) and eukaryotes (7) (see Materials and Methods for details); mitochondrial and mycoplasma sequences are not included. The numbering system derives from D. mobilis (Fig. 3). A space indicates either that the corresponding nucleotide is absent or that the sequence is too diverse to align. Y = pyrimidine. I Exceptional in Bacillus subtilis RNA.

ences in this region are not conserved but there are multiple examples of coordinated base changes to support the proposed secondary structure. The loops containing 2-4 nucleotides are separated by 4-5 base pairs with the upstream loop closest to the 16S RNA (Fig.4 A). Thus, they will lie on the same side of the double helix. Similar structural features have been found near the centres of the 23S RNA processing stems and it is shown for the D. mobilis stem in Fig. 4 B. In eubacteria, the corresponding initial processing sites also occur near the centres of the processing stems but within regular double helical regions (see Brosius et a!., 1981).

Phylogenetic implications Homology values are shown for the archaebacterial16S RNAs in Table 3. The three extreme thermophiles fall into one group with values greater than 82%; this is substan-

Table 2. Features specific to D. mobilis amongst archaebacterial 16S

D.mobilis

Archaebacteria

Eubacteria/ chloroplast

Eukaryotes

U2l C876 Loop 43/6n G 1265 GG 1477

A U 4-5n A YC

A C 4-5n A variable

U U 5n G CC

The D. mobilis sequence was aligned with those of 7 archaebacteria, 9 eubacteria/chloroplast and 7 eukaryote sequences (see Materials and Methods for details).

Table 3. Homology and Knuc value for archaebacterial 16S RNAs 1. 1. D. mobilis 2. S. solfataricus

3. T. tenax 4. H. volcanii 5. H. cutirubrum 6. He. morrhuae 7. M.formicicum 8. Me. vannielii 9. E.coli

0.153 0.158 0.333 0.339 0.336 0.264 0.258 0.459

2.

3.

4.

5.

6.

7.

8.

9.

86.2

85.7 82.9

73.1 71.0 72.2

72.7 71.1 71.2 87.9

72.9 71.4 71.4 88.1 88.0

77.7 75.2 75.4 76.6 77.8 76.3

78.2 75.6 75.1 75.6 75.0 75.3 80.8

65.7 63.7 64.4 62.5 63.2 62.9 65.5 65.2

0.194 0.366 0.364 0.360 0.301 0.295 0.496

0.347 0.363 0.361 0.298 0.303 0.483

0.132 0.130 0.280 0.293 0.52

0.131 0.264 0.305 0.506

0.284 0.299 0.512

0.222 0.462

0.467

Homology values are represented in the upper right-hand triangle. Knuc values are shown in the lower left-hand triangle.

16S RNA Gene of Desul[urococcus mobilis

.r : 168 ,

--- -,

CUCCGU / -AUG A C-G C-G G - C-lO

GC G- C

-10- AAC- G C-G C-G C-G A

C

A-U C-G C-G

- 20- C- G-20

C-G G-C G-C

A.

A g:!] C-G

AG-C

L

A-U

C-G

A-U G-C C-G

GAU • G UC - G

A-U U-A UA

C-G

G•

C-

G-C s-c A-U

U- AA GU

G-C C-G

G-C

-40

B.

G- CA

A

l -;; u.t-40 C-G

C-G G-C

A-U A-U U-A U-A C-G

-50 A - U- 50

C- G

C-G G-C

A-U

G- C

G-C

G-C

A

0

G

G-C

-60 - G- C-60 G' U

G-C G-C

5' CCCGAGAGCUUGAAUCCGAC~:~UAUUCAUUGAUGCAACACGG3' Fig. 4. Putative secondary structure of the flanking sequences of the 165 RNA gene. The putative RNA processing site is boxed (Hui and Dennis, 1985; Chant and Dennis, 1986). B. The corresponding site in the processing stem of the 235 RNA of D. mobilis.

27

tially higher than their homologi es with the halophiles Or methanogens. The S. solfataricus RNA is more homologous to the D. mobilis RNA (86%) than to that of T. tenax (83% ) whereas the D. mobilis RNA is closely homologous to both S. solfataricus and T. tenax RNAs. However, when dealing with the thermophile sequences, systematic errors may occur dur ing the homology calculation, due to their high G/C content; thus, the thermophiles tend to be grouped too closely. A comparison of the homology values for the thermoph ile RNAs with those of the halophile/meth anogen branch shows that D. mobilis is closest to the latter branch (Table 3). Thu s, D. mobilis has had a lower evoluti onary rate than either S. solfataricus or T. tenax, The rate of nucleotid e substitution Knuc (Hori and Gsaioa, 1979), was calculated from the homology values (Ta ble 3) and a phylogenetic tree was derived for the extreme therm ophiles and one extreme halophile according to McCarroll et al. (1983). Th is is presented in Fig. 5 where the lengths of the lines are proportional to a combination of evolutionary time and the rate of mutation of the sequence. The root of the tree was deduced by comparing the homolo gy values with those of the E. coli RNA (Table 3). T. tenax exhibit s the deepest branchi ng amongst the thermophil es and this correlates with the observation that D. mobilis and S. solfataricus exhibit several examples of common sequence signatures in conserved regions (Table 4). The Knuc values (Table 3) indicate that the three halophiles are most distant from the thermophiles, which reflects the formers higher rat e of evolution, with the methanogens lying in between (see also Leinfelder et al., 1985 ). Acknowledgements. We thank Professor Wolfram Zillig for providing the D. mobilis cells, and Niels Larsen for his excellent help with the sequence alignments and computer graphics. Henrik Leffers is thanked for stimulating advice. Mette Lygaard helped with the manuscript. The research was supported by a grant from the Danish Natur al Science Research Council.

Ha/obacterium yr;/cilnii

0.01 KNUC

Desulfurococcus mobilis

Thermoprofeus tenax

Fig. 5. Phylogenetic tree for the extreme thermoph iles including the extreme halophile H. uolcanii. The tree was derived as described in the text and the root was deduced bv determined the homology with the 16S RNA sequence of E. cdli (Brosius et al., 1978).

Table 4. Features common to D. mobilis and S. soltataricus within highly conserved regions D. mobilis and S. solfataricus

Other organisms

G S4'C 3hO CUG9sICAG32o

CoG GUaCAC CUClGAG 1 U U

The sequenceswerecompared with twenty three 165 RNAsequences. D. mobilis numbers are used. 1 - eubacteriaand mitochondria and 2 - archaebacteria and eukaryotes.

28

J. Kjems, R. A. Garrett,

and W. Ansorge

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Dr. Roger A. Garrett, Biostructural Chemistry, Kemisk Institut, Aarhus University, DK-8000 Aarhus C, Denmark