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Journal of Bacteriology, November 2004, p. 7821-7825, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7821-7825.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

In Vivo Production of Active Nickel Superoxide Dismutase from Prochlorococcus marinus MIT9313 Is Dependent on Its Cognate Peptidase

Thomas Eitinger*

Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, 10115 Berlin, Germany

Received 9 July 2004/ Accepted 16 August 2004


   ABSTRACT
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Metal-dependent superoxide dismutases (SODs) with a specific requirement for a manganese or iron ion for catalytic activity and copper- and zinc-dependent enzymes are essential for detoxification of superoxide anion radicals. Genome sequence analyses predict the existence of a nickel-dependent enzyme (NiSOD) as the unique SOD in oxygen-evolving marine cyanobacteria. NiSOD activity was observed in Escherichia coli when sodN and sodX (encoding a putative peptidase) from Prochlorococcus marinus MIT9313 were coexpressed.


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Toxic superoxide anion radicals (O2·–) arise from one-electron transfer to dioxygen in aerobic organisms. Superoxide dismutases (SODs; EC 1.15.1.1) are ubiquitous metalloenzymes that efficiently disproportionate superoxide to molecular oxygen and hydrogen peroxide through alternate reduction and oxidation of their active-site metal ions. Until recently, two phylogenetically independent classes of SODs were known (reviewed in references 8 and 17). Class I contains SODs with a specific requirement for a manganese or iron ion for catalytic activity (MnSOD and FeSOD) and enzymes that function with either of the two (so-called cambialistic SODs). Class II contains copper- and zinc-dependent enzymes (CuZnSODs). Members of the two classes are found in both prokaryotes and eukaryotes. In 1996, Youn and coworkers (26) described a novel type of SOD in Streptomyces species with only nickel as the catalytic metal. Based on analyses of 20 clinical and soil isolates which all contained this enzyme, Leclere and coworkers (15) concluded that the presence of cytoplasmic nickel-dependent SOD (NiSOD) is a general feature of the genus Streptomyces. NiSOD in Streptomyces coelicolor and Streptomyces seoulensis is produced as a preprotein with an N-terminal extension of 14 amino acid residues which is removed posttranslationally by a yet-unidentified peptidase. Attempts to express sodN of S. coelicolor, encoding the NiSOD precursor, in Escherichia coli failed to produce catalytically active protein. Low activity was observed when a sodN{Delta} construct lacking codons 2 to 14 was expressed (13). A portion of these SodN polypeptides may have been modified by host methionine aminopeptidase, leading to properly processed SodN. Bryngelson and coworkers demonstrated that a recombinant NiSOD precursor in which the native leader was replaced by a 47-residue peptide harboring an endoproteinase Xa cleavage site could be processed in vitro to produce fully active enzyme (2). Very recently, the high-resolution crystal structures of the NiSODs from S. coelicolor (1) and S. seoulensis (25) were reported. In the latter study, wild-type NiSOD was isolated from S. seoulensis and mutant enzymes were produced in recombinant Streptomyces lividans in which the precursors were N-terminally processed by an unknown endogenous protease. Barondeau et al. (1) purified NiSOD from the periplasm of recombinant E. coli expressing a sodN variant in which the S. coelicolor 5' leader was replaced by a Pectobacterium carotovorum pelB signal sequence. The two studies identified NiSOD as a homohexameric enzyme. The monomers have a four-helix bundle structure and contain one Ni ion bound to an N-terminal nickel hook. The Ni ion is coordinated by His-1, Cys-2, and Cys-6 (numbering of the processed form). The geometry and the oxidation state cycle between square planar Ni(II), with the amino group nitrogen of His-1, the backbone nitrogen of Cys-2, and the thiolates of Cys-2 and Cys-6 as metal ligands, and square pyramidal Ni(III) where the imidazole side chain of His-1 is the fifth ligand (1, 25). The involvement of the His-1 amino group explains the fact that Streptomyces NiSOD variants lacking residues 2 to 14 (numbering of the precursor form) but retaining the N-terminal methionine are inactive and nickel free (1).

Recently, open reading frames with significant similarity to NiSOD preproteins were identified in the genomes of marine cyanobacteria (5, 18, 21, 24). Predicted secondary structures are consistent with four-helix bundles (1, 25), and the N-terminal processing and metal coordination sites are fully conserved (Fig. 1A). The sequences immediately downstream of these putative sodN genes in the Prochlorococcus marinus strains MED4, MIT9313, and SS120, in Synechococcus strain WH8102, in Trichodesmium erythraeum strain IMS101, and in Crocosphaera watsonii strain WH8501 encode proteins with distinct similarity to each other (Fig. 1B) and, according to the PFAM database, to the serine protease S24, S26A, and S26B families. Similar proteins are also encoded in the neighborhood of sodN in S. coelicolor (13) and Streptomyces avermitilis (11).



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  		FIG. 1. Alignment of the N-terminal parts of SodN precursors (A) and complete SodX sequences (B) from Streptomyces species (S. avermitilis [Saver], S. coelicolor [Scoel], and S. seoulensis [Sseoul]) and the marine cyanobacteria P. marinus strains MED4, MIT9313, and SS120, Synechococcus strain WH8102, T. erythraeum strain IMS101 (Ter), and C. watsonii strain WH8501 (Cwat). The arrow indicates the proteolytic cleavage site established for Streptomyces and predicted for the cyanobacterial proteins. Sequences were loaded into and analyzed with the workbench GENESOAP written by R. Cramm (23). Alignments were generated with CLUSTALW and displayed with BOXSHADE.

 
In this report I shall present evidence that sodN of P. marinus MIT9313 indeed encodes a NiSOD. The enzyme can be produced in E. coli in a catalytically active state provided that the downstream open reading frame (named sodX) is coexpressed and the cells grow in the presence of nickel ion. Under these circumstances, the recombinant NiSOD restores oxygen tolerance in an oxygen-sensitive E. coli mutant lacking the endogenous FeSOD and MnSOD.

Constructs for NiSOD production. sodN and sodX were amplified using 5 µl of a concentrated live culture of P. marinus strain MIT9313 (kindly provided by Ilka M. Axmann, Institut für Biologie/Genetik, Humboldt-Universität zu Berlin, Berlin, Germany) as the template in 100-µl PCR mixtures. Primers were designed to generate a sodN product with a 5' NcoI site and a 3' end lacking the termination codon but containing a BglII site. The NcoI- and BglII-treated amplicon was inserted into a streptomycin resistance-conferring derivative of plasmid pCH675AF (4) to give psodN (Fig. 2). Expression is under the control of a lac promoter and a ribosomal binding site and results in a SodN peptide containing a FLAG epitope at the C terminus. The sodX amplicon containing 5' XbaI and 3' SacI sites was digested and inserted between the XbaI and SacI sites of psodN, yielding psodNX (Fig. 2). In this construct the sodN termination and sodX initiation codons are separated by 7 nucleotides (compared to 10 nucleotides in P. marinus MIT9313), distances that should allow translational coupling. psodN{Delta} encoding a NiSOD peptide lacking residues 2 to 20 (numbering of the precursor) was generated by inverse PCR using psodN as the template, Pfx proofreading DNA polymerase (Invitrogen), and primers with the 5'-terminal sequences CAC (His-21 codon) and CAT (complementary to Met-1 codon). The PCR product was purified, phosphorylated with T4 polynucleotide kinase, ligated, and transformed into E. coli.



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  		FIG. 2. Constructs for expression of P. marinus MIT9313 sodN in E. coli. psodN{Delta} encodes a peptide lacking residues 2 to 20. See the text for details. Arrows indicate initiation, and asterisks indicate termination codons.

 
NiSOD activity in recombinant E. coli. For functional expression analyses, the constructs were introduced into E. coli strain SG12041 (recA {Delta}lon510) (9) containing plasmid pFDX500 (orip15A Kmr lacIq) (22). The recombinants were grown overnight in 100-ml Erlenmeyer flasks at 37°C with shaking in 10 ml of Luria-Bertani (LB) medium containing IPTG (isopropyl-ß-D-thiogalactopyranoside; 1 mM), streptomycin (50 µg/ml), kanamycin (40 µg/ml), and NiCl2 as indicated in the legend to Fig. 3. Cells were harvested, concentrated 10-fold in TBE buffer (89 mM Tris, 89 mM borate, 2.5 mM EDTA), and disrupted by sonication. Particulate material was removed by centrifugation (21,000 x g, 4°C, 20 min), and SOD activity in the supernatants was determined by in gel activity staining as described previously (16). Briefly, extracts were separated on native polyacrylamide gels using TBE as the electrophoresis buffer. Gels were soaked first in 20 mM sodium phosphate buffer (pH 7.4) containing 28 µM riboflavin and 28 mM TEMED (N,N,N',N'-tetramethylethylenediamine) for 10 min and then in 20 mM sodium phosphate buffer (pH 7.4) containing 2.5 mM nitroblue tetrazolium for another 10 min. Finally, the gels were illuminated for approximately 5 min with fluorescent light. Photochemically produced superoxide radicals reduce nitroblue tetrazolium to dark blue formazan. SOD activities result in bright areas on a dark background. For detection of FLAG-tagged NiSOD by immunoblotting, native gels were electroblotted onto nitrocellulose membranes and processed with a monoclonal anti-FLAG M2 antibody-alkaline phosphatase conjugate as recommended by the manufacturer (Sigma). The results are shown in Fig. 3. It has long been known (3) that three zones of SOD activity can be separated in E. coli cytoplasmic extracts. The slow- and fast-migrating areas represent MnSOD and FeSOD, respectively, while the middle activity stems from hybrids containing subunits of both MnSOD and FeSOD. The pattern was very similar when extracts from cells containing psodN and psodN{Delta} were compared, was not significantly affected when these cells were grown in the presence of increasing concentrations of Ni2+ ion (Fig. 3), and looked essentially the same in cells lacking a recombinant plasmid (data not shown). An additional SOD activity was observed in cells harboring psodNX upon growth at Ni2+ concentrations above 100 µM. Multiple NiSOD signals which occurred independently of the presence of nickel during growth were observed by immunoblotting. This result suggested that the enzyme is present in various oligomeric states. Increasing Ni2+ concentrations led to an increase of the slower-migrating forms, which correlated with Ni2+-dependent SOD activity. The immunoblots looked different when extracts of cells containing psodN and psodN{Delta} were analyzed. The patterns were not markedly affected by the Ni2+ concentration during growth. Expression of sodN led to a single, slow-migrating form, while the product of sodN{Delta} behaved like the inactive fast-migrating form in sodNX-expressing cells. Taken together, these data demonstrated that SodX has a stabilizing effect on SodN and is required for production of active NiSOD. Although not yet proven, it is conceivable that SodX acts as an N-terminal peptidase on SodN precursors. Preliminary experiments (data not shown) militate in favor of this assumption. Denaturing polyacrylamide gel electrophoresis and Western blotting of cell extracts expressing sodN and sodX detected SodN monomers which, on the basis of electrophoretic mobility, could be assigned to the precursor and processed forms.



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  		FIG. 3. Activity staining and Western blot analysis of cell extracts of E. coli SG12041(pFDX500) containing psodN, psodN{Delta}, or psodNX. NiCl2 was added to the growth medium to final concentrations of 500 nM, 1 µM, 5 µM, 10 µM, 100 µM, 500 µM, or 1 mM or not added. Extracts were separated by native polyacrylamide gel electrophoresis, and Western blots were developed with an anti-FLAG antibody reacting with the FLAG epitope fused to SodN. The arrow points to the position of NiSOD activity.

 
NiSOD restores oxygen tolerance in an E. coli mutant. For complementation analyses, psodN, psodN{Delta}, and psodNX were transformed into the oxygen-sensitive E. coli strain QC2375 [{Delta}sodB2 (sodA-lacZ)49 {Delta}recA306; see http://www2.ijm.jussieu.fr/touati/strains.php for details]. This strain lacks MnSOD and FeSOD (20) and was kindly provided by Sam Dukan (Laboratoire de Chimie Bactérienne, IBSM, Centre National de la Recherche Scientifique, UPR 9043, Marseille, France). Transformants were selected on LB medium plates incubated under an N2 atmosphere in an anaerobic jar. The capability of the various sodN constructs to restore oxygen tolerance was first investigated by a plate assay (Fig. 4). Serial dilutions of cells grown anaerobically in liquid medium were spotted onto LB medium plates supplemented with streptomycin (50 µg/ml) and a 500 µM concentration of the indicated metal salts, and the plates were incubated at 37°C under air. As controls, metal-free plates were incubated under a N2 atmosphere and under air. Figure 4 illustrates that none of the constructs restored oxygen tolerance in the absence of metals. Metal addition had different effects. Cobalt and iron enhanced air toxicity, possibly by the Fenton reaction or by Fenton-like reactions causing oxidative stress. Manganese had a protective effect for all recombinants. This observation is in agreement with analyses of lactic acid bacteria, which maintain high intracellular manganese levels as protection against superoxide and peroxide, and may be explained by the fact that certain manganese complexes have the potential to disproportionate these reactive oxygen species (reviewed in references 8 and 12). A clear-cut result was observed in the presence of nickel. Spotting of diluted psodNX-containing cells and incubation under air resulted in colony numbers similar to those on the metal-free plate incubated under anoxic conditions. The result suggests that active NiSOD conferred oxygen tolerance. For a more detailed analysis, the E. coli QC2375 recombinants were grown microaerobically in 50-ml Erlenmeyer flasks containing 50 ml of LB medium supplemented with streptomycin (50 µg/ml) and nickel salt as indicated in the legend of Fig. 5. The cultures were incubated for approximately 20 h at 37°C with shaking and then analyzed for SOD activity by in gel staining (Fig. 5). As expected, MnSOD and FeSOD activities were not detectable in the recombinants. NiSOD activity was detectable only in cells expressing sodNX. The results were confirmed by photometric cytochrome c reduction assays (16). The assay mixtures consisted of 50 mM potassium phosphate buffer (pH 7.8), 100 µM EDTA, 50 µM xanthine, and 50 µM cytochrome c (from bovine heart; Sigma C-3006). Approximately 10 mU of xanthine oxidase (from buttermilk; Sigma X-4500) was added to produce superoxide radicals, and the rate of superoxide-based cytochrome c reduction was recorded at a wavelength of 550 nm at room temperature. One to 20 µl of the soluble cell extracts was added, and the absorption change was monitored for several minutes. SOD activity removes superoxide anions, leading to a lower rate of cytochrome c reduction. SOD activity determined in the above-described assays is given in Fridovich units per milligram of protein (one Fridovich unit is defined as the amount of enzyme that leads to a 50%-reduced rate of cytochrome c reduction). Protein was estimated by a modification of the Lowry protocol (19). SOD activity in cells containing psodN and psodN{Delta} was below the detection limit. psodNX caused significant SOD activity (Fig. 5). Interestingly, both activity staining and cytochrome c reduction assays repeatedly detected low NiSOD activity in cells containing psodNX, grown microaerobically in the absence of nickel. Under oxygen- and nickel limitation, a high-affinity ABC-type Ni2+ transporter is expressed in E. coli, and this may account for the uptake of trace amounts of the metal present in LB medium (for a review on microbial nickel transport, see references 6 and 7).



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  		FIG. 4. Restoration of oxygen tolerance in E. coli QC2375 by sodN constructs. Precultures were grown anaerobically. Ten microliters of serial 10-fold dilutions (10–1 to 10–5) were spotted onto plates containing 500 µM metal salt where indicated. Plates were incubated at 37°C under air or under a dinitrogen atmosphere. N, psodN; N{Delta}, psodN{Delta}; NX, psodNX.

 


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  		FIG. 5. SOD activity in cytoplasmic fractions of E. coli QC2375 expressing sodN constructs as determined by in gel activity staining and cytochrome c reduction assays. Cells were grown in the absence (–) or presence (+) of 500 µM NiCl2. Fifty micrograms of soluble protein was separated by native polyacrylamide gel electrophoresis. ND, below the detection limit.

 
Additional factors for NiSOD maturation. The aforementioned analyses identified sodN of P. marinus MIT9313 as a structural gene for a NiSOD and SodX as an essential accessory protein and putative peptidase. The presence of sodX-like open reading frames adjacent to sodN in other marine cyanobacteria and Streptomyces species predicts a general role for SodX proteins in NiSOD proteolytic activation. Closer inspection of the sodN region in cyanobacterial genomes identified additional candidates for NiSOD accessory genes. The segment immediately downstream of sodX and in convergent orientation in P. marinus strains MED4, MIT9313, and SS120 as well as in Synechococcus WH8102 encodes a membrane protein with significant similarity to microbial nickel-cobalt transporters, a family of transition metal permeases involved in Ni and Co uptake (4, 10). The adjacent upstream region of sodN encodes a putative FKBP-type peptidyl-prolyl-cis,trans-isomerase in tandem orientation. This enzyme may assist in trans-to-cis isomerization of Pro-5, a step considered essential for Ni-hook formation in NiSOD (1). An accessory protein (CbiX homologous protein) enhancing Streptomyces NiSOD activity was identified by Kim and coworkers (14). This protein resembles cobaltochelatases involved in cobalt insertion during coenzyme B12 biosynthesis and may function as a nickelochelatase. Homologous proteins are also present in marine cyanobacteria. The predicted protein in P. marinus MIT9313 consists of 371 amino acid residues and contains a C terminus (HHHH-X32-HDHDHDHDHDHDHDHSHDHSHAHYPYPHAEH-X15) which is ideal for metal binding.


   ACKNOWLEDGMENTS
 
I thank Celine Kretschmer for assistance with the in gel activity assays and Western blots, Edward Schwartz and Rainer Cramm for critical comments on the manuscript, and the Deutsche Forschungsgemeinschaft for financial support.


   FOOTNOTES
 
* Mailing address: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestraße 117, 10115 Berlin, Germany. Phone: 49-30-2093-8103. Fax: 49-30-4849-82923. E-mail: thomas.eitinger{at}rz.hu-berlin.de. Back


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Journal of Bacteriology, November 2004, p. 7821-7825, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7821-7825.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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