Mol Gen Genet (1993) 239:33M0

© Springer-Verlag 1993

Cloning and expression of the manganese superoxide dismutase gene of Escherichia coil in Lactococcus lactis and Lactobacillus gasseri Dipika G. Roy 1,3, Todd R. Klaenhammer 2'3, Hosni M. Hassan 1,2,3

Departments of Biochemistry 1 and Food Science 2 and Southeast Dairy Foods Research Center 3, North Carolina State University, Raleigh, NC 27695-7622, USA

Received: 8 August 1992 / Accepted: 29 November 1992

Abstract. The Escherichia coIi sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismu- tase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down- stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fu- sion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The re- combinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lacto- bacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Trans- formants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.

Key words: Escherichia c o l i - Superoxide dismutase - Fusion protein - Lactococcus - Lactobacillus

Introduction

Organisms that grow in aerobic environments produce oxygen free radicals, superoxidc radicals (O~-) and hy- droxyl radicals (OH') as by-products of normal oxygen metabolism (Fridovich 1975). These radicals are known to be toxic (Haugaard 1968; Gottlieb 1971) and mu- tagenic (Moody and Hassan 1982) to the cell. The antiox- idant enzymes, superoxide dismutases (SODs) and hy- droperoxidases, provide a cellular defense mechanism by scavenging 02 and H2Oz, respectively. Superoxide dis-

Communicated by H. Hennecke

Correspondence to: H.M. Hassan, Department of Biochemistry, Box 7622, NCSU, USA

mutases (E.C. 1.15.1.1) are metalloenzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide (McCord and Fridovich 1969). These enzymes are essential for defense against oxygen toxicity and are widely distributed in all aerobic organisms, and in some aerotolerant and obligate anaerobes (McCord et al. 1971; Hewitt and Morris 1975). There is a positive correlation between the concentration of SOD in an organism and its level of tolerance to oxygen (Tally et al. 1977). In Escherichia coli there are two types of superoxide dismutase, a manganese-containing enzyme (MnSOD) (Keele et al. 1970), and an iron-containing enzyme (FeSOD; Yost and Fridovich 1973), encoded by the soda and sodB genes, respectively. FeSOD is syn- thesized constitutively and is present in aerobically and anaerobically grown cells (Hassan and Fridovich 1977a). MnSOD is absent under anaerobic conditions and is rapidly synthesized under increased concentrations of oxygen (Gregory et al. 1973). The sodA gene, located at 88 rain on the E. coli chromosome, has been cloned (Touati 1983) and sequenced (Takeda and Avila 1986).

Synthesis of MnSOD in E. coli is under the control of complex regulatory mechanisms (Hassan 1989). The enzyme is inducible by various factors, such as oxygen, redox active compounds that generate superoxide radi- cals in the presence of air (Hassan and Fridovich 1977b, 1979), ferrous ion chelators under aerobic or anaerobic conditions (Moody and Hassan 1984, Pugh and Fridovich 1985) and strong oxidants (Schiavone and Hassan 1988b) and nitrate during anaerobic growth (Hassan and Moody 1987).

Aerotolerant microorganisms normally possess one or more superoxide dismutases; an exception is Lac- tobacillus plantarurn, which overcomes the lack of SOD by accumulating high intracellular concentrations of Mn 2+ (Archibald and Fridovich 1981a, G6tz et al. 1980) which can scavenge O i (Archibald and Fridovich 198 l a and 1981b). Several other lactic acid bacteria have been tested for the ability to scavenge 02 and many of these were found to be sensitive to oxygen (e .g.L. casei, L. bulgaricus, L. acidophilus), and lacked SOD or the Mn-scavenging system, while the more O2-tolerant

34

Table 1. Bacterial strains and plasmids

Strains Relevant Genotypes/phenotype Source/reference

Escheriehia coli

GC4468 QC779 DH5~ NC554 NC555 NC556 NC557 V850 CB1

Laetococcus lactis

LM0230e NC558 NC559 NC560 NC561 NC562

Laetobacillus gasseri

NCK100 ADH NK101 ADH NCK334 NC763 NC764 NC765 NC766 NC767

Lactobaeillus acidophilus

NCK89

Plasmids pSN(-)4 pGK12 pGKSOD pMG36e pDGH3 pDGH4

FALacU169 rpsL GC4468 with sodA-25, sodB1-A2 supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 DH5ct transformed with pDGH3 DH5c~ transformed with pDGH4 QC779 transformed with pDGH3 QC779 transformed with pDGH4 K-12, Met- Thi- Gal- NaP Rif r HsdR- EM s araD139, A (ara, leu) 7697, AlaeX74 gal U, gall(., hsr, hsm, strA

L. lactis host strain LM0230e transformed with pMG36e LM0230e transformed with pDGH3 LM0230e transformed with pDGH3 LM0230e transformed with pDGH4 LM0230e transformed with pDGH4

Bac r Bac + Adh + (~adh+)(pTRK15) Bac r Bac + Adh + (q~adh+)(pTRK15) str-lO spe-ll ATCC33323-neotype ATCC3323 transformed with pMG36e ATCC3323 transformed with pDGH3 ATCC3323 transformed with pDGH3 ATCC3323 transformed with pDGH4 ATCC3323 transformed with pDGH4

Laf- Laf + Lac- str-6 rif-7 (pPM4) (pPM27)

pBluescript SK(-) with sodA Cm r, Em r pGK12 with the E. eoli sodA Expression vector with Lactococcus promoter, Em r pMG36e with the sodA gene pMG36e with the soda gene, same construct as in pDGH3, but diffes in expression of MnSOD

Carlioz and Touati (1986) D. Touati Hanahan (1983) This study This study This study This study T. Klaenhammer C. Bloch

T. Klaenhammer This study This study This study This study This study

Luchansky et al. (1988) Luchansky et al. (1988) T. Klaenhammer This study This study This study This study This study

Muriana and Ktaenhammer (1991)

Naik and Hassan 1990 Kok et al. (1984) This laboratory van de Guchte et al. (1988) This study This study

Streptococcus strains tested were positive for superoxide dismutase (Archibald and Fridovich 1981b).

The goal of this research project was to introduce the superoxide dismutase gene into selected lactic acid bac- teria that lack this enzyme. The work presented in this report demonstrates the successful cloning and ex- pression of the sodA gene f rom E. coli into Lactococcus lactis and Lactobacillus gasseri using a Lactococcus ex- pression vector, pMG36e (Van de Guchte et al. 1989).

Materials and methods

Bacterial strains and growth media. The bacterial strains and plasmids used in this study are listed in Table 1. E. coli cells were grown at 37 ° C in Luria-Bertani (LB) medium (Miller 1972) supplemented with the appro- priate antibiotic when needed. The antibiotics were add- ed at the following concentrations: Cm, 20 gg/ml; Ap, 100 gg/ml; Kin, 50 txg/ml and Era, 200 gg/ml for E. coli.

For superoxide dismutase assays, E. coli was grown in LB with or without 1 m M MnC12 before the preparat ion of cell-free extracts. The medium was supplemented with 1% glucose (LBG) for anaerobic growth. Anaerobic growth conditions were maintained by using a Coy anaerobic chamber (Moody and Hassan 1984). Anaerob- ic cell growth was stopped by the addition of 12.5 ~tg/ml of tetracycline before removing the cells f rom the cham- ber and placing on ice.

Lactococcus lactis (LM0230e) cells were grown at 30 ° C in glucose M17 (Terzaghi and Sandine 1975) without shaking. Lactobacillus gasseri ATCC33323 cells were grown at 37 ° C in MRS medium (Difco Laboratories, Detroit Mich.) without shaking. Erythromycin con- centrations used were 1.5 ~tg/ml for L. lactis and 60 gg/ml for L. gasseri.

Materials. Methyl viologen (paraquat), 2,2'-dipyridyl, xanthine, xanthine oxidase, horse cytochrome c, and

erythromycin were purchased from Sigma (St. Louis, Mo.). DNA-modifying and restriction enzymes were ob- tained from Promega (Madison, Wis.) or BRL (Grand Island, NY). The [a-32p]dCTP (800 Ci/mmol) was pur- chased from DuPont/NEN (Boston, Mass).

Clonin9 and screening. The structural region (from nu- cleotide +41 to +901) of the sodA gene (Takeda and Avila 1986) was amplified in a Genetic Thermal Cycler Model GTC-2 (Precision Scientific, Chicago, Ill.) ac- cording to the procedure provided by the manufacturer. The sodA gene cloned in the plasmid p S N ( - )4 (Naik and Hassan 1990) was used as template. The upstream primer, 5 '-GGAGATGAATATGAGCTCTACCC-3' generated a SacI and the downstream primer 5'-GAG- GAAGCTTACGCATCAGCC-Y generated a HindIII site. The amplified product was digested with SacI and HindIII to give a fragment of 861 bp, which was ligated with pMG36e at the SacI and HindIII sites to yield the plasmid pDGH. All plasmid isolations were done as described in Sambrook et al. (1989).

E. coli strains were transformed according to the CaC12 method (Sambrook et al. 1989) and transformants were screened by plating on LB medium with 200 gg/ml of erythromycin. L. lactis and L. 9asseri cells were trans- formed by electroporation in a BioRad Gene Pulser using a 0.2 cm cuvette, according to previously described procedures (Luchansky et al. 1988; Holo and Nes 1988). L. lactis transformants were plated on M17-glucose medium with 1.5 lag/ml of erythromycin and L. 9asseri transformants were selected on MRS plates with 60 gg/ ml of erythromycin.

All erythromycin-resistant transformants were further screened (Sambrook et al. 1989) for the presence of plasmids with sodA inserts. The DNA probe was a 1.05kb AvaI fragment containing the entire sodA sequence from pSN4 (Naik and Hassan 1990), labelled with [0t-32p]dCTP by random primer extension (Feinberg and Vogelstein 1984).

Gene product analysis. E. coIi, L. lactis subsp, lactis, and L. 9asseri ATCC33323 transformants that carried the plasmid with the sodA gene were analyzed for SOD activity and the presence of MnSOD protein (SodA). Individual colonies of E. coli were initially screened for the expression of an active SOD protein by the plate- assay method (Schiavone and Hassan 1988a). Dialyzed cell-free extracts were prepared as previously described (Moody and Hassan 1984). Protein was assayed by the Folin-phenol reaction (Lowry et al. 1951).

The SOD isozymes were separated on 10% non-dena- turing slab polyacrylamide gels (Davis 1964) and stained with nitroblue tetrazolium for SOD activity (Beauchamp and Fridovich 1971). The cytochrome c method (McCord and Fridovich 1969) was used for the SOD liquid assays. Total superoxide dismutase protein was analyzed on Western blots (Towbin et al. 1979) using either denaturing or non-denaturing gels. The non-dena- turing gels were soaked in the blotting (20 mM TRIS- HC1, 0.5 mM NaC1, pH 7.5) buffer for 1 h before electro- blotting onto nitrocellulose membranes. The polyclonal

35

anti-MnSOD antibodies were used at a dilution of 1:200 and the horseradish peroxidase conjugate of goat anti- rabbit immunoglobulin at a dilution of 1:2000.

Results

Cloning of sodA in pGK12 and pMG36e

pGK12 shuttle vector. The 1.05 kb AvaI fragment from p S N ( - ) 4 (Naik and Hassan 1990), containing the ex- pression signals and the entire soda gene of E. coli was cloned into the HpaII site of the shuttle vector pGK 12 (Kok et al. 1984) to yield the plasmid pGKSOD. The plasmid, pGKSOD, was transformed by electroporation into two E. coli strains [V850 and CB1 (AsodA)] and into Laetobacillus gasseri ADH. E. coIi transformants were shown to express MnSOD as measured by SOD activity and Western blots (S. Naik and H. Hassan, data not shown). However, the sodA gene in pGKSOD was not expressed in the Lactobacillus ADH transformants. These findings suggested that E. coli sodA promoter is not recognized by the transcription/translation system of L. gasseri.

pMG36e expression vector. The expression vector, pMG36e (Van de Guchte et al. 1989) has a partial open reading frame (ORF) of eleven codons with its promoter and ribosomal binding site that originates from Lacto- coccus lactis subsp, eremoris Wg2. Immediately down- stream from the first eleven codons there is a multiple cloning site followed by a terminator. The E. coli sodA gene was amplified without its promoter or Shine- Datgarno sequence and then digested with the restriction enzymes Sad and HindIII. Cleavage with SacI elimi- nates the start codon of the sodA gene. The digested fragment was ligated into the SacI and HindIII sites in pMG36e. This construct (pDGH) codes for a fusion protein, with the first thirteen amino acids coded by the vector pMG36e and fused, in-frame, to the entire SodA protein minus its first amino acid. The ligation mix was used to transform E. coli strain DH5m DH5a was used for the initial cloning experiment, because the E. coli strain QC779, used to test the expression of sodA from the plasmid pDGH, is recA + and has been shown to cause structural modifications in plasmids (Haas and Goebel 1991). The plasmid present in erythromycin- resistant DH5~ transformants was isolated and analyzed by Southern hybridization using the sodA probe from pSN4.

Plasmids carrying the sodA insert were then used to transform the QC779 strain, which does not express endogenous Sod. Thus, any superoxide dismutase activ- ity in transformed QC779 cells must result from the sodA carried on pDGH. QC779 transformants that were eryth- romycin resistant were screened for SOD activity by the plate assay method (data not shown). Two of the SOD- positive clones, NC556 and NC557, were selected for further analyses. The two clones had been transformed with same ligation mix, but they differed in the amounts of enzyme produced. This difference could have been caused by changes within the sodA sequence generated

36

either during the PCR step or at the ligation step. The plasmids pDGH3 and pDGH4 from NC556 and NC557 were digested with SacI and HindIII and were both shown to have a 861 bp insert. Thus, subsequent experi- ments were done with both clones, NC556 and NC557, containing the pMG36e:: sodA recombinant plasmids pDGH3 and pDGH4, respectively.

Expression of SodA from pDGH3 and pDGH4 in aerobically 9rown E. coli cells

Cell-free extracts from GC4468, QC779, NC556, and NC557 were prepared and analyzed for the expression of

A 1 2 3 4

rMnSOD

MnSOD

HySOD

FeSOD

recombinant sodA gene by activity gels, Western blots, and assayed for superoxide dismutase (U/mg). Figure 1A is an activity gel of the enzymes expressed by these strains. GC4468, which is wild-type for sodA and sodB, shows the normal pattern of SOD isozymes (lane 1). The host strain, QC779, which is sod-, has neither MnSOD nor FeSOD (lane 2), while QC779 harboring the plasmid pDGH3 or pDGH4, showed a single activity band of lower mobility. This apparent recombinant protein product (rMnSOD) must be encoded by the gene carried on the plasmid (lanes 3 and 4), since QC779 has no sodA nor sodB gene. The mobility of the rMnSOD from strains NC556 (QC779/pDGH3) and NC557 (QC779/pDGH4) was lower than that of MnSOD, which may be due to the additional thirteen amino acids introduced during clon- ing.

The total amount of MnSOD antigen produced by these cells was also analyzed by Western blotting (Fig. 1B). The MnSOD antibody binds specifically to a single band confirming the presence of MnSOD in GC4468, NC556 and NC557; this binding is absent in QC779. In a 10% denaturing gel, the rMnSOD protein seems to migrate to about the same position as the wild-type MnSOD (Figure 1B), while in a more concentrated gel the rMnSOD protein has a slightly lower mobility than wild-type (data not shown). The significant difference in protein mobility on nondenaturing but not denaturing gels is probably due to changes in the electric charge caused by the additional thirteen amino acids.

B 1 2 3 4

Fig. 1. MnSOD activity and antigen in cell-free extracts from E. coli cells harboring plasmids pDGH3 or pDGH4. Cultures were grown aerobically at 37 ° C in LB medium containing 1 mM MnC12. Cell- free extracts were prepared and assayed for SOD activity and protein content on a non-denaturing 10% polyacrylamide (A), by Western blotting analysis under denaturing conditions (B). Fifty gg of total protein were loaded in each lane. Lane i, GC4468 ; lane 2, QC779; lane 3, NC556; and lane 4, NC557. MnSOD, E. eoli Mn- containing SOD; FeSOD, E. coli Fe-containing SOD; HySOD, hybrid Mn/FeSOD; rMnSOD, recombinant E. coIi MnSOD

Effects of paraquat and M n 2+ on the aerobic expression of SodA from pDGH3 and pDGH4 in E. coli cells

Under aerobic conditions, sodA in wild-type E. coli is induced by redox active compounds like paraquat, (pQ2 +) which generate superoxide radicals (Hassan and Fridovich 1977b, 1979). We asked whether this induction is lost when the sodA gene is under the control of Lactococcus expression signals. Data in Table 2 showed that addition of 0.1 mM pQ2+ resulted in a significant increase in total SOD in wild-type E. coli (sodA+), while there was no significant effect on the expression of the sodA synthesized under the control of the Lactococcus promoter (compare columns 4 and 5, Table 2). However, the total SOD activity in the wild-type strain (GC4468) and in the two clones (NC556 and NC557) increased on

Table 2. Effects of paraquat and manga- nese on the aerobic expression of pDGH3 and pDGH4 in Escherichia eoli

Strain Genotype Total SOD activity (units/mg protein)

-- Mn + Mn + Mn + paraquat

GC4468 sod + 12 21 65 QC779 sod ND ND ND NC556 QC779/pDGH3 7 26 25 NC557 QC779/pDGH4 42 118 140

Cultures were grown in LB +/--MnC12 (1 mM) at 37 ° C with constant shaking at 200 rpm. The paraquat-treated cells were grown to an initial OD6oo~0.1 before the addition of 0.1 mM paraquat. Cells were harvested at an OD6oo ~ 1.0 and dialyzed cell-free extracts were prepared and assayed as described in Materials and methods ND, not detected

Wt N C 5 5 6 N C 5 5 7 ~1 2 3 ' ~1 2 31 I1 2 31

Fig. 2. Effect of manganese and paraquat on the aerobic expression of SodA antigen in strains GC4468, NC556, and NC557. Growth conditions were the same as for Table 2. Ten gg of protein was loaded for each sample. Lanes 1, no Mn 2+ ; lanes 2, treated with 1 mM Mn 2 + ; lanes 3, treated with 1 mM Mn 2 + plus 0.1 mM para- quat

addit ion o f the cofac tor manganese (compare columns 3 and 4, Table 2).

Figure 2 indicates that the s t imulatory effect o f M n 2 + occurred at the post- t ransla t ional level, as the concentra- t ion o f M n S O D antigen did not change upon adding the cofac tor (compare lanes 1 and 2 for each strain). The addi t ion o f pQ2+ and M n 2+ together did no t affect the concent ra t ion o f M n S O D antigen in NC556 and NC557, while there was a significant increase o f de novo synthesis o f this protein in the wild-type strain (GC4468).

Expression of SodA from pDGH3 and pDGH4 in anaerobically grown E. coli cells

In E. coli, the sodA gene is normal ly repressed by anaero- biosis (Hassan and Fr idovich 1977a; Hassan 1989). Since this cis-acting regula tory elements o f the soda gene were no t included in the cloned constructs ( p D G H 3 and p D G H 4 ) , it was o f interest to s tudy the anaerobic ex- pression o f sodA f rom these plasmids to determine whether the gene is also regulated at the level o f trans- la t ion/post t ranslat ion. D a t a in Table 3 show that addi- t ion o f M n 2+ increased the activity o f r M n S O D in NC556 and NC557 while having no effect on the S o d a antigen (Fig. 3). Cell-free extracts f rom the wild-type strain g rown anaerobical ly in the presence or absence o f

37

W t f! 2 3 J '1 2

N C 5 5 6 N C 5 5 7 I I ,r

3 1 2 3

Fig. 3. Effects of manganese and 2,2'-dipyridyl on the anaerobic expression of SodA antigen in strains GC4468, NC556, and NC557. Twenty gg of protein were loaded (under denaturing conditions) for each sample. Lanes 1, no Mn 2+ ; lanes 2, treated with 1 mM Mn z+ ; lanes 3, treated with 1 mM Mn 2+ plus 0.25 mM 2,2'-dipyridyl

M n 2 + did no t conta in active M n S O D (Table 3) or SodA antigen (Fig. 3, lanes 1 and 2). The addi t ion o f the iron chelator 2,2 '-dipyridyl (0.2 mM ) increased the activity o f M n S O D in wild-type E. coli f rom 0 to 5.3 U/ rag (Table 3) with an increase in S o d a antigen (Fig. 3, Wt/ lane 3). This indicates that 2,2'-dipyridyl acts on the wild-type gene at the transcript ional and post- t ranslat ional levels, as previously shown (Schiavone and Hassan 1988b). On the other hand, addi t ion o f M n 2 + alone increased the activities o f M n S O D in NC556 and NC557 (Table 3, co lumn 3), while having no effect on SodA antigen (Fig. 3, lanes 2). These results suggested that the effect o f M n on the anerobic expression of sodA f rom p D G H 3 / p D G H 4 in E. coli is exerted at the post- t ranslat ional level. However , the addi t ion o f M n 2 + plus 2,2 '-dipyridyl to NC556 and NC557 resulted in a 2-fold increase in M n S O D (Table 3) and a slight increase in SodA antigen (Fig. 3, lanes 3) relative to that in the presence o f Mn z+ alone. This apparen t increase in SodA synthesis could be explained by a possible increase in the stability o f the recombinan t prote in in the presence o f the i ron chelator.

Expression of SodA from pDGH3 and pDGH4 in L. lactis

The sodA gene in the plasmids p D G H 3 and p D G H 4 is expressed under the control o f a Lactococcus promoter .

Table 3. Effect of manganese and 2,2'-dipyridyl on the anaerobic expression of SodA in Eseheriehia eoli cells harboring pDGH3 and pDGH4

Strain Total SOD Activity (units/mg protein)

-- Mn + Mn + Mn + 2,2'-dipyridyl

GC4468 6 (0)a 6 (0) 7 (5) NC556 b 2 42 100 NC557 b 8 58 135

Cultures were grown anaerobically in LBG +/--MnC12 (1 mM). Cells treated with the iron chelator were grown to an initial OD6o 0 ~ 0.1 before the addition of 0.25 mM 2,2'-dipyridyl. Growth was stopped at an OD6oo~0.7 by the addition of 12.5 pg/ml of tetracycline. Cells were harvested and dialyzed cell-free extracts were prepared and assayed as described in Fig. 2 and Table 2

Numbers in parentheses represent the specific activity of MnSOD in GC4468 b Numbers represent MnSOD; the host strain QC779 is sod-

Table 4. Total SOD activity expressed by Lactococcus lactis subsp. laetis

Strains Genotype Total SOD a (units/mg protein)

LM0230e Host 14 NC558 LM0230e/pMG36e 12 NC559 LM0230e/pDGH3 u 18 NC560 LM0230e/pDGH3 b 38 NC561 LM0230e/pDGH4 b 14 NC562 LM0230e/pDGH4 b 17

a Cultures were grown in M17G with 1 mM MnC12 at 37 ° C without shaking. Cells were harvested at an OD600 ~ 1.0 and dialyzed cell- free extracts were prepared and assayed as described in Materials and methods b Physical analysis of plasmids in these transformants revealed variations in plasmid size, suggesting structural alterations of these plasmids

38

A

B

1 2 3 4 5 6 7 8 9

! 2 3 4 5 6 7 8 9

~ a

*-b

~ C

~ d

~ a

~ b

~ C

but the insert appeared to be of the right size in all cases (data not shown). No further efforts were made to ac- count for the variation in levels of SOD expression among the L. lactis transformants harboring pDGH3 and pDGH4.

L. lactis has an endogenous superoxide dismutase activity which was detected in all the samples including the host strains with or without pMG36e (Fig. 4A, lanes 4 and 5). This resident SOD was identified as a man- ganese-containing SOD, based on its resistance to hy- drogen peroxide and cyanide, according to the diagnostic protocols described previously (Asada et al. 1975). Lactococcus cells transformed with the plasmid pDGH3 or pDGH4 (Fig. 4A) also synthesize the rMnSOD pro- tein (band a, lanes 6 to 9), which migrates at the same position as the rMnSOD in the E. coli samples (lanes 2 and 3). In addition to the rMnSOD, these Lactococcus transformants had an additional band (band c, lanes 6 to 9), which migrates between the rMnSOD (band a) and Lactococcus SOD (band d). This migration pattern is found only for Lactococcus cells transformed with pDGH3 and pDGH4, indicating that band c may be a hybrid molecule formed from subunits of both rMnSOD and Lactococcus SOD. Figure 4B shows a Western blot of a non-denaturing gel, identical to that used for activity staining (Fig. 4A). The E. coli MnSOD antibody does not recognize the Lactococcus SOD, but does bind to the hybrid molecule and to the rMnSOD. Thus, the hybrid enzyme appears to contain one or more subunits of the rMnSOD.

Fig. 4. Expression of SodA from the plasmid pDGH3 or pDGH4 in L. laetis subsp, lactis (A) Activity Gel (B) Western blot of a non- denaturing gel identical to the one shown in (A). Twentyfive lag of protein was loaded for the E. coli samples (lanes 1-3) and 50 lag for the L. lactis samples (lanes 4-9). Lane 1, GC4468; lane 2, NC556; lane 3, NC557; lane 4, LM0230e; lane 5, NC558; lane 6, NC559; lane 7, NC560; lane 8, NC561; and lane 9, NC562. The various SODs are designated as follows: a, E. coli recombinant MnSOD; b, E. coli MnSOD ; c, Hybrid between E. coli rMnSOD and L. lactis SOD; d, L. lactis SOD

This construct was evaluated for expression in the gram- positive lactococcal cells. Plasmid DNA from the DH5~ clones (NC554 and NC555) was isolated and used for transformation of L. lactis subsp, lactis (strain LM0230e). Transformed cells were analyzed for the plas- mid carrying the sodA gene by Southern hybridization (data not shown). Five of the transformed colonies were then analyzed for sodA expression by activity gels, West- ern blots, and SOD liquid assays (Fig. 4 and Table 4).

The data in Table 4 show that strains transformed with the plasmid pDGH3 or pDGH4 expressed more SOD activity than did the host strain carrying the vector pMG36e (NC558). However, only one transformant, NC560, showed a significant increase in the total SOD activity. The sodA plasmids in the strains NC559, NC561, and NC562, may have suffered rearrangements, resulting in lower expression of SOD. The plasmid DNA from these clones was isolated and analyzed by restric- tion digestion and electrophoresis. There were variations in the plasmid sizes obtained from the different clones,

Expression of SodA from pDGH3 and pDGH4 in L. gasseri

We also introduced pDGH3 and pDGH4 into the L. 9asseri neotype strain ATCC33323. The eryth-

A 1 2 3 4 5 6

r M n S O D

B 1 2 3 4 5 6

-,-r M n S O D

Fig. 5. Expression of sodA from the plasmid pDGH3 or pDGH4 in Lactobaeillus 9asseri ATCC33323 A activity gel B Western blot (de- naturing conditions). Lane 1, host strain ATCC33323; lane 2, NC763; lane 3. NC764; lane 4, NC765; lane 5, NC766; lane 6, NC767. Each sample contained 200 lag of total protein

39

Table 5. Expression of pDGH3 or pDGH4 in Lactobacillus gasseri

Strain Genotype Total SOD activity (units/mg protein)

334 Host 0.0 NC763 ATCC3323/pMG36e 0.0 NC764 ATCC3323/pDGH3 0.9 NC765 ATCC33323/pDGH3 0.9 NC766 ATCC3323/pDGH4 0.8 NC767 ATCC33323/pDGH4 0.9

Cultures were grown in MRS medium at 37 ° C without shaking. Cells were grown to an OD60o~ 1.0, before harvesting. Cell-free extracts were prepared and assayed as described in Materials and methods

romycin-resistant transformants were analyzed for SOD expression. A single band of activity was observed in L. gasseri transformed with pDGH3 or pDGH4, while no activity is seen in the host or in L. gasseri transformed with the vector pMG36e (Fig. 5A, lanes 1 and 2). The results of the Western blot (Fig. 5B) confirm the results of the activity gel (Fig. 5A) and demonstrate that sodA is expressed in L. gasseri ATCC33323. The total specific SOD activity (Table 5) produced was low. This may be due to poor expression of the Lactococcus P32 promoter in Lactobacillus. Neither pDGH3 nor pDGH4 could be successfully transformed into the L. gasseri strains ADH or into the Lactobacillus acidophilus strain NCK89.

Discussion

Lactic acid bacteria are important microorganisms wide- ly used in the food and dairy fermentation industries. Their widespread use and significance in food conver- sions and preservation have provided the opportunity to develop new strains with specialized roles. Lactic acid bacteria that colonize the intestinal tract could be used as a delivery vehicle for therapeutic agents. Lactic acid bacteria that express the antioxidant enzyme superoxide dismutase could also be used to prevent lipid peroxida- tion of dairy products. Such bacteria delivered to the gastrointestinal tract might also provide a defense mech- anism against inflammatory diseases of the bowel. In this study, we have successfully cloned and expressed the soda gene of E. coli in Lactococcus and Lactobacillus bacterial strains that are model hosts for subsequent evaluations of such applications.

The transcription and translation signals of E. coli genes seem not to be recognized by lactic acid bacteria. Thus, when the plasmid containing the entire soda gene of E. coli, pGKSOD, was introduced into L. gasseri ADH, it failed to be expressed. However, when the struc- tural region of sodA was cloned in the vector pMG36e, in which the expression of the gene is under the control of Lactocoecus expression signals, the resulting chimeric gene was expressed in E. coli and in the lactic acid bac- teria, L. lactis subsp, lactis LM0230e and L. gasseri ATCC33323. Transcription and translation of this chimeric gene was shown to be regulated by the Lacto-

coccus expression signals and, therefore, was not subject to the regulatory elements found in wild-type E. coll. This construct is thus useful for future studies on post-transla- tional regulatory mechanisms of sodA in E. coli.

L. lactis subsp, lactis contains a native Sod protein, identified as MnSOD, which did not cross react with antibodies against the E. coli MnSOD (Fig. 4B). How- ever, subunits of the two proteins (i.e. rMnSOD and L. Iactis MnSOD) seemed to be compatible at the pri- mary/secondary structural levels as they were able to interact and form an active heterologous protein (Fig. 4). A similarly active heterologous SOD (i.e. hybrid SOD) is found in aerobically grown wild-type E. coli and is composed of subunits from the resident FeSOD and MnSOD (Fig. 1; Hassan and Fridovich 1977a).

The E. coli sodA gene with a Lactococcus promoter was also expressed in Lactobacillus gasseri ATCC33323. The protein produced in this host shows SOD activity, as demonstrated by activity gel and liquid SOD assay and is recognized by the antibody against the E. coli MnSOD. The weak expression (Fig. 5) may be due to inefficient activity of the Lactococcus promoter in a Lac- tobacillus host. Future work will include efforts to in- crease the expression of MnSOD in Lactobacillus by using other promoters. We also tried to transform other strains of Lactobacillus, such as NCK101 and NCK89, with the plasmids pMG36e, pDGH3 and pDGH4, but failed to recover any transformants.

The wild-type soda gene in E. coli was inducible by air, paraquat and 2,2'-dipyridyl, in agreement with previous reports (Hassan 1989). On the other hand, the soda gene under a Lactococcus promoter was expressed under both aerobic and anaerobic conditions and was not inducible by paraquat, but the rMnSOD protein seemed to be more stable in the presence of 2,2'-dipyridyl (Table 3 and Fig. 3). These data suggest that induction of the wild-type sodA by paraquat and 2,2'-dipyridyl occurs at the transcriptional or translational levels, as previously reported (Hassan and Moody 1987; Hassan 1989). The co-factor Mn 2+ is necessary for an active MnSOD. Addition of MnCI2 increased the total SOD activity, but not the amount of protein produced (Tables 2, 3 and Fig. 2, 3). This indicates that Mn regulates MnSOD activity at the post-translational level, in agree- ment with reports by Touati (1988) and Beyer and Fridovich (1991).

Acknowledgements. This work was supported by grants from the Southeastern Dairy Food Research Center and by DCB-8910153 from NSF. We wish to thank Dr. D. Touati for QC779, Dr. C. Bloch for CB1, Dr. J. Kok for the plasmids pGK12 and pMG36e, Dr. D. Clare for MnSOD antibodies, and Evelyn Durmaz and Graciela DeAntoni for their contributions to the transformation of L. Iactis and L. gasseri, respectively.

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