ARCHIVES

OF BIOCHEMISTRY

AND

Vol. 283, No. 1, November

BIOPHYSICS

15, pp. 210-216,199O

Characterization of a Superoxide Dismutase Gene from the Archaebacterium Methanobacterium thermoautotrophicum’ Masashi

Takao,*

Atsushi

Oikawa,?

and Akira

Yasuity2

*Department of Bioscience, School of Hygienic Science, Kitasato University, Sagamihura tResearch Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980, Japan

228 and

Received May 17,1990, and in revised form July 24,199O

A gene encoding superoxide dismutase (SOD) was cloned from the archaebacterium Methanobacterium thermoautotrophicum, the first example from an anaerobic bacterium. The deduced amino acid sequence showed overall similarity to sequences of known Mnand Fe-SODS from aerobic organisms. Judging from a detailed sequence comparison, the cloned SOD gene is classified as Mn-SOD. By comparison of Mn-SOD sequences among various species it was suggested that archaebacterial superoxide dismutase is a direct descendant of a primordial enzyme. Between a putative promoter and the start codon there is an inverted repeat sequence which is also found in the counterpart of Halobacterium halobium. o 1990 Academic PESS, IIN.

Protection against superoxide is an important cellular defense system. One of the most characterized mechanisms is the disproportionation of 0, into H,O, and 0, catalyzed by superoxide dismutase [SOD,3 (l)]. There are three classes of SOD with respect to its metal cofactor: copper-zinc (CuZn-SOD), manganese (Mn-SOD), and iron (Fe-SOD). In general, CuZn-SOD is found in eukaryotes, Fe-SOD is found in eubacteria and archaebacteria, and Mn-SOD is found in both bacteria and eukaryotic mitochondria. From comparisons of amino acid sequences and the crystal structure of SOD, it was concluded that Mn-SOD and Fe-SOD originated from a 1 This work was supported by a grant-in-aid for “specific research on molecular mechanism of photoreception” (Grant 01621001) from the Ministry of Education, Science and Culture (Japan) to A.Y. * To whom correspondence should be addressed at The Research Institute for Tuberculosis and Cancer, Tohoku University, Seiryomachi 4-1, Aobaku, Sendai 980, Japan. Fax: 022-2757324. 3 Abbreviations used: SOD, superoxide dismutase; PCR, polymerase chain reaction; ORF, open reading frame.

common ancestor and are distinct from CuZn-SOD [for reviews, see (2,3)]. Eubacterial aerobes contain either Mn-SOD or FeSOD or both and many aerobes show induction of the SOD by oxidative stress (2). Some strict anaerobes have been shown to contain SOD (4,5), although several anaerobes do not show SOD activity (6). Because of the complexity of this eubacterial SOD distribution it is not yet clear whether some anaerobes acquired their SOD genes after the appearance of atmospheric oxygen or a primordial anaerobic organism of ‘urkaryote’ nature already possessed SOD. The primary sequence data of Mn-SOD and Fe-SOD increased in the past 5 years, but they were only for aerobic organisms. Recently, archaebacterial Mn-SOD sequences were determined from the cloned genes of two closely related halobacteria Halobacterium halobium (7, 8) and Halobacterium cutirubrum (9) and they showed similarities to eubacterial and eukaryotic SODS. However, since both halobacteria are aerobic, a possibility remains that lateral gene transfer between archaebacteria and eubacteria occurred after accumulation of oxygen on the earth (9,10). To investigate the origin of the archaebacterial SOD gene, we attempted here to clone SOD genes from other archaebacterial species. By polymerase chain reaction (PCR) with synthetic primers designed from the conserved sequence regions among known Mn-SODS, a part of the SOD gene from Methanobacterium thermoautoa strict anaerobe, was amplified and then the trophicum, complete SOD gene from this organism was obtained and analyzed. MATERIALS

AND

METHODS

Materials. Restriction enzymes and DNA modifying enzymes were purchased from Boehringer-Mannheim, New England Biolabs, and TOYOBO. Taq polymerase was from TaKaRa. Multiprime labeling

210 All

0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.

Methanobacterium

thermoautotrophicum

kit (Amersham) and DNA sequencing kit (TOYOBO and U.S.B.) were used for making cY-32P-labeled DNA probe and for sequencing, respectively. Escherichia coli DH5a was used for transformation. Genomic DNAs from Sulfolobus soPreparation of genomic DNA. lufaturicus and Sulfolobus acidocaldarius cells were prepared by a standard method (11). To obtain the genomic DNA from M. thermoautotrophicum, cells were crashed by a french-press and subsequently extracted with phenol. The genomic DNA was fractionated by sucrose density gradient centrifugation to obtain the DNA fragments longer than 10 kb. Cloning of the SOD gene by PCR. Four synthetic oligonucleotide primers containing restriction sites at their 5’ end were made by using a DNA synthesizer (Applied Biosystems Model 381A). Genomic DNA was mixed with a pair of primers and subjected to a PCR amplification (12) with 30 cycles. We selected the annealing temperatures of primers to denatured DNA with 55°C as the “high annealing temperature” and 37°C as the “low annealing temperature.” Amplified fragments were recovered from low melting point agarose gel (Bio-Rad), digested with restriction enzymes corresponding to the primer end, and cloned in pUC118. M. thermoautoIsolation of genomic clone. Using a PCR-amplified trophicum DNA fragment as a hybridization probe, genomic DNA of this bacterium was analyzed by Southern blotting. PstI-restricted genomic fragments to which the probe hybridized were isolated from a low melting point agarose gel and cloned in pUC118. This PstI library was screened by the colony hybridization technique using the same probe. A Bg211-Hind111 library containing genomic DNA fragments flanked by BglII and Hind111 sites was also constructed and screened in the same manner. The isolated clones from the libraries were Nucleotide sequencing. subcloned in pUC18 or pUC118 using restriction sites found in the cloned fragments and sequenced using a universal or a reverse primer according to the dideoxy sequencing method (13). Some sequences were determined by the use of synthetic primers. The nucleotide sequence shown in this paper was confirmed by sequencing in both strands. DNA and deduced amino acid sequences were analyzed with the GENETYX software package. RESULTS

Genomic DNAs from three archaebacteria, S. solfuturicus, S. acidocaldarius, and M. thermoautotrophicum were subjected to PCR-amplification to obtain a part of the SOD gene. From the known Mn-SOD sequences, three conserved regions were selected and oligonucleotides corresponding to the amino acid sequences were synthesized as primers for PCR (Fig. 1A). Four oligonucleotide primers with all codon variations were then synthesized. The pair of primers 1 and 3 amplified DNA of an expected size from the M. thermoautotrophicum genome at the high annealing temperature (Fig. 1B). The amplified M. thermoautotrophicum fragments were digested with restriction enzymes recognizing the sites at the primers and cloned in a plasmid vector. In contrast, no amplified fragment was detected from the S. solfataricus and S. acidocaldarius genomes using primers 1 and 3 as well as the other pairs, primers 1 and 2 or primers 3 and 4, even at the low annealing temperature (Fig. 1B and data not shown). The results suggest that Sulfolobus SOD, if exists, differs significantly in amino acid sequence even within the above conserved regions.

SUPEROXIDE

A

DISMUTASE 1

M-SOD

211

GENE 200

100

III

ml

1111 I

II

Illl

II 1

T

KqiiiJ

A PCR -1+ primer

B - 470 bp -

primer

5oobo

C Bg

-

51

PHC

SHC H

I

I I

II

-

+ t+-----

-t--c----

P

I

-f

FIG. 1. Cloning of archaebacterial SOD gene. (A) Positions and corresponding amino acid sequences of synthesized primers. Relative positions of conserved amino acids among eight known Mn-SOD sequences (see Fig. 4) are indicated as vertical bars in Mn-SOD of about 200 amino acids. Sense primers (1,4) and antisense primers (2,3) encode boxed amino acid sequences with all codon variations. At the 5’ end each primer contains an EcoRI site (primer 1, 4), an XbaI site (primer 2) or a BamHI site (primer 3). (B) Agarose gel electrophoresis after amplication of archaebacterial genome. Using a set of primer 1 and 3, PCR was carried out by using genomic DNAs from S. acidocaldark (lane l), S. solfataricus (lane 2), and M. thermoautotrophicum (lane 3). An amplified M. thermoautotrophicum fragment of 470 kb is indicated. (C) Restriction map and sequence strategy of M. thermoautotrophicum SOD gene. Using a probe derived from a PCR-amplified fragment (bold line), a 2-kb BglII-Hind111 fragment and a 1.4-kb PstI fragment were cloned from partial genomic libraries. Restriction sites used for subcloning are: B, BgZII; H, HindIII; Hc, HincII; P, P&I; S, SmaI; Sl, SalI. Not all HincII sites are indicated. Sequenced region and direction is indicated by horizontal arrows.

Seven clones were randomly selected from transformants harboring the PCR-amplified M. thermoautotrophicum DNA. The restriction maps of the cloned DNA fragments were identical among the seven clones. They showed identical restriction maps. The deduced amino acid sequence from the determined nucleotide sequence of a clone exhibited an overall similarity to known Mn-SODS and Fe-SODS. Since a Southern analysis of the genomic DNA of M. thermoautotrophicum, by using the cloned fragment as a probe, detected an intense single band (result not shown), the amplified fragment did not originate from a contamination of foreign DNA. Thus we concluded that the PCR-amplified fragments were derived from an SOD gene of M. thermoauto-

212

TAKAO,

OIKAWA,

39 99 159 219 279 339 399 451 51; 5;; 614 6;; 7i: 101 819 121 879 141 939 161 999 181 1059 201 1119 1179 1239 1299 1359 TccGGGnnlGnlTCTGnlGnlClGGnnlnGGGnnnlGG~IGCnln~CnnGG~lG~Cl 1419 GGAGGMCTGCRG

FIG. 2. Nucleotide and deduced amino acid sequences. The nucleotide sequence of the cloned M. thermoautotrophicum Pat1 fragment and the deduced amino acid sequence for SOD is shown. In the upstream region of SOD a putative promoter sequence is boxed and a potential ribosome binding sequence is dotted. An inverted repeat found in the upstream sequence of SOD gene is marked by arrows. The inverted repeat (arrows) near the 3’ end in the SOD gene is a potential termination sequence. In the downstream region two elements forming a direct repeat are indicated by arrows. Double lines are putative start and termination codons of a 138-bp-long ORF present on the opposite strand (see text). A putative promoter and a ribosome binding sequence for the 138-bp ORF are boxed and dotted, respectively.

trophicum. The genomic Southern analysis further suggested that the bacterium has a unique SOD sequence hybridizing to the probe. To obtain the full length sequence and to avoid choosing a wrong sequence due to the error-prone Taq polymerase used in PCR (14) we cloned a 1.4-kb genomic fragment from the PstI library and a 2-kb one from the BgZII-Hind111 library (Fig. 1C). The nucleotide sequence of the cloned genomic DNA was determined as shown in Fig. 2. It contained an open reading frame (ORF) with the expected size of SOD, but starting at GTG. Taking this GTG as the start codon, the N-terminal sequence of the ORF becomes five residues longer than that of H. halobium SOD. No other potential start codon (ATG or GTG), however, exist in the neighborhood. Supporting this start position, an eight

AND

YASUI

nucleotide stretch complementary to the 16 S rRNA 3’ sequence [AUCACCUCCUoH, (15)] was found immediately upstream of the GTG. This sequence is a putative ribosome binding sequence as found in most M. thermoautotrophicum structural genes (15). The deduced sequence of the ORF comprises 205 amino acids with M, of 24,096 (including the first methionine). Although the M. thermoautotrophicum SOD sequence shows the highest resemblance to H. cutirubrum Mn-SOD (43.8%), it is equally similar to Bacillus stearothermophilus Mn-SOD (41.9%), human Mn-SOD (41.8%), and Pseudomonas oualis Fe-SOD (40.5%) as shown in Table I. Ligands for a metal and structurally and functionally important residues are conserved as discussed later. Recently we demonstrated that the H. hulobium SOD gene is cotranscribed with an unknown ORF (ORF151) and a gene encoding photolyase, which repairs pyrimidine dimers produced by uv-light in DNA by using visible light (8). In order to know if the cloned SOD gene is also linked to a photolyase gene in M. thermoautotrophicum, we determined and analyzed a 779-bp sequence upstream of the SOD start codon and 295 bp downstream of the stop codon. The photolyase from this organism was already purified and its molecular weight was reported as 60 kDa (16). No candidate for photolyase ORF was found either in the upstream or in the downstream sequences of the SOD gene in M. thermoautotrophicum. Even in the far upstream region, around BglII and Sal1 sites in Fig. lC, there is no photolyase-like sequence (sequence data not shown). Thus, unlike H. halobium, physical linkage of photolyase and the SOD gene is not the case in the M. thermoautotrophicum genome (Fig. 3A). The putative archaebacterial promoter sequence TTTA(A/T)A (17) was found 72 bp upstream of the start codon of the cloned gene with a slight variation, TTGAAA. We previously found a similar putative promoter sequence TTTAAC starting at 44 bp upstream of H. haZobium sod1 (8). Extensive homology between M. thermoautotrophicum and H. halobium sequences was found just in front of the start codons of the SOD genes as shown in Fig. 3B. The sequence comprises an inverted repeat of GGTGG (CCACC). It should be noted that this sequence, however, was not found in the promoter region of another halobacterial SOD gene (9) and of any other eubacterial SOD gene. In the 3’ flanking region we found a direct repeat starting at 54 bp downstream of the stop codon. One element contains 45 bp and shows perfect identity to the other except for one substitution. Both repeated elements are included in a 138-bp-long ORF found in the opposite strand. A promoter-like sequence AAATTT and a potential ribosome binding sequence AGGAG are situated at 45 bp and 12 bp upstream of the putative ATG start codon of the ORF, respectively (Fig. 2).

Methanobacterium

thermoautotrophicum

SUPEROXIDE

TABLE

DISMUTASE

GENE

213

I

Pairwise Comparison of Mn-SOD and Fe-SOD Sequences (percentage identity)” Arch. (Mn)

Arch. (Mn)

Eub. (Mn)

Euk. (Mn)

Eub. (Fe)

Mt

Hh

Mt Hh Hc

40.9 43.8

82.4

EC BS Tt

40.7 41.9 36.5

SC Hum Zm EC Pl PO

Eub. (Mn) Hc

EC

BS

36.3 40.9 33.5

40.7 41.4 34.0

59.3 53.1

62.6

33.8 41.8 38.4

33.0 37.9 36.2

33.0 38.4 34.3

39.3 44.6 43.0

40.2 36.8 40.5

35.1 35.8 35.8

35.1 35.2 36.3

38.7 42.8 43.3

Euk. (Mn) Tt

SC

Hum

40.2 50.5 42.9

44.1 49.8 46.5

48.6 48.3

55.4

50.2 50.0 52.7

37.8 41.7 42.0

37.1 37.6 35.4

38.7 41.8 43.7

Eub. (Fe) Zm

EC

Pl

34.5 38.5 36.3

75.1 67.9

65.5

PO

-

a Employed sequences are: Mt, Methanobacterium thermoautotrophicum; Hh, Halobacterium halobium; Hc, Halobacterium cutirubrum; Ec(Mn), E. coli sodA; Bs, Bacillus stearothermophilus; Tt, Thermus thermophilus; SC, Saccharomyces serevisiae; Hum, human; Zm, Zea maize: Ec(Fe). ., E. coli sodB: Pl. Photobacterium leiopnathi: PO. Pseudomonas ovalis. Sequence data were taken from the EMBL database and Swiss Plot protein database.

DISCUSSION

Assignment

of Metal Class

The deduced amino acid sequence of the cloned gene is similar to SOD sequences of other organisms (Fig. 4). Although both Mn-SOD and Fe-SOD were derived from a common origin, some residues are specific for respective classes of SOD. On the basis of the amino acid sequences of five Mn-SODS and three Fe-SODS and data from X-ray diffraction of B. stearothermophilus MnSOD (18), five residues have been proposed as primary

, 500bp,

A

3’

5’ -

-

>O.GkRNA

r2.8kRNA

B TTGAAA+39b+--

GGTGG~TAT~GCAAAGTAGGGG~

II II ii\\\ If TTTAAC--@lbp+--CGGTGGATCCACCm -c-

FIG. 3.

//////

Comparison of gene structure. (A) SOD gene structure of M. thermoautotrophicum (top) and H. halobium (bottom). Relative SOD coding regions are coordinated. The position of a putative promoter sequence is indicated by the arrowhead. The longest ORFs in respective frames of the M. thermoautotrophicum sequence (except for SOD) are shown by bold lines. H. hulobium transcripts with lengths of 2.8 and 0.6 kb are indicated by arrows [ref. (8)]. (B) Homology between M. thermoautotrophicum (top) and H. halobium (bottom) SOD promoter regions. The putative promoter is shown in the leftmost sequence. Arrows indicate inverted repeat.

candidates for distinguishing between Mn- and Fe-SOD (19). Three of them (Gly76, Gly77, Phe84) characterizing Mn-SOD are conserved in the M. thermoautotrophicum sequence. The other two residues at positions 154 and 155, which are Gln and Asp, respectively, in most Mn-SODS, and Ala and Gly in all Fe-SODS, are substituted with His and Asn, respectively, in the M. thermoautotrophicum sequence. The Gln-to-His substitution also occurs in H. halobium, H. cutirubrum, and rat Mn-SODS. In most Mn-SODS the residue Gln154 interacts with Tyr34 (18), while the substituted His could also interact with this Tyr. Therefore, this substitution may not change the 3D-configuration of Mn-SOD. As for Asp155 conserved in most Mn-SODS, the ionic pairing with Arg or Lys at position 72 has been proposed (18). The paired substitutions in M. thermoautotrophicum, Asp to Asn at position 155 and the basic amino acid to Ser at position 72, result in a polar interaction, which might partly compensate the ionic interaction between Asp and Arg (Lys). From these aspects, we tentatively assigned the cloned SOD gene to the Mn-type. SODS from anaerobes have been characterized (6) and purified (20-22), including that from the archaebacterium Methanobacterium bryantii (23), but the amino acid sequence has not been determined so far from the anaerobes. All the purified SODS from anaerobic bacteria were classified as the iron-type. If our classification based on the deduced amino acid sequence is true, the sequence is the first example of an Mn-type SOD from an anaerobe. Characterization of Archaebacterial SOD Sequences The sequence alignment (Fig. 4) points out several specific features of archaebacterial SOD. Some func-

214

TAKAO, 10

tin tin nn tin tin tin tin tin tin Fe Fe Fe

tit Hh HC EC BS Tt SC Hum zm EC Pl PO

nn lln nn nn Iln nn tin nn Fe Fe Fe

Ht. Hh HC EC BS Tt SC “Urn zn EC Pl PO

tln lln

nt Hh

tin Ill-8 Hn nn tln nn Fe Fe Fe

EC BS Tt SC Hun zn EC Pl PO

nn

nn

20

OIKAWA,

AND

30

YASUI 4G

50

60

70

SQHELPSLPYDYDALEPHISEQVV

SFELPALPYAKDALAPHISAETIEYHYGKHHQTYVTNLNNLIKGT-AFEGKSLEEII-R------SSEGG AFELPALPFAHNALEPHISQETLEYHYGKHHNTYVVKLNGLVEGT-ELAEKSLEEII-K------TSTGG AFELPPLPYAHDALQPHISKETLEYHHDKHHNTYVVNLNNLVPGTPEFEGKTLEEIV-K------SSSGG

SGUAULVLKSGWAULVVNSGUAULVKDSGUAFIVKNL SGUGULGFNSGUVULALDVFNNAAQVUNHTFYUNCLAP--NAGGEP-TGKVAEAIAASFGSFADFKAQFTDAAIKNFGSGUTULVKN7-TGEVAAAIEKAFGSFAEFKAKFTDSAINNFGSSUTULVKNVFNNAAQVUNHTFYUNCLAP--NAGGE IFNNAAQVUNHTFYUNCLSP--DGGGQP-TGALADAINAAFGSFDKFKEEFTKTSVGTFGSGUAULVK--

nc

SDG-KLAIVSTSNAGTPLTT------DATPLLTVDVUEHAYYIDYRNARPGYLEHFWALVNWEFVAKNLAA ANGS-LAIVNTSNAGCPITEE-----GVTPLLTVDLWEHAYYIDYRNLRPSYMDGFWALVNUDFVSKNLAA ADGS-LALCSTIGAGAPLTS------GDTPLLTCDVWEHAYYIDYRNLRPKYVEAFWNLVNUAFVAEEGKTFKA

FIG. 4. Sequence alignment of Mn-SOD from three kingdoms and Fe-SOD from eubacteria. The alignments are based on the previous one [ref. (S)]. The amino acid positions are numbered from the first residue of E. coli Mn-SOD to facilitate comparison with the data of Parker and Blake (19). Triangles are the proposed residues which distinguish between Mn- and Fe-SOD. Residues of halobacterial Mn-SODS, which are different from those invariant among the other Mn-SODS and Fe-SODS, are boxed. Residues of Mn-SOD invariant exclusively within the same kingdom are marked by white letters. The archaebacterial residue 154 is not marked because the same amino acid is found in rat Mn-SOD (31). Abbreviations of species are the same as in Table I.

tions have been assigned to invariant residues among eubacterial and eukaryotic Mn-SODS and eubacterial FeSODS (19). Four residues defined as metal ligands (His26, Hi&l, Asp175, His179) and another four residues (His30, His31, Trp133, Tyrl81) predicted as composing active sites are all conserved in three archaebacterial sequences. Therefore, the catalytic function and mechanism of the archaebacterial SOD should be quite similar, if not identical, to the eubacterial and eukaryotic SODS. Assuming the cloned gene encodes a Mn-SOD, there are many amino acid residues of Mn-SODS conserved exclusively in each of three primary kingdoms, archaebacteria, eubacteria, and eukaryotes. There are 14 positions for archaebacteria, 18 for eubacteria, and 7 for eukaryotes (Fig. 4). These values are much larger than those found within interkingdom species; numbers of exclusively invairiant residues did not exceed 5 between sequences of species from different kingdoms. Therefore, it is reasonable to assume that the bias of the invariant residue distribution should reflect the limitation

of amino acid changes occurring after branching to the three primary kingdoms. Although lateral transfer from the eubacterial SOD gene has been proposed as an evolutionary root of the halobacterial cognate gene (lo), the result of the above analysis appears to contradict this possibility. Rather, it strongly suggests that archaebacterial SOD is a direct descendant of a primordial enzyme of urkaryote nature.

Conserved Amino Acids in Halobacteria Halophilic lineages are thought to have evolved from an anaerobic methanogen branch (one major archaebacterial branch) following adaptation to high salt environments and aerobic environments (24). Since H. cutirubrum Mn-SOD has optimum activity at 2 M salt (25), while E. coli Mn-SOD and Fe-SOD diminish their activities with increasing ionic strength (26), some amino acid residues of halobacterial SOD should represent a molecular adaptation to the high salt optimum. The deduction of the amino acid sequence of the M. thermoau-

Methanobacterium

thermoautotrophicum

totrophicum SOD gene enabled us to find out the amino acid sequences conserved exclusively in halobacteria. By comparing the amino acid sequences, six residues were found as candidates for the adaptation to high salt conditions as indicated in Fig. 4. These residues of the two halobacteria are different from those invariant among M. thermoautotrophicum and organisms of the other two kingdoms. Interestingly, a substitution of Thr for Lys at position 29 was found in both halobacterial Mn-SODS. An acetylation study (26) indicates that the lysine residue(s) is responsible for the diminished activity of E. coli SODS at increased ionic strength. The structural study (18) suggests that Lys29 is the residue which plays a role of electrostatic guidance to bring 0, to the catalytic moiety. Although the substitution found in halobacteria may cause the loss of this guidance factor, this substitution may be necessitated for the adaptation to a high salt optimum. Upstream Sequences, Candidates for Regulatory Elements Several bacteria are known to have inducible SOD. In H. halobium a 0.6-kb transcript of SOD is induced by aerobic stress [see Fig. 3A and Ref. (S)]. Since M. thermoautotrophicum cells require a strict anaerobic condition to grow, we could not investigate the inducibility of this bacterial SOD by addition of oxygen as we did for H. halobium (8). However, as suggested in the E. coli sodA gene (27), the induction of the SOD gene may be mediated via a regulatory element near its promoter. In the upstream region of both M. thermoautotrophicum and H. halobium SOD genes, there is a palindromic sequence consisting of a GGTGG (CCACC) stem and a loop of two or three A/T. The sequences of these inverted repeats are thus candidates for a transcriptional regulatory element of these archaebacterial Mn-SODS. The inducibility of SOD was recently characterized from another halobacteria, H. cutirubrum and Halobacterium volcani (10). To our surprise, no inverted repeat sequence was found in the promoter region of the H. cutirubrum SOD gene (9). Furthermore, no homologous element was found in the nucleotide sequence upstream of the SOD genes between H. cutirubrum and H. halobium nor between H. cutirubrum and M. thermoautotrophicum. Assignment of authentic regulatory elements awaits the functional analysis of these SOD promoter regions. Gene Organization The tandem arrangement of the photolyase and SOD genes seen in H. halobium (8) was not found in the neighborhood of the M. thermoautotrophicum SOD gene, or other bacterial SOD or photolyase genes. The arrangement of both genes in H. halobium may be acquired after halophilic lineages evolved from the methanogen branch

SUPEROXIDE

DISMUTASE

GENE

215

(24). The deduced amino acid sequence of potential ORF upstream of H. cutirubrum SOD analyzed from the reported sequence (9) does not show homology to any known photolyases, indicating that the photolyase gene of H. cutirubrum should be present at a different locus. [H. cutirubrum also exhibits photoreactivation (28) and thereby possesses a photolyase gene.] These facts suggest that the tandem gene arrangement of H. halobium occurred as a relatively recent event or there is another SOD gene in Hxutirubrum (9), which is tandemly arranged with the photolyase gene as in H. halobium. Our recent result of a Southern analysis suggests the latter case. Evolutional

Origin of SOD in Anaerobic Organisms

A high similarity in the primary structure of SODS among strictly anaerobic and aerobic archaebacteria, eubacteria, and eukaryotic mitochondria strongly suggests their common origin. According to geological estimation (29), the partial pressure of oxygen in mid-Precambrian period was about 3 Pa. The aqueous oxygen concentration in equilibrium with the atmospheric oxygen at the time is calculated, using oxygen solubility, as 40 nM at 20°C (22 nM at 1OOOC).Because superoxide could be produced from molecular oxygen photochemically by solar radiation and chemically by catalytic action of flavins possibly present in the ancient organisms, and because, according to Tyler’s estimation (30), the intramitochondrial concentration of superoxide in the present-day organisms is kept around 8 pM by Mn-SOD, our results suggest that organisms in mid-Precambrian period were already under evolutional pressure which forced the creation of SOD activity. ACKNOWLEDGMENTS We thank Drs. Y. Koga and S. Takayanagi for providing us archaebacterial cells. Technical assistance by Mr. K. Horaguchi and Miss J. Kikuchi is acknowledged. REFERENCES 1. Fridovich, I. (1974) Adu. Enzymol. 41,35-97. 2. Hassan, H. M. (1988) Free Radic. Biol. Med. 5,377-385. 3. Touati,

D. (1988) Free Rudical Biol. Med. 5,393-402.

4. Hewitt,

J., and Morris,

5. Carlsson, J., W&hen, biol. 6,280-284.

J. G. (1975) FEBS I&t. J., and Beckman,

6. Gregory, E. M., Moore, W. E. C., and Holdernan, Environ. Microbial. 35,988-991. Salin, M. L., Duke, M. V., Oesterhelt, Gene 70,153-X9. Takao, M., Kobayashi, T., Oikawa, Bucteriol. 171,6323-6329.

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35,171-175. Dale, J. P., and Greenaway, J. P. (1984) in Methods in Molecular Biology (Walker, J. M., Ed.), Vol. 2, pp. 197-200, Humana Press, NJ. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491. Sanger, F., Nicklen, S., and Coulson, A. (1977) PFOC. N&Z. Acad. Sci. USA 74,5463-5467. Tindall, K. R., and Kunkel, T. A. (1988) Biochemistry 27,60086013. Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R., and Wolfe, R. S. (1979) Microbial. Rev. 43,260-296. Kiener, A., Husain, I., Sancar, A., and Walsh, C. (1989) J. Biol. Chem. 264,13,880-13,887. Zillig, W., Palm, P., Reiter, W.-D., Gropp, F., Piihler, G., and Klenk, H.-P. (1988) Eur. J. Biochem. 173,473-482. Parker, M. W., and Blake, C. C. F. (1988) J. Mol. Biol. 199,649661. Parker, M. W., and Blake, C. C. F. (1988) FEBS Lett. 229,377382.

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20. Hatchikian, 161. 21. Kanematsu,

E. C., and Henry,

Y. A. (1977) Biochimie

S., and Asada, K. (1978) Arch. Biochem.

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S., and Asada, K. (1978) FEBS Z&t. 91,94-98.

23. Kirby, T. W., Lancaster Jr., J. R., and Fridovich, Biochem. Biophys. 210,140-148. 24. Woese, C. R. (1987) Microbial. 25. May, B. P., andDennis,

Reu. 51,221-271.

P. P. (1987) J. Bacterial.

26. Benovic, J., Tillman, T., Cudd, A., and Fridovich, Biochem. Biophys. 221,329-332. 27. Takeda,

Y., and Avila,

I. (1981) Arch.

H. (1986) Nucleic

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