Appl Microbiol Biotechnol (1992) 37:216-224

Applied AFwrobiology Biotechnology © Springer-Verlag 1992

Lysozyme expression in Lactococcus lactis Maarten van de Guehte*, Fimme Jan van der Wal, Jan Kok, and Gerard Venema Department of Genetics, Centre of Biological Sciences, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Received 16 September 1991/Accecpted 8 January 1992

Summary. Three lysozyme-encoding genes, one o f eukaryotic and two o f prokaryotic origin, were expressed in Lactococcus lactis subsp. Iactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence o f correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes o f the E. coli bacteriophages T4 and ). were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame o f lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

Introduction Traditionally, lactic acid bacteria are used in a variety of dairy and other fermentations. They play an important role in the development of flavour and texture, and in the conservation o f the products. However, growth o f spoilage bacteria cannot always be prevented by the mere acidification o f the product resulting from the production o f lactic acid. Therefore, food preservatives are often added. For example, during the manufacturing of G o u d a cheese, nitrate is added to prevent late blowing, a cheese defect that it characterized by an abnormally open texture and unattractive flavours, caused by the outgrowth of Clostridium tyrobutyricum spores. As a (partial) substitute for nitrate, hen egg white lysozyme (HEL) can b e used (Crawford 1987). This enzyme is reported to have antimicrobial activity against several bacteria involved in food spoilage and food-borne disease, including C. tyrobutyricum and Lys* Present address: National Food Biotechnology Centre, University College, Cork, Ireland Correspondenceto: G. Venema

teria monocytogenes (Hughey and Johnson 1987; Hughey et al. 1989). In the research presented here, three genes encoding different lysozymes, with a possible application in the prevention of food spoilage, were examined for whether they can be expressed in Lactococcus lactis. Recent years have seen rapid progress in rendering lactococci amenable to genetic manipulation. Transformation systems and cloning vectors have been developed (Kok et al. 1984; van der Lelie et al. 1988; van der V o s s e n et al. 1988) and gene expression signals have been isolated and characterized (de Vos 1987; van der Vossen et al. 1987). These tools have been used for the expression o f several heterologous genes (de Vos et al. 1989; van der Guchte et al. 1989, 1990, 1991). Recently, we reported on improved gene expression in L. lactis by the use of translational coupling (van de Guchte et al. 1991). In the work presented here we examined whether this strategy could be used to enhance the expression o f two lysozyme genes o f bacteriophage origin in L. lactis.

Materials and methods Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and Bacillus subtilis were grown in TY broth (Rottlander and Trautner 1970) or on TY broth solidified with 1.5% agar. In B. subtilis protoplast transformation, DM3 was used as the plating medium (Chang and Cohen 1979). L. lactis was grown in glucose M17 broth (Terzaghi and Sandine 1975), or on glucose M17 broth solidified with 1.5% agar. Sucrose (0.3 M) was added to these media to osmotically stabilize electroporated L. lactis cells. Erythromycin was used at a concentration of 100 Ixg/ml for E. coli and 5 I~g/ml for B. subtilis and L. lactis. DNA manipulations and transformation. Plasmid DNA was isolated by the method of Birnboim and Doly (1979). Bacteriophage T4 DNA (T4dC(+), amC87(42-), amE51(56-), denB-s19, unf39(alc)) (Takahashi et al. 1978) was obtained from Amersham International (Amersham, UK). Restriction enzymes, Klenow enzyme, and T4 DNA ligase, were purchased from Boehringer (Mannheim, FRG) and used according to the instructions of the supplier. Taq DNA polymerase was obtained from Promega (Ma-

217 Table 1. Bacterial strains and plasmids

Bacterial strain or plasmid

Relevant feature(s)

Source or reference

830 supE +, supF +, rE, m~, metBL21 (F-, hsdS, gal, rff, mff) DE3 lysogen carrying the T7 RNA polymerase gene in the chromosome under control of the inducible lacUV5 promoter araD139, AlacX74, A(ara, leu)7697, galU. galK, strA

Hohn (1979) Studier and Moffat (1986)

Bacteria Escherichia eoli BHB2600 BL21(DE3)

MC1000

Lactococcus lactis subsp, lactis Plasmid-free strain IL1403 Bacillus subtilis PSL1

Casadaban and Cohen (1980) Chopin et al. (1984)

r-, m - , stp, recE4

Ostroff and P6ne (1983)

pAP19

Ap r, carrying the bacteriophage 2. R gene

plL253

Em r, high copy number vector for use in L. lactis

plL37

Em r, pIL253 derivative containing the HEL coding sequence and expression signals from pMG37 Em r, lactococcal expression vector

Jespers et al. (unpublished) Simon and Chopin (1988) This work

Plasmids

pMG36e pMG36eHEL pGM37 pMLR0,1,2,5 pMT40-42, pMT44-45 pT712 pTTR pTC6

Em ~, pMG36e derivatice encoding a mature hen egg white lysozyme (HEL) fusion protein Em r, PGM36eHEL derivative encoding mature HEL Em r, various pMG36e derivatives carrying the )~ R gene Em ~, pMG36e derivatives encoding T4 lysozyme Ap r, expression vector containing a T7 promoter Apt; pT712 derivative carrying the A R gene Em r, pMG36e derivative in which ORF32 and lacZ are translationally coupled

dison, Wis., USA). A Bio-med thermocycler 60 (B. Braun, Melsungen, FRG) was used for polymerase chain reaction (PCR)-mediated DNA amplification. Site-directed mutagenesis was performed using the pMa/c gapped duplex method described by Stanssens et al. (1989). Oligonucleotides were synthesized using an Applied Biosystems 381A DNA synthesizer (Applied Biosysterns, Foster City, Calif., USA), and had the following 5 ' ~ Y sequences: 1, CCC GGG TCG ACT TAG GAG GTA "IrA TGA ATA TAT TTG AAA TGT TAC GTA TAG A; 2, AGA TCT GCA GTT ATA GAT T I T TAT ACG CGT CCC AAG TGC CA; 3, ATG AAT ATA T I T GAA ATG TTA CGT ATA GA; 4, TTC AAA TAT ATT CAT T r c AAA ATT CCT CCG AAT ATT TTT TrA CCT ACC; 5, GCC TCC TCA TCC TCT TCA TCC TC; 6, T I C AAA TAT ATT CAT TFI" T I T CCT CCT TGA GGA TCG ATC CCC GGG (all used for the construction of plasmids pMT40, pMT41, and pMT44); 7, GCT CAC ATC GTC CAA AGA CTT TCA TTT CAA AAT TCC TCC GAA TA (used to delete ORF32 in the construction of pMG37); 8, TCT GCT GAA ACG ATT GCC CCG GGA AAA TIC CTC CGA ATA T (used to change the sequence GAAA TG into CCCGGG in the construction of pMT45 and pMLR2); 9, CGT TGA TTA TTG ATT TCT ACC ATT TCA AAA TTC CTC CGA AT (used to delete ORF32

van de Guchte et al. (1989) van de Guchte et al. (1989) This work This work This work Tabor and Richardson (1985) This work van de Guchte et al. (1991)

in the construction of pMLRS). Unless stated otherwise, all plasmids were constructed using E. coli as a host for transformation following the method of Mandel and Higa (1970). Protoplasts of B. subtilis were prepared and transformed using the procedure of Chang and Cohen (1979). Plasmids were introduced into L. lactis subsp, lactis by means of electroporation (van der Lelie et al. 1988). DNA sequence analysis and in-vitro transcription and translation. Relevant parts of newly constructed plasmids were checked by DNA sequencing. Thb sequence analyses were performed according to Tabor and Richardson (1987) using the T7 polymerase system (Pharmacia, Uppsala, Sweden) on denatured plasmid DNA. The prokaryotic DNA-directed translation kit from Amersham International was used for in-vitro transcription and translation. Proteins were visualized by autoradiography after separation on sodium dodecyl sulphate (SDS)-polyacrylamide (15%) gels. Molecular mass standards were obtained from Amersham International. Production of ,~,-lysozyme-specific antibodies. To produce 2,-lysozyme-specific antibodies, the A R gene was overexpressed in E.

218

coli using the T7 RNA polymerase overexpression system. The gene was recloned in pT712 (Tabor and Richardson 1985), in which the SacI-EcoRI fragment of pAP19 (Jespers et al. unpublished data) was inserted downstream of the T7 promoter to give pT7R. In E. coli BL21(DE3) carrying plasmid pT712 or pT7R, T7 RNA polymerase was induced by the additon of isopropyl-fl-Dthiogalactoside (IPTG, final concentration: 0.4 mM) to a culture with an optical density at 600 nm (OD6o0) of 0.3-0.4. After incubation for 1 h at 37 ° C, rifampicin was added to a final concentration of 200 lxg/ml. After an additional 1 h incubation, cells were collected by centrifugation, and resuspended in sample buffer for SDS-polyacrylamide gel electrophoresis (PAGE) according to Laemmli (1970). Samples were applied to SDS-polyacrylamide (15%) gels, and the position of the )1.lysozyme relative to a "Rainbow" MW marker (Amersham International) was determined. The ~. lysozyme was electro-eluted from unstained gels, and concentrated in a "Biotrap" (Schleicher & Schuell, Dassel, FRG) as indicated by the manufacturer. The concentrated material was used to immunize a rabbit. )!.-Lysozyme-specific antibodies were obtained, as is evident from Fig. 6A. Immunoprecipitation, SDS-PAGE and Western blotting. Proteins produced in vitro were immuno-precipitated as described by Edens et al. (1982) using a polyclonal rabbit anti-HEL antiserum (van de Guchte et al. 1989). SDS-PAGE was performed according to Laemmli (1970). Western blotting was performed according to Towbin et al. (1979). Samples for SDS-PAGE and Western blotting were prepared as described previously (van de Guchte et al. 1989). The prestained SDS-PAGE molecular mass standards of Bio-Rad (Richmond, Calif., USA) were used as a reference on gels used for Western blotting. A polyclonal rabbit anti-HEL antiserum (van de Guchte et al. 1989) was used to detect HEL. )!.Lysozyme was detected using the 2-1ysozyme-specific antibodies described above. Assay oflysozyme activity. Lysozyme activity was assayed in either of two ways. For E. coli, a qualitative freeze-and-thaw assay was used. Ceils from 1 ml of an overnight-grown culture were pelleted by centrifugation and resuspended in 50 ixl TY broth. The suspension was frozen at - 2 0 ° C and subsequently thawed at 37 ° C. E. coli strains producing enzymatically active lysozyme lysed after this treatment. For L. lactis, a quantitative assay was used. Cells from 25 ml of an overnight culture were collected by centrifugation and resuspended in 1.5 ml of 50 mM TRIS-HC1 buffer (pH 7.0) containing 100 mM NaC1. To 1.4 ml of this cell suspension, 0.8 ml of glass beads (0.1 mm in diameter) was added, and the cells were disrupted using a "Shake it, Baby" cell disrupter (Biospec Products, Bartlesville, Okla., USA) (two 5-min cycles at maximum speed setting and 4 ° C). Cell debris was removed by centrifugation in an Eppendorf centrifuge. The protein content of the lysate was determined according to Bradford (1976). Lysozyme activity was determined in a spectrophotometric assay using lyophilized E. coli B cells (Sigma, St. Louis, Mo., USA) as a substrate. Lyophilized cells (650 mg) were suspended in 400 ml of 50 mM TRIS-HC1 (pH 7.0) and mixed with 80 ml chloroform by stirring for 10 min. The CHC13 and the water phase were allowed to separate, after which the CHC13 was removed. Immediately following this treatment, the E. coli cell suspension was used in the assay. In a microplate (Greiner 655101; Greiner, Solingen, FRG) four blank wells were filled with 50 Ixl of 50 mM TRIS-HC1 (pH 7.0), 100 mM NaC1, and eight wells were filled with 50 I.tl of the cell lysate. To each well 200 lxl of the E. coli cell suspension was added using a multipipette (t= 0). The OD450 was measured at 5-s intervals using a Titertek Multiskan MCC/340 (Flow Laboratories Int., Lugano, Switzerland). The OD readings between t= 10 and t ~ 25 s were used to calculate the AODa5o/min. In this way, for each cell lysate eight values were obtained, of which the highest and the lowest value were omitted. From the blanks the highest and the lowest value were also omitted, and the mean of the remaining two values was subtracted from the mean value of the cell lysate. The figures obtained in this way were related to a cal-

ibration curve of HEL from Sigma and, together with the protein content of the lysate, a measure of lysozyme activity ~xpressed as units of HEL equivalents per milligram protein was obtained.

Results Hen egg white lysozyme Plasmid constructions. F r o m the p l a s m i d p M G 3 6 e H E L (van de G u c h t e et al. 1989), e n c o d i n g a m a t u r e H E L fusion protein, a deletion derivative that e n c o d e s the m a t u r e H E L was c o n s t r u c t e d b y site-directed m u t a g e n esis using oligonucleotide 7 as described in Materials a n d methods. The resulting p l a s m i d was designated p M G 3 7 . The E c o R I - X m n I f r a g m e n t o f p M G 3 7 , containing a p r o m o t e r , the s e q u e n c e e n c o d i n g m a t u r e H E L , a n d a transcription terminator, was subsequently r e c l o n e d in B. subtilis in the high c o p y n u m b e r v e c t o r p I L 2 5 3 (Simon a n d C h o p i n 1988), cut with EcoRI a n d S m a I , to give pIL37. In-vitro transcription and translation. Plasmids p M G 3 6 e H E L a n d p M G 3 7 were subjected to in-vitro transcription a n d translation, f o l l o w e d b y i m m u n o p r e cipitation using a rabbit a n t i - H E L antiserum. A u t o r a d i o g r a p h y s h o w e d that p M G 3 7 specified a p r o t e i n the size of, a n d i m m u n o l o g i c a l l y related to, m a t u r e H E L (results n o t shown). H E L gene expression in B. subtilis and L. lactis. Plasmid p M G 3 7 was i n t r o d u c e d in L. lactis subsp, lactis IL1403. In contrast to results obtained with strain I L 1 4 0 3 ( p M G 3 6 e H E L ) , in cell lysates o f overnightg r o w n I L 1 4 0 3 ( p M G 3 7 ) no l y s o z y m e c o u l d be detected b y W e s t e r n blotting (results n o t shown). I n lysates o f B. subtilis PSL1 t r a n f o r m e d with p M G 3 7 isolated f r o m L. lactis I L 1 4 0 3 ( p M G 3 7 ) , the m a t u r e H E L was readily detectable (results n o t shown), indicating that no deleterious r e a r r a n g e m e n t s h a d o c c u r r e d in the l y s o z y m e gene in L. lactis. The gene was subsequently r e c l o n e d o n the high c o p y n u m b e r vector plL253 to give pIL37. This p l a s m i d gave rise to l y s o z y m e p r o d u c t i o n in B. subtilis (results n o t shown) as well as in L. lactis (Fig. 1), as was evident f r o m the results o f Western blotting. N o lysoz y m e activity c o u l d be detected in L. lactis, however. To e x a m i n e w h e t h e r this might be c a u s e d b y the a b s e n c e o f correctly f o r m e d disulphide b o n d s in the protein, samples o f IL1403(plL37) were p r e p a r e d for SDSP A G E in the p r e s e n c e or absence o f 50 mM dithiothreitol (DTT). Samples o f c o m m e r c i a l l y available purified H E L were treated identically. After electrophoresis the proteins were visualized b y Western blotting. The c o m mercial H E L a n d that was p r o d u c e d b y L. lactis migrated to identical positions after t r e a t m e n t with DTT. W h e n t r e a t m e n t with D T F was omitted, however, the proteins f r o m the two different sources m i g r a t e d to different positions (Fig. 1), suggesting that the p r o t e i n h a d n o t a d o p t e d the correct c o n f o r m a t i o n in L. lactis.

219

Fig. 1. Western blot of lysates of Lactococcus lactis (plL253) (lanes 2 and 5), and L. lactis (plL37) (lanes 1, 6 and 7). Lanes 3 and 4 contain hen egg white lysozyme (HEL; Sigma). Samples in lanes 4-7 were treated with the reducing agent dithiothreitol (DTT) as described in Materials and methods. Samples in lanes 1 to 3 were prepared without the addition of DDT. The arrows mark the position of the HEL produced by L. lactis PCR reactions

O)

~

Templotes

SD ATG. . . . T4,,mea~lene---" . . . . . . . - - . , ~ . ,

T4 genome

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P32 SD ATG - ORF32 - - ~

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EcoRl 1

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®

i

~k e

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EcoRI ® Pstl ATG---T,)• l ~ene --'~'l

pvu I i ( P32 SD ATG-ORFI32 SD ATG

®

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product

cl) product

v

Fig. 2. Schematic outline of the polymerase chain reactions (PCR) used for the construction of plasmids pMT40 (a), pMT41 (b), and pMT44 (c). P32, lactococcal promoter 32 (van der Vossen et al. 1987); ORF32, open reading frame 32 (van der Vossen et al. 1987); SD, Shine Dalgarno sequence; ATG, translational start codon; lacZ, E. coli lacZ gene; 1, 2, 3, 4, 5, 6, amplification primers the sequences of which are given in Materials and methods (see text for details)

Bacteriophage T4 lysozyme Plasmid constructions. T h e E. coli bacteriophage T4 e gene, encoding T4 lysozyme, was cloned from the T4 genome in the lactococcal expression vector p M G 3 6 e (van de Guchte et al. 1989) using the polymerase chain reaction (PCR). Oligonucleotides were designed on the basis of the known D N A sequences of the T4 e gene (Owen et al. 1983), and of pMG36e. Two different strategies were followed (Fig. 2). The first strategy (a) was aimed at the cloning of the T4 e gene with its ribosome

b jnd~gsi:te~as~:a, Sa!T-Psti fragment in the multiple cloning site o f pMG36e. The second strategy (b) was designed to clone the T4 lysozyme coding sequence directly downstream of the ribosome binding site o f ORF32 present in pMG36e. Strategy (a) employed one PCR amplification step, using T4dC D N A as a template, and two oligonucleotides. Oligonucleotide 1 contained, in the 5 ' ~ 3 ' direction, a SalI recognition sequence, 14 nucleotides of the 5'-non-coding sequence preceding the T4 e gene and comprising the Shine-Dalgarno (SD) sequence, and the first 29 nucleotides of the T4 e coding sequence. Oligonucleotide 2 contained, in te 5'--.3' direction, a PstI recognition sequence, and 31 nucleotides complementary to the 3'-end of the T4 e gene. After amplification of the T4 e gene using these primers, the PCR product was purified on an agarose gel, cut with SalI and PstI, and cloned into the vector pMG36e cut with the same two enzymes. Strategy (b) involved three PCR amplification steps. In PCR bl), T4dC D N A was used as the template. O1igonucleotide 2, described above, was used together with oligonucleotide 3, consisting o f the first 29 nucleotides of the T4 e coding sequence, to amplify this coding sequence. In PCR step b2), p M G 3 6 e was used as the template. Oligonucleotide 4 contained, in the 5'-+3' direction, 15 nucleotides complementary to the start of the T4 e coding sequence, and 33 nucleotides complementary to the non-coding sequence directly upstream of ORF32 in pMG36e. This oligonucleotide was used in combination with oligonucleotide 5 located upstream of promoter 32 to amplify the promoter area o f pMG36e contiguous with a small piece of the T4 e coding sequence. The partially overlapping products of PCR steps b l and b2 were purified on an agarose gel, and subsequently used in PCR step b3 together with the oligonucleotides 2 and 5 described above. In this way, the promoter area of pMG36e and the T4 e coding sequence were amplified as one unit. The product o f this PCR step was purified on an agarose gel, cut with PvuI and PstI, and used to replace the PvuI-PstI fragment o f pMG36e carrying promoter 32. In both strategies, (a) and (b), the ligation mixtures were used to transform E. coli to erythromycin resistance. Among the transformants, those expressing the T4 e gene were selected by means o f a freeze-and-thaw assay. The PCR-generated inserts o f a number o f positive clones were checked by D N A sequencing. On average, PCR amplification appeared to have introduced one base substitution or deletion in these clones. Plasmid pMT40 (Fig. 3), generated according to strategy (a), contained the correct T4 e coding sequence, preceded by the T4 e SD sequence from which one nucleotide had been deleted. Plasmid pMT41 (Fig. 3) was reconstructed from restriction fragments of two different clones, and contained exactly the sequence we intended to obtain following strategy (b). In pMT40, translation of ORF32 is terminated by the presence of a translational stop codon 7 bp upstream of the start codon of the T4 e gene. In contrast, an in-frame fusion between these two open reading

220

pMT40

EcoRI

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I

EcoRI

XmnI ..I',. i "v"

Hindnr

I

I

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GGAGGAATTTTGAAAT._._G-.G66 - TA'GGAGTATTAT._._~G

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EcoRI

I

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I

' ~-. . .t._.______~ ............................ r__..___.__.__.3 GGAGGAATTTTGAAAT__.GG

pMT42

XmnI

I

T

~

EeoRI

EcoR I

Hind TIT

I

I

I

T

r~..__._--- -J L------__~l GGAGGAATTTTGAAAT.__GG

pMT44

EcoRI

EcoRI

Hind11I

I

I

I

I

i

XmnI

. . . . . . . . . . . . . . . . . . . . . . . . . . .

Xmnl

T

"l

r-'-'-" GGAGGAATTTTGAAATG-55-AAGGAGGAAAAAAAT.._._~GA

pMT45 PvuI,~EcoR I

EcoRI I

EeoRI I

XmnI

4.1 kbp PvuI " Nhel ori

I

GGAGGAATTTTCCCGGG -55- AAGGAGGAAAAAAAT.__GGA

XmnI

pMT

HindTIT

pwO1

frames was created in pMT42 by the introduction of a frame shift in ORF32 (Fig. 3). To this purpose, pMT40 was cut with AccI, the sticky ends were filled in with Klenow enzyme, and religated. ORF32 and the T4 e gene in pMT44 (Fig. 3) were translationally coupled with a spacing between stop and start codons that was optimal for the translational coupling of ORF32 and the E. coli l a c Z gene (van de Guchte et al. 1991). This configuration was realized using the PCR b l product described above, and two additional PCR amplification steps (Fig. 2). In PCR step cl,~plasmid pTC6 (van de Guchte et al. 1991) was used as the template in a reaction with oligonucleotide 5 described above, and oligonucleotide 6. The latter contained, in the 5'--+3' direction, 15 nucleotides complementary to the start o f the T4 e coding sequence, and 35 nucleotides complementary to the sequence directly preceding the l a c Z gene in pTC6. The partially overlapping products o f P C R b l and cl were purified on an agarose gel, and subsequently used in PRC step c2 together with the oligonucleotides 5 and 2. The product of this reaction was purified on an agarose gel, and the X m a I - E c o R I fragment containing the start of the T4 e sequence was used to replace the corresponding fragment in pMT40, result-

Xrnnl .c~)

Fig. 3. Schematic representation of plasmids pMT40, pMT41, pMT42, pMT44, and pMT45. These plasmids share the EcoRI-XmnI part represented

at the bottom of the figure with pMG36e (van de Guchte et al. 1989). This part of the plasmids contains an erythromycin resistance marker (Emr), and the replication functions of the cryptic L. lactis subsp, cremoris Wg2 plas~nid pWV01 (Kok et al. 1984). The EcoRI-XmnI fragment containing promoter 32 and the T4 e gene is drawn •separately for each of the plasmids. SD sequences are doubly underlined. Translational start codons are singly underlined. Relevant translational stop codons are overlined. ~ , lactococcal promoter 32; ,*, translated ORF32; ~:C>, non translated ORF32; z~, T4 e gene; T, transcription terminator (Kok et al. 1988)

ing in the formation of plasmid pMT44 (Fig. 3). Finally, the sequence GAA TG comprising the translational start codon in pMT44 was changed into C C C G G G by sitedirected mutagenesis using oligonucleotide 8 as described in Materials and methods, in order to prevent the translation of ORF32. The modified plasmid was designated pMT45 (Fig. 3). Bacteriophage T4 e #ene expression in L. lactis. Plasmids pMT40 through pMT42, pMT44 and pMT45 were introduced into L. lactis IL1403. In lysates of the L. lactis strains carrying these plasmids, the lysozyme activity relative to that of H E L was determined using lyophilized E. coil cells as a substrate. The results are presented in Fig. 4. A very marked difference was observed between the lysozyme activities in strain IL1403(pMT40), in which the T4 e gene is preceded by O R F 32 and the slightly modified T4 e SD sequence, and IL1403(pMT41), in which the T4 e gene is directly preceded by the ribosome binding site of ORF32. The latter strain reached an expression level ten times higher than that of the former. This expression level could be raised by a factor o f 1.5 through translational coupling to ORF32 in IL1403(pMT44). The depend-

221

o i.

o~ .EE

~

600

pMT42, which contains an in-frame fusion between ORF32 and the T4 e gene, gave rise to a lysozyme activity level 1.5 times that of IL1403(pMT40), but only onetenth of that of IL1403(pMT44).

518000

400 328000

278000

Bacteriophage A lysozyme 200 2350

m

==

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31000

Plasmid constructions. The SacI-ClaI fragment from the plasmid pAP19 (Jespers et al. unpublished data), containing the lysozyme-encoding A R gene, was recloned in the lactococcal expression vector pMG36e (van de Guchte et al. 1989) cut with SacI and AccI, to give pMLR0 (Fig. 5). In pMLR0, the A R gene and the vector-derived open reading frame 32 (ORF32) constitute a two-cistron system. The stop codon introduced in the reading frame of ORF32 and the start codon of the ~ R gene are separated by 3 bp. To examine the potential translational coupling of these two cistrons, three derivatives of pMLR0 were made (Fig. 5). First, the SspIXbalI fragment containing the ribosome binding site and the start of ORF32 was removed to give pMLR1. The XbaI Y-protruding end was filled in with Klenow

48OO0

7J

pMG36e pMT40 pMT#2 pMT41 pMT44 pMT45

Fig. 4. Lysozyme activities measured in lysates of L. lactis IL1403 carrying the plasmids indicated on the abscissa, expressed as units of HEL equivalents per milligram protein

ence of this enhanced expression on the translation of ORF32 was demonstrated by the difference in lysozyme activity obtained with pMT44 and pMT45. In pMT45, translation of ORF32 was prevented, and the expression level of the T4 e gene concomitantly decreased to approximately 50% of that obtained with pMT44.

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GGAGGAATTTTGAAAT._.G_G

- Xmnl

Fig. 5. Schematic representation of plasmids pMLR0, pMLR1, pMLR2, and pMLRS. The plasmids share the EcoRI-XmnI part represented at the bottom of the figure with pMG36e (van de Guchte et al. 1989). This part of the plasmids contains an erythromycin resistance marker (Emr), and the replication functions of the cryptic L. lactis subsp, cremoris Wg2 plasmid pWV01 (Kok et al. 1984). The EcoRIXmnI fragment containing promoter 32 and the 2 R sequence is drawn separately for each of the plasmids. SD sequences are doubly underlined. Translational start codons are singly underlined. Relevant translational stop codons are overlined. ~ , lactococcal prometer 32; =~, translated ORF32; ~:4:>, non-translated ORF32; ~>, 2 R gene; T, transcription terminator (Kok et al. 1988). Only one of the SspI sites present in the pMLR plasmids is indicated

222

enzyme, and ligated to the blunt end generated by SspI. Secondly, the entire ORF32, from the ATG start codon up to the last nucleotide preceding the ATG of the 2. R gene, was deleted by means of site-directed deletion mutagenesis, to give pMLR5. Thirdly, the D N A sequence GAAA TG, comprising the translational start codon of ORF32 in pMLR0, was changed into CCCGGG, thereby preventing translation of this ORF. The new plasmid was designated pMLR2. Functional expression of the bacteriophage ~ R gene in L. lactis. The 2`-lysozyme-specific antibodies were used to examine whether the 2` R gene could be expressed in L. lactis. In 1ysates of IL1403(pMLR0) the 2` R gene product was detected using Western blotting (Fig. 6B). In IL1403(pMLR5), however, 2` lysozyme could not be detected (Fig. 6). The plasmid pMLR5 appeared to be structurally unstable in L. lactis. When the plasmid was isolated from L. lactis, and used to retransform E. coli, no lysozyme activity was detected in a freeze-and-thaw assay, whereas the original plasmid gave rise to cell lysis. Restriction enzyme analysis revealed that in some

cases small parts of the ,t. R gene fragment had been deleted, whereas in others the alterations were not detectable in this analysis (data not shown). Activity measurements showed that in IL1403(pMLR0) a biologically active lysozyme was produced. The same observation was made in IL1403 carrying plasmid pMLR1 or pMLR2. The relative lysozyme activity, as compared to that of HEL, was measured using lyophilized E. coli cells as a substrate. The results are presented in Fig. 7, and show that in all cases the lysozyme activity was dearly above the background level displayed by IL1403(pMG36e). The highest expression level was obtained in the two-cistron system present in IL1403(pMLR0). Deletion of the first part of ORF32 had a marked effect on the expression of the 2` R gene in L. lactis: the ativity observed with pMLR1 was 38% of that observed with pMLR0. After correction for background activity observed in IL1403(pMG36e), the ratio between the two was 26%. When translation of ORF32 was prevented by changing the D N A sequence comprising the translational start codon of ORF32, as was the case in pMLR2, the lysozyme activity was reduced even further. The ratio of the activities observed with pMLR2 and pMLR0 was 24%, and after correction for the background activity only 10%.

Discussion

Fig. 6. Western blot of lysates of A Escherichia coli BL21(DE3) carrying pT7R (lane 1) or pT712 (lane 2) (both after induction of T7 RNA polymerase), and B L. lactis IL1403 carrying pMLR0 (lane 1) or pMLR5 (lane 2). Lane 3, A, contains prestained molecular mass standards, of which the 17-kDa band is indicated. The arrows indicate the position of the ~ R gene product

%

16

14500

x

"~' 12 ~

8 5500

g

4

-I-

g

o

pMG36e pMLROpMLR1

pMLR2

Fig. 7. Lysozyme activities measured in lysates of L. lactis strains carrying the plasmids indicated on the abscissa, expressed as units of HEL equivalents per milligram protein

The expression in L. lactis of three different lysozyme genes, one of eukaryotic and two of prokaryotic origin, was studied. The HEL produced, however, appeared to be inactive, probably due to the absence of correctly formed disulphide bonds. Incorrect folding of mature HEL, resulting in an enzymatically inactive protein, has also been reported in E. coli under conditions of overproduction (Imoto et al. 1987). No disulphide bonds are present in the functionally related bacteriophage T4 lysozyme (Matthews et al. 1981b). This prompted us to examine whether the gene encoding this enzyme could be expressed in L. lactis as a possible alternative to HEL in the suppression of food spoilage bacteria. A low level of expression of the T4 e gene was observed in IL1403(pMT40) relative to that obtained in IL1403(pMT41). This low level expression may result from translational interference caused by translation of the upstream ORF32. Alternatively, the slightly modified T4 e ribosome binding site present in pMT40 may be inferior to that of ORF32, which directly precedes the T4 e gene in pMT41. Translational coupling of the T4 e gene to ORF32, as in pMT44, served to raise the expression level by a factor of 1.5 compared to that observed in IL1403(pMT41). The same result could not be obtained by a simple translational gene fusion between ORF32 and the T4 e gene, as shown by IL1403(pMT42). This configuration, however, raised the lysozyme activity by a factor of 1.5 when compared to IL1403(pMT40), which may result from improved translation initiation or enhanced product stability. Since it is conceivable that the specific activity of the fused protein may be less than that of auth-

223 entic T4 lysozyme, the actual increase in expression may have been higher than that measured on the basis of the enzyme activity. Translational coupling, as present in pMT44, served to increase T4 lysozyme production by a factor of 1.5, whereas fl-galactosidase production could be increased by a factor of 2.9 using the same configuration of translational coupling (van de Guchte et al. 1991). This may indicate that the efficiency of translation initiation of the T4 e gene was higher than that of the lacZ gene, and could not be improved much further by translational coupling to ORF32. Further improvements may be achieved by exchanging P32-ORF32 with other lactococcal expression signals providing more efficient initiation of translation. In addition to the bacteriophage T4 e gene, a second bacteriophage-derived lysozyme-encoding gene, the R gene, was expressed in L. lactis. Expression of the R gene was found to be strongly dependent on the configuration in which the gene was present. In pMLR0, the ~, R gene was translationally coupled to the upstream ORF32. In the absence of translation of ORF32, the expression level of the R gene was lowered, and showed promoter-to-gene distance dependence: IL1403(pMLR1) showed a higher activity level than IL1403(pMLR2). For unknown reasons the plasmid pMLR5, derived from pMLR0 by removing the complete ORF32 sequence, was structurally unstable in L. lactis. Therefore, we were unable to examine whether translational coupling served to improve translation of the ~ R gene in pMLR0 as compared to the configuration in which ORF32 was absent. The finding that translational coupling occurred while the stop codon of ORF32 and the start codon of the 2 R gene were separated by three nucleotides is at variance with the absence of such coupling between ORF32 and E. coli lacZ in an analogous configuration (van de Guchte et al. 1991). Translational coupling between ORF32 and lacZ was only observed when the stop and start codons were either contiguous or overlapping. When the respective stop and start codons became separated by one to three nucleotides, translational interference rather than translational coupling was observed. The translational coupling in the expression of the 2 R gene observed here, may be explained by assuming that, as postulated in many other cases of translational coupling, translation of ORF32 served to remove mRNA secondary structures occluding the ribosome binding site (RBS) of the A.R gene. This would facilitate the access of the ribosomal subunits to the RBS and thus account for the enhanced expression of the ~ R gene. The maximum activity levels obtained with the T4 e and ~ R genes, expressed as units of HEL equivalents per milligram protein, differed considerably. According to Koteswara Rao and Burma (1971), the specific activities of HEL, T4 lysozyme, and 2 lysozyme relate as 1:250:10, whereas Bienkowska-Szewczyk and Taylor (1980) state that ~. lysozyme is 200-fold more active then HEL (both using E. coli as a substrate). Therefore, we cannot discriminate between the possibilities that the

observed differences in the activity levels of 2 and T4 lysozyme result from a difference in the amount of active enzyme present, or from a difference in the specific activities of the enzymes. The observation that the lysozymes of the bacteriophages T4 and 2 can be produced intracellularly in L. lactis without lethal effects is consistent with earlier observations made in E. coli (Garret et al. 1981; Perry et al. 1985). Bacteriophage T4 lysozyme is very similar to HEL in several respects and, in fact, both genes are thought to have diverged from a common ancestor (Griitter et al. 1983; Matthews et al. 1981a). Although differing in primary structure, the enzymes are similar in the conformation of their backbones, in substrate binding, and in their presumed mode of action (Jollrs and Jollrs 1984; Matthews et al. 1981a). Therefore, in principle, T4 lysozyme may be an alternative to HEL in the prevention of C. tyrobutyricum spore germination during cheese ripening. Preliminary experimental results indicate that the lysozyme of bacteriophage T4 in fact causes the lysis of C. tyrobutyricum cells (unpublished results). Since the bacteriophage ~ R gene product attacks the same bond in the peptidoglycan (Bienkowska-Szewczyk and Taylor 1980; Taylor et al. 1975), the ;~ lysozyme may also be an alternative to HEL for suppression of food spoilage bacteria during dairy fermentations. The applicability of these enzymes, produced intracellularly in L. lactis, requires their release form the cell during the early stages of cheese ripening. Since L. lactis strains show considerable differences with respect to autolysis (McKay and Baldwin 1990), this may be accomplished by choice of the host strain. Moreover, thermolytic L. lactis strains (MacKay and Baldwin 1990) may be used to constitute a delivery system for the intracellularly produced enzymes. Thus the production of these enzymes by L. lactis may render the addition of nitrate in cheese-making redundant, without the substantial financial consequences involved in alternative methods such as the addition of the purified enzyme, or bactofugation of the cheese milk. Acknowledgements. This work was supported by BCZ Friesland, Leeuwarden,The Netherlands.We thank Henk Mulder for preparation of the figures.

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