Cloning, sequencing, expression and characterization ... - iubmb - Wiley

Vol. 47, No. 5, May 1999

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL pages 803-814

CLONING, SEQUENCING, EXPRESSION AND CHARACTERIZATION OF THE MANGANESE SUPEROXIDE DISMUTASE GENE FROM VIBRIO ALGINOLYTICUS Yu-Chiau Shyu, Chi-Chien Chiu and Fu-Pang Lin'

Institute t?fMarine Biotechnology, National Tairvan Ocean University, I(eelung, Tairvan 202, R.O.C. "To whom correspondence should be addressed: Institute of Marine Biotechnolog3~ National Taiwan Ocean University, No. 2 Pei-Ning Rd., Keelung 202, Taiwan, R. O. C. Tel: 886-2-24622192 Ext. 5505; Fax: 886-2-24622320; E-maih [email protected]

Summary The soda gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector~ The flail-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the soda showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60~ and was insensitive to inhibitors such as H202, NaN3 and diethyldithiocarbamic acid. Key Words: Mn-superoxide dismutase (Mn-SOD), Vibrio alginolyticus.

Introduction Superoxide dismutases (SODs ; EC 1.15.1.1 ) are metalloproteius that catalyze the dismutation of superoxide radical ( 0 2 - ) to oxygen and hydrogen peroxide against oxidative damage(I). Three types of SODs with either copper/zinc, manganese, or iron as a prosthetic metal have been identified (2).

Biochemical characteristics of these three SOD isozymes have indicated

that CuZn-SODs are sensitive to CN', diethyldithioearbamie acid and H202, but insensitive to azide, whereas Mn-SODs are resistant to CN-, diethyldithiocarbamic acid, H202and azide inhibition, Fe-SODs are insensitive to CN" and diethyldithiocarbamic acid, but sensitive to H202 and azide inhibition. The different inh~ition patterns by these inhibitors make it possible to distinguish which type of SOD from crude homogenates (3). SODs are present in both prokaryotes and eukaryotes in aerobic or anaerobic environments. In general, CuZn-SOD is mainly found in eukaryotes, Mn-SOD is present in both bacteria and eukaryotic mitochondria, and Fe-SOD is found in both eubacteria and arehaebacteria. The considerable amino acid homology and similar crystal structure of Mn-SOD and Fe-SOD indicated that both types of these two SODs originated from a common ancestor, whereas CuZn-SOD is distinct and structurally unrelated to both Mn-SOD and Fe-SOD (4, 5). 803

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Vol. 47, No. 5, May 1999

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Vibriosis has been determined as one of the major threats in fish and shellfish marine aquaculture (6,7). Various Vibrio species have been reported to cause large die-offs in cultured penaeide in Taiwan (8-12). The white spot syndrome (WSS) in the carapace has been found as the major symptom of the 1994 suii~a~eroutbreaks in Taiwan. And Vibrio alginolyticus was thought to be a major role inthe die-offofthis WSS syndrome (12). The isolation, characterization and virulence of a pathogenic F. alginolyticus from diseased tiger prawns with WSS syndrome in the carapace has been reported (13), An extraceUular zlk~llne protease of F. alginolyticus was reported as a virulent factor for these die-offoutbreaks. F. alginolyacus is not only a fi~h pathogen bacteria but also known as a human pathogenic Fibrio.

DNA manipulations Chromosomal DNA from F. alginolyticus was prepared by the method as desenq~ed by Marmur (15). Plasmid DNA preparation, restriction analysis and PCR were performed by the standard techniques (16, 17). DNA was recovered from agarose gel with Qiaquiek gel extraction kit (Qiagen, Germany). Recombinant plasmids eoustmetion, DNA ligation and transformation were carried out by the methods described by Ausubel et. al., and Sambrook et. al. (16, 17). Cloning of the soda gene by PCR A pair of degenerate primers encoding the well conserved region of known Mn-SODs were designed and synthesized with an Applied Biosystems 380A DNA synthesizer. Genomie DNA was mixed with the degenerate Mn-SOD primer pairs (primer 1 and 2) and subjected to a PCR amplification with 35~40 cycles. The temperatures of 55~ and 40~ were selected as the high and low annealing temperature, respectively. Amplified PCR products were analyzed by 0.8% agarose gel eleetropboresis and DNA fragments were recovered from the gel by the Qiaquiek gel extraction kit (Qiagen, Germany). The isolated and purified DNA fragments were cycle sequenced with a forward and a reverse sequencing primer by the Sanger's dideoxy chain-termination method (18) using the Thermo-Sequenase kit (Amersham, Ohio, USA). The sequence data were analyzed by using Sequence Analysis Sofrware Package of the Genetics Computer Group, version 8.0.1 (University of Wiscousin, USA) (19). Two gene specific primers (primer 3 and primer 4) were then designed based on this nucleotide sequence of the DNA fragment amplified from the degenerate Mn-SOD primer pairs. The full-length of F. alginolyticus soda was accomplished by applying the technique of random primed gene walking PCR (20). Expression of F.. alginolyticus sodA gene in E. coli Plasmid pVAsodA was constmeted by inserting a PCR product encoding a E alginolyticus full-length soda gene genomie DNA using the N- and C- terminal primer pairs (primer 5 and primer 6) into the pET20b (+) vector at the NdeI and XhoI sites in which the N- and C- terminal of the Mn-SOD were located, respectively. E. coli BL21 (DE3) pLysS cells harbouring plasmid pVAsodA were induced by 0.1raM isopropyl-13-D-thiogalactopyranoside 0PTG) at midexponential phase (OD~00 0.4-0.6) and incubated for further 2 h at 37~ (21). The cells (from a 50 ml culture) were centrifuged, washed and resuspended in 6ml binding buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaC1, 5 mM imidazole). Cell lysates were obtained by sonicating in a Sonieator XL2020 (Heat System-Ultra Sorties, lne) at 20~30% power for 3 rain at 4~ Cell debris was removed by eenlrifugation at 10,000xg for 15 mill and the protein concentration was measured by the dye-binding method of Bradford (22), with bovine serum albumin (Bio-Rad Laboratories) as 804

Vol. 47, No. 5, May 1999

BIOCHEMISTRY a n d MOLECULAR BIOLOGY INTERNATIONAL

the standard. The supernatant fraction was applied to a His-Bind Resin column (Novagen, Madison, W'ts. USA) and the recombinant M_n-SOD was purified by the metal ion affinity chromatography (23). The homogeneity of purified VAMn-SOD was estimated by 12% SDSPAGE (24).

Enzyme assay SOD activity was determined by the xa~thine oxidase-2- (4- iodo-phenyl)-3- (4nitrophenol)-5-phenyltetrazolinm (I.N.T.) method (25). Unit of SOD activity was measured by comparing with that of standard SOD provided from the RANSOD kit (RANDOX Laboratories. Ltd. UK. ). The unit of SOD activity was defined as a half-maximal inh~ition ofthe rate of xanthine oxidase mediated reduction ofnitroblue tetrazoliun~ SOD activity was also visualized after eleetrophoresis in nondenaturing polyacrylamide gels as an achromatic zone from inh~ition o f photochemical reduction ofnitroblue tetrazolium to formazan blue (26). Recently PCR( polymerase chain reaction) has been used as a diagnostic tool for several human pathogenic Vibrio species identification. PCR-methods targeting enterotoxin genes (ctxA,

ctxB) of Vibrio cholera, hemolysin genes (tdh,trh) of

Vibrioparahaemolyticus and cytolysin

gene (hlyA) of Vibrio vulnificus have been reviewed by Olsen et. al. (14). To investigate the possibility of employing the superoxide dismutase gene as a strain specific gene probe instead of the tedious and time-eousumlng conventional test for Vibrio strain identification, it was attempted to clone the sod gene from several Vibrio species. A partial sodA gene (Mn-SOD gene) from several Vibrio species was obtained sueeessfuUy by PCR with degenerate primers designed from the eouserved regions among known Mn-SODs (F-P, Lin, unpublished results). In this paper, the complete sodA gene from V. alginolyticus was cloned, sequenced and expressed in the heterologous E. cob host. The recombinant V. alginolyticus Mn-SOD (VAMn-SOD) with a Histagged C-terminal was efficientlypurified from the crude E. coli cell lysate by the metal ion affinity chromatography. The biochemical characteristics of this recombinant VAMn-SOD was presented.

Materials and Methods

Bacterial strains and plasmids Microorganism was isolated from the marine coast ofKeelung, Taiwan. I~brio alginolyticus was identified according to the procedures of Bergey's Manual of Bacteriology and was used as a source of chromosomal DNA for soda gene cloning. Escherichia coli Novablue { end~l hsdR17 (rk12-n~12+) supE44 thi-1 recA1 gyrA96 relA1 lac IF' proA+B+lacIqZ /% M15:: TnlO(TetR)]} (Novagen, Madison, W'ts. USA), was used for the cloning experiments, and E. coli BL21 (DE3) pLysS [F-opmT hsdSB (rs-n~-)gal dcm (Lclts857 indl Sam7 nin5 lacUV5-T7 genel) pLysS (CmR)] (Novagen, Madison, Wis. USA) was used for the expression and purification of the recombinant VAMn-SOD. The plasmid pET20b (+)(Novagen, Madison, W'ts. USA) was used as both the cloning vector and the expression vector. 805

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Media and growth conditions V. alginolyticus was grown in L-broth (1% peptone, 0.5% yeast extract, 1% NaC1) at 25 ~ for 36 hrs. E. coli were grown in the same medium except the growth temperature was 37~ The ant~iotic of ampicillin was used at 100 ~tg/ml. Thermostability studies The purified recombinant enzyme (5 units, SOD) was incubated at various temperatures in 20 mM Tris-HC1 buffer (pH 7.9) for 10 rain and the residual SOD activity was immediately measured by the method described above (25). Inactivation by Inhibitors Purified enzymes (4 units, SOD) were treated with various inhibitors at different concentrations in 20 mM Tris-HCl buffer (pH 7.9) at 25~ for 40 min. The remaining SOD activity was monitored by the activity assay as described (25). Nucleotide sequence accession number The nucleotide sequence reported here has been deposited in the GenBank data base under the accession number AF013768. Results

Cloning and sequencing of the V. alginolyticus soda gene Nucleotide sequences of two degenerate Mn-SOD primers (primer 1 and primer 2) as well as gene specific primers (primer 3-6) were shown in Table 1. A gene fragment of approximate 0.5 Kb was produced at the annealing temperature of 45~ by 35 cycles PCR using the primerl and primer 2 (Fig. 1, lanel). The nucleotide sequences of this 0.5 Kb DNA was obtained by cycle sequencing and was analyzed by GCG, version 8.01 software against the GenBank database. A partial open reading frame was deduced and exhibited a considerable amino acid homology to known Mn-SODs and Fe-SODs (data not shown). The full-length gene was then completed by the combination of gene specific primers (primers 3, primers 4) and the method of Random Primer Gene Walking PCR (Fig. 1, lane2).

Nucleotide and deduced amino acid sequence analysis The nucleotide sequence of the full-length E alginolyticus soda DNA revealed a complete ORF of 603 bp encoding a polypeptide of 201 amino acids with a calculated Mr of 22672 Da. A typical bacteria ribosome binding site (Shine-Dalgamo sequence) (27), AGGAGG, was found 9 bp upstream from the presumptive start codon (ATG). A palindromic loop was located 13 bp downstream from the stop codon (TAA) and might be acting as a transcriptional terminator (Fig. 2). A search of GenBank database revealed that the cloned V. alginolyticus M_n-SOD gene showed a significant amino acid homology to other reported Mn-SOD and Fe-SOD genes (Table 2). Extremely identical amino acid sequences conservation were found in the regions for a metal ligand binding, the structural and functional residues of Mn-SOD or Fe-SOD proteins (Fig. 3). 806

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Table 1. Oligonucleotides used for V. alginolyticus sodA cloning )rimer

aucleotide sequence

( 5' ~ 3' )

~rimer 1 VIA CC(A/T) TA(T/C) (C/G)C(T/A) TA(T/C) GA(T/A) GC(T/A) "VIA GAA CCA CAT AT primer 2 AA (A/G) TA(A/G) TA(A/T) GC(A/G) TG(T/C) TCC CA(A/T) ACA TC primer 3 GTG TTG TGG TGT TTA TCA TGA TGC primer 4 CAC TTC AAC CGC GAA TCA AGA TTC ACC A primer 5 GGA ATT CCA TAT GAC TTA CAC ATT ACC AG iprimer 6 CCG CTC GAG AGC T I T TGC GAA ATA TTC GTT TAC TTT GTC CCA G

Table 2. Pairwise Comparison of Mn-SOD and Fe-SOD Sequences (percentage identityf Eub.(Mn)

Eub.(Mn)

Arch.(Mn) Euk.(Mn)

Eub.(Fe)

Va Bs Tt Ee Hh He Sc Hum Zm Ee PI Po

Arch.(Mn)

Euk.(Mn)

Va

Bs

Tt

Ec

Hh

He

Se

67.8 59.5 57.4 41.8 40.8 41.3 51.0 48.1 50.8 46.5 51.3

62.6 59.3 33.5 40.9 40.2 50.5 42.9 50.2 50.0 52.7

53.1 33.5 34.0 44.1 49.8 46.5 37.8 41.7 42.0

36.3 40.7 39.3 44.6 43.0 38.7 42.8 43.3

82.4 33.0 37.9 36.2 35.1 35.8 35.8

33.0 38.4 34.3 35.1 35.2 36.3

48.6 48.3 37.1 37.6 35.4

Hum Zm

Eub.(Fe)

Ec

P1

Po

55.4 38.7 34.5 41.8 38.5 75.1 43.7 36.3 67.9 65.5

"Employed sequences are: Va, Hbrio alginolyticus; Bs, Bacillus stearothermophilus; Tt, Thermus thermophilus; Ee (Mn), E. coli sodA; Hh, Halobacterium halobium; He, Halobacterium cutirubrum; So, Saccharomyces cerevisiae; Hum, human; Zm, Zea maize; Ec (Fe), E. coli sodB; P1, Photobacterium leiognathi; Po, Pseudomonas ovalis. Sequence data were taken from the EMBL database and Swiss Prot protein database.

Expression and purification of the recombinant VAMn-SODfrom E. cob SDS-PAGE analysis of protein extract obtained from in vivo expression of VAMn-SOD in

E. cob indicated that an induced protein with an apparent Mr of 27 KDa, which is closely matched to the one calculated from the deduced ORF. An abundant recombinant VAMn-SOD protein was produced after 0.1 mM IPTG induction for 2 hr at 37~

(Fig. 4, lane 4). There was no significant

effect on protein induction level by the increase concentration of IPTG (Fig.4, lane5-7). An efficient one step affinity purification using the His-Bind Resin for the recombinant VAMn-SOD and SOD activity staining on native PAGE were presented (Fig. 5A and B). 807

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0/ Fig.1. Cloning of Vibrio alginolyticus soda gene. Agarose gel electrophoresis at~er PCR amplification of E alginolyticus genomie DNA.Using a set of primer 1 and primer 2, PCR was carried out by using genomic DNA from V. alginolyticus (lane 1). The PCR product of flaillength soda was amplifted by primer 5 and primer 6 (lane2). L HindnI marker (lane M).

Biochemical characterization of the recombinant VAMn-SOD The pttrified VAMn-SOD recombinant enzyme was consisted of one type of subunit of Mr 27 kDa as estimated by SDS-PAGE (Fig. 4, lane4). The recombinant VA_Mn-SOD was fully active at temperatures range from 25~ to 60~ (Table 3). lnba"oitorsof SOD did not affect significantly on the VAMn-SOD activity at severaldifferent concentrations tested (Table 4). Discussion

The soda gene cloned from V, alginolyticus exhibited the protein with the overall homology to other Mn-SODs from Bacillus stearothermophilus (67.8%), Thermus thermophilus (59.5%).

E.coli (57.4%) (Table2). The distinct biochemical characteristics of Mn-SOD such as the resistance to inlu~oitors,NAN3, I-I202 and diethyldithiocarbamate was found in the recombinant VAMn-SOD protein encoded from the cloned soda gene. Furthermore, the recombinant VAMnSOD showed a significant resistance to 5 mM KCN inhibition (data not shown), implicating the VAMn-SOD might be a Mn-typr SOD. Although the N-terminal amino acid sequences of the recombinant VAMn-SOD has not been determined, the Mr. of the recombinant the VAMn-SOD 808

Vol. 47, No. 5, May 1999


TTTTTTTTC GTCGTATATCATTAGGCTTGACATGCTCGAGTATTTCTTAATCTCAGTTTTAGCTACCGGAGATCTTGTTGCCAAGCTGG TCAC~CAAGTCGTTGTTGTTTTGGCAAATTATGTGTTTAGTAAATTTTTGAT7TTCAATACGA~AAAAAGTGAGAAAAAG TAAGCATATTTTTTGAACAAAGTCTGATAGTCTGATAATGTTACTTATGTAAACATTATCATTGAGGAGGATTTTTTTT ATG ACT TAC AEA TTA CCA GAT TTA CCT TAC GCA TAC GAT GCA TTG GAA CCA TAT ATT GAT M T Y T L P D L P Y A Y D A L E P Y l D GAA GAA ACG ATG CAT TTG CAT CAT GAT AAA CAC CAC AAC ACT TAT GTA ACA AAC TTA AAT E E T M H L H H D K H H N T Y V T N L N GCA GCG ATT GAA AAA CAT CCT GAA TTA GGT GAA AAA ACA GTT GAA GAA TTA TTA GCA GAC A A I E K H P E L G E K T V E E L L A D TTT TCT TCT GTA CCT GAA GAT ATT CAA ACA GCG GTT CGC AAC AAT GGC GGC GGC CAT GCT F S S V P E D [ Q T A V R N N G G G H A AAC CAC ACG TTC TTC TGG GAA ATC TTA GGC CCA AAT GCC GGT GGC GAA CCT ACT GGG GCA N H T F F W E [ L G P N A G G E P T G A ATC AAA GAG GCA ATT GAA ~AA ACT TTT CGC AGC TTT GAA GAC TTT AAA GAA GAA TTT AAA I K E A I E E T F G S F E D F K E E F K ACT GCT GCA ACT GGA CGT TTT GGT TCA GGT TGG GCA TGG TTA GTT GTT AAA GAC GGT AAA T A A T G R F G S G W A W L V V K D G K CTA GCA ATC ACT TCA ACC GCG AAT CAA GAT TCA CCA TTG ATG GAT GGT CAA ACA CCT GTA L A t T S T A N Q D S P L M D G Q T P V TTA GGT TTA CAT GTT TGG GAA CAT GCG TAT TAC TTA AAA TAC AAA AAT GTT CGT CCA GAT L G L D V W E H A Y Y L K Y K N V R P D TAC ATC AAT GCT TTC TGG ACT CTT GTT AAC TGG GAC AAA GTA AAC GAA TAT TTC GCA AAA Y I N A F W S V l N W D K V N E Y F A K GCT TAA ACAAACTATAGGAAGAAGf~CTGACTAAG~CTTCTTTTTTGTGGATAGAAAGAAATTTCGTTATTGCTA A * ACGCTTACATAATCCGCTATAATGAAATCGACGGAA

Fig.2. Nucleotide and deduced amino acid sequences o f l~. alginolyticus Mn-SOD gene. In the upstream region of Mn-SOD gene, a putative nq~osome binding site was underlined. The start and stop eodons of the 201 amino acid Mn-SOD gene were indicated (in boldface). A putative palindrome structure was indicated by the arrow head symbol.

estimated by SDS-PAGE was 27KDa which is consistent to the calculated Mr. from the deduced ORF of the cloned sodA gene. Since only two types of SOD have been reported in Vibrio species and the amino acid sequence homology between the VAMn-SOD and the reported Mn-SODs from the GenBank, as well as the major biochemical characterization of the VAMn-SOD on the thermostability and sensitivity to inhibitors, it was concluded that the gene cloned from V. Since the vibriosis has been a major threat in fish and shellfish marine aquaculture (6,7), a fast and reliable Vibrio strain identification method was necessary to improve the traditional 809

Vol. 47, No. 5, May 1999


I

[0

20

30

40

50

60

WTYTLPD LPYAYDALEPYIDEETWHL~HDKHHNTYVTNLNAMEKHPELGEKTVEELLADFSSVPED PFELPA LPYPYDALEP ~IDKETINIH ~TKHHNTYVT NLNAALEGIIPDLQNKSLEELLSNLEALPES HIDAKTWEIll HQKHHGAYVTNLNAALEKYPYLHGVEVEVLLRHLAALPQD Tt ................................ PYPFKLPD LGYPYEALEP Ec .................................. SYTLPS LPYAYDALEPHFDKQTWEIH~TKHHQTYVN NANAALESLPEFANLPVEEL ITKLI)QLPAD AENR ETGDHASTAG Hh ................................ WSQHELPS LPYDYDALEP IIISEQVVTWI[ HDTHHQSYVD GLNSAEETL. . . . . . . Hc ................................. SEYELPP LPYDYDALEP filSEQVLTIH HDTImQ(;YVN GINDAEETL. . . . . . . AENR ETGDHASTAG Sc ........ iF AKTAAANLTKKGGLSL~TT ARRTKVTLPDLK~DFGALEP Y|SGQINELH YTKHHQTYVN GFNTAVDQFQELSDLLAKEPSPANARKilA Hum .......... ~LSRAVCGTSRQLAP~LGYLGSRQKHSLPDLPYDYGALEP HINAQI~QLH HSKHHAAYVNNLNVTEEKYQ EAL. . . . . . . AKGDVTAQ[A Zm WALRTLASKKVLSFPFGGAGRPLAAAASARGVTTV.TLPD LSYDFGALEP AISGEIWRLH HQKHHATYVANYNKALEQLETAV. . . . . . . SKGDASAVVQ Fe Ec .................................. SFELPA LPYAKDALAP HISAETIEYH YGKHHQTYVTNLNNL[KGT. AFEGKSLEEI IRSSEG. . . . Fe PI .................................. AFELPA LPFAINALEP HI~ETLEYH YGKHHNTYVVKLNGLVEGT. ELAEKSLEE! IKTSTG. . . . Fe Po .................................. AFELPP LPYAHDALQP HISKETLEYH HDKHHNTYVV NLNNLVPGTPEFEGKTLEEI VKSSSG. . . .

in in in in in in in in in

Va . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bs ...................................

Va

90 I00 II0 [20 130 I40 150 lfD * iQTAVRNNGGGHANHTFF~E ILGP...NAG GEPTGAIKEA IEETFGSFED FKEEFKTAATGRFGSGWAWLVVKD...GKL AITSTANQDSPLW. . . . . . D IRTAVRNNGGGHANHSLF~T ILSP...NGG GEPTGELADA INKKFGSFTA FKDEFSKAAA GRFGSGWAWLVVNN...GEL EITSTPNQDS P I i . . . . . . E IQTAVRNNGGGHLNHSLFIRLLTP...GGA KEPVGELKKA [DEQFGGFQA LKEKLTQAAWGRFGSGWAWLVKDPF..GKL HVLSTPNQDN PVW. . . . . . E KKTVLRNNAGGHANHSLFIKGLKK...GTT LQ..GDLKAA IERDFGSVDN FKAEFEKAAA SRFGSGWA~LVLKG...DKL AVVSTANQDSPLWGEAiSGA ALGDVTHNGCGHYLHTWFIEHWSPD...GG GEPSGALADRIAADFGSYEN WRAEFEVAAGA..ASGWALLVYDP.VAKQL RNV&VDNHDEGALW. . . . . . ALGDVTHNGSGHILHTLF~QSWSPA...GG DEPSGALADRIAADFGSYENWRAEFEAAASA..ASGWALLVYDS.HSNTL RNVAVDNHDEGALW. . . . . . IQQNIKFHGGGFTNHCLF~E NLAPF.SQGGGEPPTGALAKA [DEQFGSLDE LIKLTNTKLA GVQGSGIAF1VKNLSNGGKLDVVQTYNQD. . . . TV...TG LQPALKFNGGGHINHSIFWTNLSP~.NGGGE.PKGELLEA IKRDFGSFDK FKEKLTAASV GVQGSGWG~LGFN.KERGHLQ[AACPNQD. . . . PL.QGTI" LQAAIKFNGGGHVNHSIFWKNLKPISEGGGEPPHGKLGWAIDEDFGSFEA LVKKWNAEG~ALQGSG~VILALD.KEAKKV SVETI'ANQD. . . . PLVTKGA ,..GVFNNAAQV~NHTFY~N CLAP.._NAG GEPTGKV~EA IAASFGSFADFKAQFTDAAI KNFGSG~T~LVKNSD..GKLAIVSTSNAGTPL . . . . . TTD ...GVFNNAAQVINHTFYINCLAP...NAG GEPTGEVAAAIEKAFGSFAE FKAKFTDSA[ NNFGSSWTWL VKNAN.,GSLAIVNTSNAGCPI . . . . . TEE ...G[FNNAA QVWNHTFYIN CLSP,,.DGG GQPTGALADAINAAFGSFOK FKEEFTKTSV GTFGSGIA~LVK,AD..GSL ALCSTIGAGAPL . . . . TS 70

80

[70

[80

~n In gn in in Wn in in Wn Fe Fe Fe

Bs Tt Ec Hh Hc Sc Hum Zm Ec PI Po

in in tn ~n in in In in in Fe Fe Fe

Va GQTPVLGLDVtEHAYYLKYK NVRPDYINAF WSVIN~DKVN EYF..AKA Bs GKTPILGLDVWEHAYYLKYQNRRPEYIAAF WNVVNWDEVAKRYSEAKAKTt GFTPIVGIDVWEHAYYLKYQNRRAOYLQAIWNVLNWDVAEEFFKKA. . . . Ec SGFP|iGLDV WEIIAYYLKFQNRRPDYIKEF WNVVNWDEAA~RFAAKK--Hh GSHPILALDVWEHSYYYDYGPDRGSFVDAFFEVIDWDPIA ~NYDDVVSLF E Hc GSHPILALDVWEHSYYYDYGPDRGSFVDAFFEVVDWDEPT ERFEQAAERF E Sc PLVPLVAIDAWEHAYYLQYQNKKADYFKAI WNVVNWKEASRRFDAGKI-Hum GLIPLLGIDVWEHAYYLQYKNVRPDYLKAI WNVINWENVTERYWACKK-Zm SLVPLLGIDVWEHAYYLQYKNVRPDYLNNI WKVWNWKYAGEVYENVLA-Ec .ATPLLTVDVWEHAYYIDYRNARPGYLEHFWALVNWEFVAKNLAA. . . . . P[ GVTPLLTVDLWEHAYYIDYRNLRPSYWDGFWALVNWDFVSKNLAA. . . . . . Po GDTPLLTCDV~EHAYYIDYRNLRPKYVEAF~NLVNW~FVA EEGKTFKA--

[90

200

210

Fig.3. Sequence alignment of Mn-SOD from three Kingdoms and Fe-SOD from eubacteria. The amino acid positions are numbered from the first residue ofE. coli M_n-SOD to facilitate comparison with the data of Parker and Blake (28). The metal ligands and active sites are indicated by ~r and V , respectively. Abbreviations of species are the same as in Table2. Sequence data were taken from the EMBL database and Swiss Prot protein database. biochemistry and microbiology tests on Vibrio species identity. The diagnostic PCR techniques have been applied in the identification of several Vibrio strains such as V. cholera, V. parahaemolyticus, and V. vulnificus by their specific toxin producing genes (14). The possl~o'dityof Mn-SOD gene from V. alginolyticus serving the same purpose was examined by carrying out the cross-PCR using the N-terminal and C-terminal primer pairs (primer5 and 6, Table1). Among the atginolyticus was a soda gene. It was the first example of a Mn-type SOD gene cloned from the Vibrio species. 810

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Table 3. Effect of temperature on the activity of the recombinant V. alginolyticus Mn-SOD Temperature

Relative activity a (%)

(~ 25

104

37

I00

45

108

60

105

80

16

99

2

a. SOD activity was monitored by the xanthine oxidase-I.N.T method (25). The relative activity at a given temperature was calculated by dividing the absolute rate by that at 37~ Results represent the average of three independent experiments.

Table 4. Effects of various iahib~tors on the recombixlant VAMn-SOD aetNity,

Inhibitors

Residual activity (%)a

None

100

Diethyldithiocarbamic acid 1 mM

78

5 mM

82

10 mM

80

5 mM

84

10 mM

78

20 mM

80

0.2 mM

78

0.4 mM

77

0.6 mM

78

NaN3

H202

~. Residual activity is expressed as a percentage of the activity of an untreated sample against samples treated with various concentration of inhibitors.

811

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M

1 2

3

4

5

6

7

97.4

66.2 42.7 31 21.5 14.4

Fig.4. Expression of the recombinant V. alginolyticus M_n-SOD in E. cob BL21 (DE3) pLysS. Cells were harvested after induction with 0-~0.8mM IPTG for 2 hrs at 37~ Proteins were separated by 12%SDS-PAGE. LaneM, a low range protein MW marker (kDa), lanel, E. coli BL21 (DE3) pLysS extract, lane2, E. cob BL21 (DE3) pLysS containing pET20b (+), lane3-7 are samples after induction with 0, 0.1, 0.2, 0.4, 0.8 mM IPTG for 2 hrs, respectively. Each lane was loaded with a I0 }xgtotal protein.

A

B

1 2 3

1 2 3

Fig.5. Activity analysis of the purified recombinant K alginolyticus Mn-SOD expressed in E. coli BL21 (DE3) pLysS. Cells were harvested aider 0.1ram IPTG induction for 2 hrs at 37~ Proteins were separated by 10% Native-PAGE. panel A, coomassie brilliant blue stainln~, panel B, activity staining, lane1, cell extract; lane2, purified recombinant Mn-SOD from E. coli BL21 (DE3) pLysS; hne3, purified and dialyzed recombinant M_n-SOD from E. eoli BL21 (DE3) pLysS. 812

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Vibrio species tested, only V. alginolyticus produced a distinct sodA gene PCR product, whereas V. damesela, V. metschnikovii,

V. mimicus, V. parahaemolyticus, V. cholera, K proteolyticus

and V. vulnificus did not (F.P.LIn unpublished results). Though the sodA gene encoded a Mn-SOD

protein was conserved in many aerobes and anaerobes, the distinct nueleotide sequences region might be utilized as gene specific primers for strain-identification purpose. When more sod nueleotide sequences from Vibrio species were available, it would be a signit~eant step to speed up the Vibrio strain confirmation by this diagnostic PCR. Furthermore, a partial putative sod PCR

product was amplified by PCR using the same primer pairs (primer 1 and 2, Table 1) ~om other Vibrio species such as K cholera, K proteolyticus, V. metschnikovii, E mimicus and E parahaemolyticus, indicating the usefulness of this primer pairs. Their full-length genes were

underway in this laboratory. In summary, the sodA gene encoding a Mn-SOD of V. alginolyticus has been cloned by

PCR techniques and overexpressed in the heterologous E. coli host using pET20b (+) expression system. The recombinant VAMn-SOD protein was active and showed consistent characteristics to the reported M_n-SODs. A strain specific gene probe by using sodA from Vibrio strains could be further developed when other ;qbrio soda gene would be cloned and sequenced.

Acknowledgement This work was supported by a research grant NSC 87-2313-B-019-002 ~om the National Science Council, Taiwaa, R. O. C.

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