Appl Microbiol Biotechnol (1991) 36:358-363

App//ed

Microbiology Biotechnology © Springer-Verlag 1991

Short contribution

Molecular cloning and nucleotide sequence determination of the Bacillus stearothertnophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli John K. Brehm, Steve P. Chambers, Kevin J. Brown, Tony Atkinson, and Nigel P. Minton Molecular Genetics Group, Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK Received 8 April 1991/Accepted 26 July 1991

Summary. The gene (sod) encoding Bacillus stearothermophilus Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequence determined. With the exception of the posttranslationally cleaved N-terminal methionine residue, the predicted amino acid sequence exhibits complete identity to the previously determined amino acid sequence. The recombinant M n S O D was shown to be functionally active in E. coli both in vitro and in vivo, and was expressed to 49% o f the soluble cell protein by coupling its transcription to the E. coli trp promoter. The sequenced region o f D N A was also found to encompass a second open reading frame. The putative encoded polypeptide exhibited no significant primary sequence homology to any currently characterised protein.

the resolution o f 24 nm, have been published for the B. stearothermophilus enzyme (Parker and Blake 1988) and indicate that the polypeptide chain of the monomer is organised into two domains, one with an all ahelix structure and the other with an a-helix/fl-pleat structure with the manganese atom b o u n d between the domains. SODs from various sources are currently o f great interest as potential therapeutic treatments for oxidative damage. Their use in a clinical setting for the treatment o f a wide variety of disorders have been proposed (see Beck et al. 1988). Indeed, currently bovine C u / Z n S O D is being utilised for the treatment o f inflamed tendons in horses and for treating osteoarthitis in man (Puhl et al. 1984). In the present communication we describe the cloning of the gene encoding the M n S O D o f B. stearothermophilus N C A 1503, the determination o f its entire nucleotide sequence and its overexpression in Escherichia coli.

Introduction The molecular dismutation of O~- to hydrogen peroxide (H202) and oxygen (02) is catalysed by a ubiquitous class of metalloenzymes termed superoxide dismutases (SODs). On the basis of their metal ion content, three classes o f SOD are recognised: C u / Z n - , Fe-, and Mncontaining enzymes (Fridovich 1975). While all three forms catalyse the same reaction, the Fe-containing SODs (FeSOD) are largely confined to procaryotes and the C u / Z n enzymes ( C u / Z n S O D ) predominantly to eucaryotes. Mn-containing SODs (MnSOD) are universally present (Geller and Winge 1984). The Bacillus stearotherrnophilus enzyme is a typical prokaryotic MnSOD, comprising two identical subunits each o f 203 residues with one atom of Mn per dimer. Although relatively thermostable and with a wide active p H range (Atkinson and Bown, unpublished) the enzyme is not as thermostable as the tetrameric MnSOD's o f Thermus spp. (Sato et al. 1987). Crystallographic coordinates, to

Offprint requests to: N. P. Minton

Materials and methods Bacterial strains and growth conditions. The bacterial strains utilised were B. stearothermophilus NCA 1503 (NCIB 8924, ATCC 7954), and the E. coli strains TG1 (K12 A[lac-pro] supE thi hsdD5 F'-traD36 proA +B + lacl~Z M15), W5445 (pro leu thi thr supE44 lacy tonA hsdM hsdR rpsL), E. coli QC781 (F-, lac-4169 ~[sodA::MudlIPR13123 rpsL CmR), QC773 (GC4468 O[sodBkan]l-A2 KmR, and QC799 (soda sodB, CmR KmR). B. stearothermophilus was grown as previously described (Atkinson et al. 1979). E. coli was routinely cultured in Luria broth (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaC1), supplemented, where appropriate, with 50 Ixg/ml of ampicillin (Ap), 15 Ixg/ml of tetracycline (To), 30 l~g/ml of kanamycin (Km) or 5 p.g/ml of chloramphenicol (Cp). The medium used for recombinant E. coli ceils, at both the laboratory and pilot scale, contained glucose, 1.4%; NH4SO4, 0.25%; KH2PO4, 0.3%; K2HPO4,0.2%; Na-citrate, 0.005%; yeast extract (Difco, West Mosley, Surrey, UK), 0.5%; MgSO4-H20, 1%; 100 ~M MnSO4 and trace elements, 1.0%. The trace element stock was; EDTA. Na2, 0.5%; FeC13•6 H20, 0.05%; ZnO, 0.005%; CuC12.2H20, 0.001%; CoNO3.6H20, 0.001%; NH4MoTO24, 0.001%. Cultures were grown at 37° C, pH 7.0, with an air flow rate of 1 vvm.

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Fig. 1. Restriction enzyme map of the expression vectors pMTL1003 and pMTL1013. The respective components of the illustrated vectors are as follows. The pSC101-derived DNA encompassing the partition function par (PAR) represents nucleotide 3 to 244 of the sequence of Miller et al. (1983) and has been inserted between the HaeII site at position 470 of pMTL20 (Chambers et al. 1988) and nucleotide 475 (fusion point A). A synthetic trp promoter element (5'-AGCTTACTCCCCATCCCCCCAGTGAATTCCCCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAGCTFGGC-3Ohas been fused between nucleotides 421 and 284 of pMTL20 (fusion points B and C, respectively), and rrnB-derived DNA (nucleotide 6566 to 6840, Brosius et al. 1981), carrying the double transcriptional terminators T1 and T2, inserted between the HaeII site at position 2853 of pMTL20 and position 2367 (fusion point D). The polylinker is that of pMTL20. Restriction enzyme sites, marked with an asterisk were created by site-directed mutagenesis as follows: EcoRV, pSC101 sequences 83 and 84 changed from A to T and T to C, respectively; NdeI, pMTL20 nucleotides 261 and 262 changed

from C to A and G to C, respectively, and NruI pMTL20 nucleotide 418 changed from C to T. The latter mutagenic change simultaneously destroyed a unique PvulI site (equivalent to that present at nucleotide position 421 of pMTL20), pMTL1013 was construtted by inserting the illustrated DNA fragment encoding the pBR322 tet gene between the indicated DraI and SspI sites of pMTL1003. Prior to its insertion, site-directed mutagenesis was employed to destroy certain restriction enzyme recognition sequences (labelled I to 5), without affecting the codon sense of the tet gene. The sites removed, and the changes made (relative to the pBR322 nucleotide sequence, Balbas et al. 1986) were: (1) ClaI and HindlII (nucleotides 27 and 28, A to T and T to A, respectively); (2) EcoRV (nucleotide 187, T to C); (3) BamHI (nucleotide 379, T to C); (4) SphI (nucleotide 565, T to C), and (5) Sall (nucleotide 656, C to T). The B#III site at the 5'-end of the tet gene was created by fusion of the sequence 5'-GATCTGTTAATI'CGAGCTCGCCC-Y to pBR322 nucleotide 4362, and the Bcil site at the 3'-end by fusion of the sequence 5'-GATCAGTTGATCCCC-3' to pBR322 nucleotide 1429

Recombinant procedures. The vectors employed in this study were phages M13mp18/19 (Yanisch-Perron et al. 1985) and plasmids pAT153 (Twigg and Sherratt 1980) and pMTL1003/pMTL1013 (Fig. 1). pMTL1003 is derived frpm pMTL4 (Chambers et al. 1988), replicates from a mutant ColE1 replicon (600 copies per cell; Minton et al. 1988), encodes the pUC8-derived bla and lacZ' (Yanisch-Perron et al. 1985), and incorporates the pSC101 partition function (par; Miller et al. 1983), the E. coli rrnB double terminator (Brosius et al. 1981) and the pMTL20 polylinker cloning region (Chambers et al. 1988). Transcription of lacZ' is under the control of a synthetic trp promoter, pMTL1013 differs from pMTL1003 only in that the bla gene (Ap R) has been replaced with the pBR322 tet (Tor~) gene. Procedures used for the isolation of chromosomal and plasmid DNA, and the transformation of E. coli have been previously described (Minton et al. 1983). Restriction endonucleases and DNA-modifying enzymes were used in the buffers and under the conditions recommended by the supplier (Northumbria Biologicals, Northumbria, UK). M13 tern-

plates containing randomly derived DNA inserts were generated by a sonication procedure (Minton et al. 1986), and subjected to nucleotide sequence analysis using the dideoxynucleotide method and "sequenase R'' enzyme (Tabor and Richardson 1987). Nudeotide sequence data generated was analysed using the computer software of DNASTAR (Madison, Wise., USA). DNA/DNA hybridisation of restriction fragments and in-situ colony hybridisations were as described by Barstow et al. (1986). Hybridisations using the 5'-end-labelled 50-mer oligonucleotide probe were carded out at a temperature of 55° C for 2 h or more, followed by several washes, of 5 min duration, at 45 ° C.

Determination of superoxide dismutase activity. Harvested cells, resuspended in 0.05 original volumes of 50 mM phosphate buffer (pH 7.8), were disrupted by sonication, and cell debris removed by centrifueation at 100000 for 5 min. SOD assays, a modification of the method of McCord and Fridovich (1969), were performed in 1 ml of 0.05 M potassium phosphate buffer, pH 7.5,

360

containing 10-4M EDTA, pH 7.5, in a 1-cm light path cuvette thermostatted at 30° C. The reaction mixture contained 2.5 X 10-5 M ferricytochromeC and 7 x 10-3 M xanthine (sodium salt) and sufficient xanthine oxidase to produce a rate of reduction of ferricytochromeC at 550 nm of approximately0.1 absorbance units/min. Under these conditions,the amount of SOD required to inhibit the rate of reduction of ferricytochrome C by ~ 50% (i.e. to a rate of 0.05 absorbance units/min) is defined as one unit (U) of activity. This method gives an activitylevel approximately tenfold higher than that of the original since the generation of superoxide in the systemis no longer rate limited. Protein concentration was determined by the method of Bradford (1976). Results and discussion

Cloning of the B. stearothermophilus sod gene To allow the detection of the B. stearothermophilus MnSOD structural gene by D N A / D N A hybridisation experiments, a 50'-mer antisense oligonucleotide (5'GTGTTGTGGTGTTTCGTGTGGTGGATGTTCATCGTrTCTTTGTCGATGTG-3~ was synthesised, complementary to amino acids 17 through 34 of the previously determined primary sequence of the purified enzyme (Brock and Walker 1980). The nucleotide bases used in positions of codon degeneracy corresponded to those most frequently used in B. stearothermophilus genes (Barstow et al. 1986). Southern blot experiments demonstrated that under the conditions employed (see Materials and methods), appropriately radiolabelled oligonucleotide hybridised strongly to various restriction fragments of B. stearothermophilus NCA 1503 genomic DNA, including; a 2.45-kb BclI, 5.1-kb ClaI, 6.8-kb EcoRI, 3.4-kb HindlII, 20-kb PstI, 3.2-kb Sail, 3.5-kb SstI and a 17-kb XhoI fragment. Having demonstrated the specificity of the oligonucleotide probe, a plasmid library of the B. stearothermophilus genome was constructed by ligating sized (approx. 8 kb), partially digested (Sau3a) chromosomal DNA with BamHI-cleaved pAT153 DNA, and transforming the resultant ligation mixtures into E. coli W5445. Of the 4,125 Ap R, Tc s transformants obtained, analysis of the plasmids of 50 random representatives indicated 92% contained inserts. Each ApRTc s recombinant clone was individually screened by in-situ colony hybridisation, using the radiolabelled oligonucleotide as a probe. The probe was shown to hybridise strongly to two different recombinant clones. Plasmid DNA was isolated from each clone and designated pBCM1 and pBCM2. Restriction analysis showed that the insert of pBCM1 was 6.85 kb, while that of pBCM2 was 4.7 kb in size. Furthermore, the insert of pBCM1 entirely encompassed that of pBCM2 (data not shown).

ated from both pBCMl-derived fragments, by: (i) subcloning the 1.6-kb EcoRI-SstI fragment into M13mpl8 and M13mpl9, and (ii) subjecting the 3.0-kb HindlII fragment to the sonication procedure. A 2040-bp portion of the sequence obtained is illustrated in Fig. 2, and was determined on both DNA strands. It's translation revealed the presence of an open reading frame (ORF) of 615 bp beginning with an AUG codon (nt 387) and terminating with a UAA codon (nt 1001). The deduced polypeptide was 204 amino acids in length and, with the exception of the N-terminal Met, exhibited perfect conformity to the experimentally determined protein sequence of MnSOD (Brock and Walker 1980). A nucleotide sequence beginning at 438 and ending at 488 exhibited near perfect complementarity to the oligo-probe utilised to identify the gene. The three positions at which mismatch occurred (nt 465, 477 and 483) all resulted in neutral G.T pairing, accounting for the observed efficient binding of the oligo to B. stearothermophilus-derived DNA encoding sod. The translational initiation codon was preceded by a sequence (5'CAAAAGGAGGAGA-3') exhibiting strong complementarity to the 3'-termini of the B. subtilis 16S rRNA (3'-UCUUUCCUCCACU-53. A sequence exhibiting dyad symmetry occurs immediately 3' to the translational stop codon (nt 1007 to 1036), and probably represents a Rho-independent transcriptional terminator. The putative RNA stem-loop structure formed would have a AG of 23.0 kcal. A second putative ORF was identified 3' to sod, initiating with an AUG codon at nt 1133 and preceded by a sequence exhibiting reasonable complementarity to the B. subtilis 16S rRNA. The encoded putative polypeptide (ORF B) exhibits no exceptional homology to any protein currently found in the PIR database although it may be significant, given the catalytic function of SOD, that one of the closest matches is that of the "i" polypeptide of cytochrome oxidase. The structural gene had a G + C content of 53.1%, and exhibited a similar codon usage to that of the sod gene (data not shown). During the course of this work the equivalent sod gene from another strain of B. stearothermophilus (ATCC 12 980) was cloned and its nucleotide sequence determined (Bowler et al. 1990). Alignment of the two sequences (See Fig. 2) reveals four nucleotide differences, all of which occur external to the sod coding region. It is notable that the two differences that reside 3' to the sod gene are encompassed by the putative transcriptional terminator. These changes would reduce the stability of a transcribed RNA transcript from a AG of -23.0 kcal (strain NCA 1503) to -16.8 kcal (strain ATCC 12980). It is also apparent that at least 39 codons of the downstream ORF of strain NCA 1503 are also conserved in the genome of strain ATCC 12980.

Determination of the sod nucleotide sequence Southern blot analysis of pBCM1/2 showed that both plasmids contained a common 3.0-kb HindlII and a 1.6-kb EcoRI-SstI fragment that hybridised to the oligonucleotide probe. M13 template clones were gener-

Expression of the MnSOD sod gene in E. coli To elicit the overexpression of sod in E. toll, the gene was isolated from pBCM2 as a 1.3-kb NruI-HindIII

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Fig. 2. Nucleotide sequence of the Bacillus stearothermophilus gene encoding manganese superoxide dismutase (MnSOD). The illustrated region is a 2040-bp fragment derived from pBCM1. Of the two (ORFs), ORF A corresponds to the sod gene and ORF B to the unidentified putative gene. Possible ribosome binding sites preceding both ORFs are underlined and labelled S.D. The region of dyad symmetry, which may correspond to the transcriptional

terminator of sod, is indicated by facing arrows above the sequence. The sequence obtained from strain ATCC 12980 (Bowler et al. 1990) has been aligned below the relevant portion of our sequence (nucleotides 1 to 1259), with nucleotide identity being indicated by a colon. The dash (position 1027) indicates the absence of a nucleotide

fragment (Fig. 2) and cloned, by an indirect route, into the S i n a i site o f pMTL1003 (Fig. 1). T w o recombinant plasmids (pBCM3 and pBCM4), representing the two possible orientations o f insertion o f the cloned fragment, were chosen for further study. In the case o f pBCM3, s o d was orientated such that its expression

could be enhanced by transcriptional read-through from the vector trp promoter. T w o analogous plasmids p B C M 5 (equivalent to pBCM3) and p B C M 6 (equivalent to p B C M 4 ) were generated by using pMTL1013 in place o f pMTL1003. Cells harbouring all four plasmids were grown in LB, supplemented with 100 ~M MnSO4,

362 degrees of native SOD activity. In this case transcription from the trp promoter was induced late in the exponential phase following tryptophan depletion from the media. Although the soda host (QC781) produced equivalent levels of SOD to the wild-type Eo coli host TG1, the two strains QC773 and QC799 defective in sodB, produced significantly lower levels of recombinant SOD (5000 and 4000 U/mg, respectively). Evidence that the enzyme produced is functionally active in vivo was obtained by showing that the presence of pBCM3 partially alleviated the growth inhibitory effect of 10-SM methyl viologen (Paraquat, a commercial weed killer known to generate superoxide free radicals, Carlioz and Touati 1986) on the soda mutant strain QC781 (data not shown). Small-scale fermentation of E. coli TG1 containing pBCM3

Fig. 3. Sodium dodecyl sulphate-polyacrylamidegel electrophoresis (SDS-PAGE) of total cell extracts of TG1 cells carrying pBCM3. Total cell extracts were derived from 10-h samples subjected to SDS-PAGE: lane 1, purified B. stearothermophilus MnSOD; lane 2, molecular mass markers; lane 3, soluble cell lysate of TG1 carrying pBCM3 and transcription from the vector trp promoter induced in late exponential phase by the addition of indole acrylic acid (20 txg/ml). Cells were removed from the cultures for estimation of SOD activity at hourly intervals. The maximum level of MnSOD produced by cells carrying pBCM3 and pBCM5 was 62 275 and 55 590 U / ml culture, respectively. This equates to 12210 and 10250 U / m g soluble protein, respectively, and was attained after 10 h. By reference to the specific activity of pure MnSOD (25000 U/mg), this represented 49% (pBCM3) and 41% (pBCM5) of the soluble cell protein. Confirmation of these levels was obtained by densiometric scanning of Coomassie blue stained gels following sodium dodecyl sulphate-polyacrylamide gel electrophoresis of total cell extracts (see Fig. 3). That high expression was due to the vector trp promoter was indicated by the low level of SOD produced (10.9 and 15.2 U / m l culture) by cells harbouring pBCM4 and pBCM6. The ability of E. coli to support high level of expression of the B. stearothermophilus sod gene was consistent with the observation that its encoding region makes little use of modulator codons (a single C G G and a G G A codon are used), exhibits a codon bias characteristic of highly expressed E. coli genes (Grosjean and Friers 1982), and is preceded by a near to consensus ribosome binding site. The levels of sod expression directed by pBCM3 were examined in a range of E. coli hosts with varying

Although the level of expression of recombinant SOD obtained in batch culture was of a high order of magnitude, it was of interest to see whether high production rates could be translated to conditions more closely resembling those employed for commercial production of recombinant proteins. Accordingly, an 8-1 pilot-scale culture was carrried out using the media described in Materials and methods. The inoculum for the seed was provided by freshly transformed cells plated out onto LB supplemented with tryptophan (100 ~tg/ml) and ampicillin (100 ixg/ml) for promoter repression and selection, respectively. The seed was provided by a 500-ml 2 x LB culture supplemented with MnSO4, ampicillin and tryptophan. The seed was grown at 37°C at 200 rpm for 7 h. Once inoculated, the culture was allowed to go its full course before harvesting, relying on tryptophan starvation to switch on the trp promoter late in the cultures exponential phase of growth. Cells were harvested by centrifugation, and the cell paste bagged, flash frozen and stored at - 8 0 ° C until extracted. The level of SOD expression obtained from the pilot-scale cultures were consistent with those obtained in shakeflask experiments. Following purification, characterisation of the purified recombinant SOD identified the protein as a dimer, with each subunit having a molecular mass of approximately 21 kDa. In conclusion, we have demonstrated that high copy number vectors carrying the E. coli trp promoter may be used to direct efficiently the expression of the B. stearothermophilus sod gene, such that the encoded product represents nearly 50% of the soluble cell protein. The over-produced protein is both active in vitro and in vivo. These high levels will expendite the use of MnSOD from this organism in human therapy, where its extreme stability and low antigenicity offer considerable advantages over SOD enzymes from other sources. Nucleotide sequencing of the region of the B. stearothermophilus has indicated the presence of a second gene distal to sod, the function of which is unknown. It is interesting to note that the whole of this region is conserved in another thermophilic bacillus, B. caldo-

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tenax ( C h a m b e r s et al., u n p u b l i s h e d ) , a n d at l e a s t t h e first 39 c o d o n s in a s e c o n d B. stearothermophilus strain. Acknowledgements. We wish to thank Nicola Minion for the preparation of this manuscript, and D. Touati (Institute Jacques Monod, Paris) for supplying the sodAB strains.

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Molecular cloning and nucleotide sequence determination of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli.

The gene (sod) encoding Bacillus stearothermophilus Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequ...
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