Gene, 96 (1990) 149-150 Elsevier

149

GENE 03817

The

superoxide dismutase-encoding gene of the obligately anaerobic bacterium Bacteroides gingivalis*

(Recombinant DNA; met~oenzyme; oxidative stress; transposon mutagenesis; genetic complementation; regional divergence; evolution)

Koji Nakayama Department of Microbiology, Faculty of Dentistry, K yushu University 61, Fukuoka 812 (Japan) Received by Y. Sakaki: 11 June 1990 Revised: 13 August 1990 Accepted: 22 August 1990

SUMMARY

The gene (sod) encoding the superoxide dismutase (SOD) of the obligately anaerobic bacterium Bacteroides gingivalis was cloned. The amino acid (aa) sequence of the SOD, deduced from the nucleotide sequence of the sod gene, basically resembled that of known Fe-SODs. However, the aa sequence of the B. gingivalis SOD was found to be intermediate between those of Fe-SOD and Mn-SOD in a limited region around the putative second ligand, where major differences between the aa sequences of Fe-SOD and Mn-SOD are known to exist.

B. gingivalis, an obligately anaerobic bacterium, possesse~ a unique SOD, which is activated by either Fe or Mn (Amano et al., 1990). This class of SOD may represent an evolutionary intermediate of Fe- and Mn-gODs (Hassan, 1989). To clone and sequence the sod gene, recombinant plasraids contedning i.t were isolated from a chromosomal B. gingivalis ATCC33277 gene library using the genetic complementation of an Eschen'chia coli sodA sodB double mutant for aerobic growth on minimal medium (Carlioz and Touati, 1986). Restriction analysis ol'the recombinant plasraids (pKD210, pKD211 and pKD212) revealed that the cloned fragments in these plasmids shared a common segCorrespondence to: Dr. K. Nakayama, Department of Microbiology, Faculty of Dentistry, K~ushu University 61, 3-1-1 Maidashi Higashi-ku, Fukuoka 812 (Japan)Te1.(81-92)641-1151, ext. 4253; Fax(81-92) 641-3770. * On request, the author will supply detailed experimental evidence for the conclusions reached in this brief note. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000 bp; nt, nucleotide(s); SOD, superoxide dismutase; sod, gene encoding SOD; "In, transposon. 0378-1119/90/$03.50 © 1990 Elsevier SciencePublishers B.V.(BiomedicalDivision)

ment. Determination of SOD activity and paraquat sensitivity of the sodA sodB mutants containing the plasmids confirmed the expression of the sod gene in E. coll. TnlO00 mutagenesis and subcloning of pKD210 located the sod gene to a region of approx. 1 kb covering a 0.45-kb EcoRlBgilI fragment within the common segment. The nt sequence of a 734-bp region covering the EcoRl,BgllI fragment was determined (Fig. 1). A single open reading frame with the capacity of encoding a 191-aa protein was found, the ATG start codon and the stop codon being located at nt 100 and nt 673, respectively. Comparison of the deduced aa sequence with those of Fe- and Mn-SODs in eubacteria reported so far revealed that it had several characteristics of Fe- and Mn-SODs. (1) The sequence consists of 191 aa, in accordance with the fact that Fe- and Mn-SODs are composed of about 200 aa; (2)the aa residues which are supposed to serve as metal ligands (His 27, His 74, Asp ts7 and His ~61of the B. gingivalis SOD) are found in the typical positions in the aa sequence; (3)several other highly conserved residues of Fe- and Mn-SODs, which are dispersed in their aa sequences, are also seen in the aa sequence of the B. gingivalis SOD. Recently, Amano et al. (1990) have reported that three

150 1o

20

30

40

50

60

CATCATGTTCTTATA.~TGATTAAAAAGTTGTCGGTGTCGGTTTTACACTGCTCCGATACA 70 80 90 [00 IlO 120 AATTATTAAAAGACTTAATTACAATAAAAATCAGACGTTATGACTCACGAACTCATTTCC MetThrHisGluLeulleSer 5 130 140 150 160 170 i180 CTGCCTTATGCGGTCGATGCACTGGCTCCTGTTATCbGCAAAGAAACAGTGGAATTCCAC LeuProTy~AlaValAspAlaLeuAlaProVallleSerLysGluTh~ValGluPheUis 10 15 20 25 190 200 210 220 230 240 CACGGTAAGCACCTGAAGACCTATGTGGACAACCTCAATAAGCTCATCATCGGCACGGAA HisG1yLysHisLeuLysThrTyrValAspAsnLeuAsnLysLeulleIleG1yThrGlu 30 35 40 45 250 260 270 280 290 300 TTTGAAAACGCAGACTTGAATACCATCGTACAAAAGAGCGAAGGCGGTATCTTCAACAAT PheG1uAsnA1aAspLeuAsnThrIleVaIGlnLysSerGluGlyGlyI1ePheAsnAsn 50 55 60 65 310

320

330

340

350

360

GCCGGCCAAACCCTCAACCACAATCTCTATTTCACTCAGTTCCGTCCGGGCAAAGGAGGA AlaGlyGlnThrLeuAsnHisAsnLeuTyrPheThrG1nPheArgProGlyLysGlyGly 70 75 80 85 370 380 390 400 410 420 GCACCGAAAGGCAAACTGGGGGAAGCTATCGACAAACAATTCGGCTCATTCGAAAAGTTC AlaProLysGlyLysLeuGlyGluAlalleAspLysGlnPheGlySerPheGluLysPhe 90 95 100 105 430 440 450 460 470 480 AAAGAGGAGTTCAACACAGCCGGTACCACCCTCTTTGGTTCGGGCTGGGTATGGCTTGCA LysGluGluPheAsnThrAlaGlyThrThrLeuPheGlySerGlyTrpValTrpLeuAla 110 115 120 125 490 500 510 520 530 540 TCCGATGCCAATGGCAAACTGTCCATCGAGAAGGAACCCAATGCCGGCAATCCCGTGCGC SerAspAlaAsnGlyLysLeuSerIleGluLysGluProAsnAlaGlyAsnProValArg 130 135 140 145 550 560 570 580 590 600 AAAGGGTTGAACCCTTTGCTCGGATTCGACGTATGGGAGCACGCATATTkTCTGACTTAC LysGlyLeuAsnProLeuLeuGlyPheAspValTrpGluHisAlaTyrTyrLeuThrTyr 150 155 160 165 610 620 630 640 650 660 CAGAATCGTCGTGCCGACCACCTCAAAGATCTTTGGAGTATCGTTGACTGGGATATTGTA G1nAsnArgArgAlaAspHisLeuLysAspLeuTrpSerIleVaIAspTrpAspI1eVal 170 175 180 185 670 680 690 700 710 720 GAATCTCGGTATTAAGAACTCCATGGGTGCACTTTGCACCAATACATAAGGAAGCCTCTG

GluSerArgTyr*** 190 730 CCAAGAACCGATCG

740

Fig. I. Nucleotide sequence of B. gmgivalissod gene. D N A ~agments of pKD210 were excised with the restrictionenzymes BglII,EcoRI, and Kpnl, and cloned into the appropriatesitesof pUCI8 for sequencing by the dideoxy chain-terminationmethod using an D N A sequencingkitwith SequenaseT M (United States Biochemical, Cleveland, O H ) and [a-3SS]dCTP (> 37 TBq/mmol, Amersham Jap~m, Tokyo, Japan). Both strands were sequenced. The lastdigitsof the numerals are alignedwith the corresponding nt. The sequence data bare been submiRed to DDBJ/EMBL/GenBank under accession number/.)~i~2,~

SOD isozymes found in B. gingivalis are formed from a single apoprotein, and that the sequence of the N-terminal 12 aa of the purified protein was Met-Thr-His-Glu-Leu-IleSer-Leu-Pro-Tyr-Ala-Val. This sequence is identical to the aa sequence deduced from the nt sequence of the sod gene reported here. It is thus higi'dy likely that the SOD apeprotein the~ have purified is the product of the sod gene described here. They also mentioned in their paper that the apoprotein, prepared by dialysis in the presence of guanidium chloride plus 8-hydroxyquinoline to remove the

metal cofactor from SOD, is reactivated by either Fe or Mn. On the other hand, it was proposed that the interdomain relocation of an active-center Gin residue (Gin 7° and Ala 14a in Fe-SODs correspond to Gly and Gin in Mn-SODs, respectively), or the following substitutions (Gly69 ~ Ala, Leu76---~ Phe, and pho77---, Tyr) at the active site, around the second ligand, could account for the specificity for the metal cofactors (Caflioz et al., 1988; Parker and Blake, 1988). Judging from the position of the active-center Gin residue, identity of the highly conserved residues, and the absence of a spacer region between aa 58 and 59, the B. gingivalis SOD seems to be basically of the Fe-type. However, its aa sequence around the third and fourth ligands is more similar to that of Mn-SODs. Furthermore, its aa sequence is intermediate between those of Fe-SOD and Mn-SOD in the vicinity of the second ligand, where a considerable difference is seen between Fe-SODs and Mn-SODs. Thus, Gly 69 and Leu 76 in the B. gingivalis SOD are identical to the corresponding residues of Mn-SODs, whereas Gin ~° and Tyr ~7 are identical to those of Fe-SODs. In addition, Phe 78 in the B. gingivalis SOD corresponds to Trp at the corresponding sites of Fe- and Mn-SODs. This characteristic sequence around the second ligand may be related to the weak selectivity ofB. gingivalis SOD for metal co factors.

ACKNOWLEDGEMENTS

I thank Drs. S. Yonei and F. Yoshimura for the kind gifts of bacterial strains and Prof. H. Nakayama for his helpful advice and critical reading of the manuscript. I am also grateful to Ms. C. Yoshida for general laboratory support.

REFERENCES Amano, A., Shizukuishi, S., Tamagawa, H., Iwakura, K., Tsunasawa, S. and Tsunemitsu, A.: Characterization of superoxide dismutases purified from either anaerobically maintained or aerated Bacteroides gingivalis. J. Bacteriol. 172 (1990) 1457-1463. Carlioz, A. and Touati, D.: Isolation ofsuperoxide dismutase mutar~ts in E.~'cherichia coil: is superoxide dismutase necessary for aerobic life? EMBO J. 5 (1986) 623-630. Carlioz, A., Ludwig, M.L., St~dlings, W.C., Fee, J.A., Steinman, H.M. and Touati, D.: Iron superoxide dismutase: nucleotide sequence of the gene from Escherichia coil K12 and correlations with crystal structures. J. Biol. Chem. 263 (1988) 1555-1562. Hassan, H.M.: Microbial superoxide dismutases. Adv. Genet. 26 (1989) 65-97. Parker, M.W. and Blake, C.C.F.: Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett. 229 (1988) 377-382.

The superoxide dismutase-encoding gene of the obligately anaerobic bacterium Bacteroides gingivalis.

The gene (sod) encoding the superoxide dismutase (SOD) of the obligately anaerobic bacterium Bacteroides gingivalis was cloned. The amino acid (aa) se...
235KB Sizes 0 Downloads 0 Views