Cloning and expression of the Lactococcus lactis subsp. Cremoris SK11 gene encoding an extracellular serine proteinase

169

Gene, 85 (1989) 169-176 Elsevier GENE 03336

Cloning and expression of the Lac~ococcus luctis subsp. cremoris SK11 gene encoding an extracellular serine proteinase (Recombinant DNA; cell-envelope attachment; milk protein; lactic streptococci; structural instability; overproduction; deletion mapping; truncated proteinase)

Willem M. de Vos, Pieter Vos, Hans de Haard and Ingrid Boerrigter Molecular Genetics Group, Department of Biophysical Chem&ry, Netherlands Institute for Dairy Research (NIZO), Ede (The Netherla~~) Received by J.-P. Lecocq: 12 May 1989 Revised: 20 August 1989 Accepted: 31 August 1989

SUMMARY

The Lacrococcus lactis subsp. cremoris SK1 1 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades asi-, /3- and rc-casein, was identified in a rlEMBL3 gene library in Escherichiu colt’ using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-de~cient derivatives of L. lactis subsp. lads 712 and L. Iactis subsp. cremoris SK1 1, respectively. The proteinase deficiency of these hosts was complements to wild-type (wt) levels by the cloned SK1 1 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSKll1. The expression of recombinant plasmids containing 3’ and 5’ deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. Iactis subsp. la&s. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-te~~ly truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade as,-, @-and Ic-casein.

INTRODUCTION

The mesophilic lactococci L. factis subsp. la& and cremoris (pre~ously denominated S~ept~eocc~ Correspondenceto: Dr. W.M. De Vos, NIZO, P.O. Box 20, 6710 BA Ede (The Netherlands) Tel. 31-8380-59558; Fax 31-8380-50400. Abbreviations: Ap, ampicillin; Cm, chloramphenicol;

HRPO,

lactis and Streptococcus cremoris), which are used in industrial dairy fermentations, contain large cellenvelope-located serine proteinases that initiate proteolytic degradation of the milk protein casein (Geis horse radish peroxidase; Km, kanamycin; kb, kilobase or 1000 bp; ORF, open reading frame; PA, polyacrylamide; PrtP, a cell-envelope-located proteiuase which degrades as,-, b- and ic-caseiu from L. kactirsubsp. cremor& SKI 1; prtP,gene encoding PrtP; *, resistance; SDS, sodium dodecyl sulfate; wt, wild type.

0378-I1t9/89/$03.50 Q 1989 Elsevier Science Publishers B.V.(BiomedicalDivision)

170

1985; Thomas and Pritchard, 1987). In spite of their functional similarities, a remarkable genetic and biochemical heterogeneity exists between the proteinases of different L. Iactis strains. All known lactococcal proteinase genes are located on plasmids, which in various strains differ largely in size and genetic organization (De Vos, 1987). In addition, the biochemical properties of the proteinases from different strains vary considerably (Thomas and Pritchard, 1987). On the basis of differences in caseinolytic specificity the proteinases have been classified into two main groups: the PI-type proteinase which predominantly degrades j?-casein and the PIII-type proteinase which degrades ccsi-, /?- and rc-casein (Visser et al., 1986). Most genetic studies have focused on the PI-type proteinase genes. The L. Zactissubsp. eremoti Wg2 proteinase-encoding gene has been cloned and expressed in the proteinase-deficient 1;. lactis subsp. lactis derivative MG1363 (Kok et al., 1985). Nucleotide sequence analysis of the 5706bp Wg2 proteinase-encoding gene revealed that the cloned DNA fragment lacked the last 130 codons of the 3’ end. The truncated expression product, however, retained et al.,

wt activity and cell-envelope location (Kok et al., 1988a,b). Recently, it has been reported that a fragment of the 56-kb lactose-prote~ase-en~od~g plasmid pLP712 of L. /act& subsp. lactis 712, which specifies a proteinase with PI-type specificity, was cloned in strain MG1363 (Gasson et al., 1987). The proteinase pr~uction of the resulting construct, however, was found to be strongly reduced which was attributed to the incomplete cloning of this proteinase-encoding gene. In contrast to the abundance of PI type proteinase, the PHI-type proteinase has only been found in the strains L. Iactis subsp. cremoris AM1 and SK11 (Visser et al., 1986). Both strains are related (De Vos, 1986) and contain the proteinase-encoding 78-kb plasmid pSKll1 (De Vos and Davies, 1984). In this paper we describe the cloning and expression of the SK1 1 proteinase (prtP) gene in proteinasedeficient L. lactis derivatives. The expression, specificity and location of the proteinase, encoded by the complete SK1 1 prtP gene or by 5’ and 3’ deletions, was investigated in the lactococcal hosts to identify the regions involved in expression and cellenvelope location. One of the clones resulted in a

TABLE I Bacterial strains and plasmids Strain or plasmid E. coli MC1061 K803 4359 L. la& s&p. factis NCDO 4109 MG1363 MG1820

L. la&is subsp. crenwris SK112 SK1 128

Relevant genotype or phenotype

Reference

hsdR, araD 139, A(ara&u)7697, dlacZ74, gal& ~sL hsdR, gal, met, supE hsdR, supE, tonA (P2)

Casadaban et al. (1980) Wood (1966) Kam et al. (1980)

Lac + Prt + multiplasmid strain Lac- Prt- plasmid derivative of NCDO 712 Lac + derivative of MG1363 containing the lactose miniplasmid pMG820

Gasson (1983) Gasson (1983) Maeda and Gasson (1986)

Lac + Prt + multiplasmid strain Lac + Prt - derivative of SK1 12

De Vos et al. (1984) De Vos and Davies (1984)

ApR, 2.8 kb, ColEl replicon ApR, 2.8 kb, ColEl replicon CmR, KmR 4.3 kb, pSH71 replicon CmR, KmR, 2.8 kb, pSH71 replicon

Vieira and Messing (1982) Vieira and Messing (1982) De Vos ( 1986) De Vos (1986)

Plasmids PUO puc13 pNZI2 pNZ 122

171

well-expressed, C-terminal truncated proteinase that is fully secreted into the growth medium and still shows the capacity to degrade xsi-, /I- and K-casein. A preliminary report describing some aspects of this work has appeared previously (De Vos, 1986).

MATERIALS AND METHODS

(a) Bacterial strains, plasmids and bacteriophages The relevant properties of the bacterial strains and plasmids used in this study are listed in Table I. Bacteriophages IEMBL3 (Frischauf et al., 1983) and 12L47.1(Loenenand Brammar, 1980) were used in combination with the E. coiz’hosts K803 and 4359. (b) Media and DNA transfer E. co&strains were grown on L-broth based media (Maniatis et al., 1982). Media based on Ml7 broth (Difco) supplemented with 0.5% lactose or glucose were used to grow L. lactis. If appropriate, the media contained 5Opg Ap/ml or 1Opg Cm/ml. For the assay of proteinase production lactic streptococci were grown in pasteurized, reconstituted non-fat milk supplemented with 0.5% glucose or 0.1% casiton (Difco) when required. In vitro packaging and transformation of E. coli was performed essentially as described in Maniatis et al. (1982). Protoplasts of L. la& subsp. lactis were transformed as described by Kondo and McKay (1984). Plasmid DNA was introduced into L. lactzk subsp. cremoti by electroporation at 5000 V/cm and 25 PF using a Gene Pulser Electroporator essentially usiug conditions described by the manufacturer (Biorad) and described initially for Lactobacillus casei (Chassy and Flickinger, 1987).

In all constructions phage or plasmid DNA purified by CsCl gradient centrifugation was used. The prote~ase-enco~g plasmid pSKlll(78 kb) was separated from other plasmids present in the multiplasmid strain L. lack subsp. cremoris SK112 by preparative gel electrophoresis on a 0.8% lowmelting-point agarose (Biorad) gel. The pSKll1 DNA was recovered from the gel and used to construct a physical map. In addition, the pSKll1 DNA was subjected to digestion with limiting amounts of Suu3A to generate fragments with an average size of 20 kb, which were used to construct a gene bank in IEMBL3 digested with BamHI + EcoRI as described by Frischauf et al. (1983). All enzymes were used according to the instructions of the suppliers (Bethesda Research Laboratories and Boehringer). DNA synthesis was carried out using a Cyclone DNA synthesizer (Biosearch). (d) Proteinase purification and immunological methods Purification of the proteinase was performed by preparative electrophoresis on a neutral 10% PA gel and gel slices were assayed for proteinase activity using [methyl-*4C]fi-casein (Exterkate and De Veer, 1985). PA gel slices ~ont~~g approx. 20 pg proteinase were used to immunize rabbits and the IgG fraction of the obtained serum was purified by DEAE chromatography. Phage libraries were screened with a gene expression kit using antibodies labeled with HRPO in a sandwich technique following the instructions specifled by the supplier (Boehringer). Western-blot analysis of proteins separated on SDS-15% PA gels (Laemmli, 1970) was performed using the HRPO-labeled antibodies or flailed antibodies in ~omb~ation with HRPOlabeled goat anti-rabbit antibodies (Biorad).

(c) DNA meth~ology

RESULTS AND DISCUSSION

Plasmid DNA and bacteriophage DNA were isolated from E. coli according to established protocols with minor mod~cations (Maniatis et al., 1982). Plasmid DNA was isolated from lactic streptococci grown to mid-log phase using a modification of the alkaline extraction procedure (De Vos et al., 1984).

(a) Identifhxtion of the Lac~ococcus Zuctis subsp. cremoris SK11 proteinase gene in a gene library The prote~ase-enc~~g plasmid pSKlll(78 kb, Fig. 1) is the largest plasmid present in all industrially used, multiplasmid, strains of L. 1actA.v subsp.

172

cremoris SK1 1 (De Vos et al., 1984). Plasmid DNA

of pSKll1 was isolated from strain SK1 12 and used to construct a simple physical map (Fig. 1). In addition, a library of partial Suu3A fragments of pSKll1

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Fig. 1. Plasmid pSKll1 and derived phages and plasmids. The physical map of the natural L. la&s subsp. cremoti SK112 proteinase-encoding plasmid pSKll1 is shown with a detailed map of its enlarged proteinase region including the presumed location and orientation of the prtP gene. The DNA fragments cloned in phage I (IEMBL3 and 147.1) or plasmid vectors (pNZl2 and pNZ122) are indicated by the hatched and open areas, respectively, and are all flanked by Sal1restriction sites. Plasmids pNZ54 and pNZ55 represent two orientations of the plasmids obtained after transformation ofL. iact& MG1363 with the 4.5-kb Sal1 fragment from 1NZ501 (previously multiplied by subcloning in the unique Sal1 site of 147.1) ligated with SalIlinearized pNZl2. Plasmids pNZ511 and pNZ512 were constructed by isolating the 6-kb HindHI-Sal1 fragment from lNZ243 DNA and providing it with a HindHI-PstI-Sal1 polylinker by ligation with an excess ofHindIHdigestedpUC9 DNA. After subsequent digestion with Sal1 the resulting 6-kb Sal1 fragment was multiplied by subcloning in the unique Sal1 site of 1I47.1 and finally cloned into L. lacrhMG1363 using the SalIlinearized vector pNZ122. Clones differing with respect to orientation of the insert were obtained and designated pNZ5 11 and pNZ512. pNZ521 was constructed by replacing the 1.3-kb BglIISal1 fragment of pNZ511 by the 3.1-kb BglII-XhoI fragment of plasmid pPS 101, containing the righthand part of the SK1 1 prtP gene. pPSlO1 is a plasmid containing a 7-kb PsrI-EcoRV fragment of INZ501 inserted into PstI-SmaI site of pUCl3X, a pUCl3 derivative in which the unique EcoRI site is replaced by a synthetic XhoI adapter (P.V. and W.M. de V., unpublished results). Restriction sites for BamHI (B), Sal1 (S) and XhoI (X) are indicated on pSKll1 DNA and additional sites for BglII (Bg), EcoRI (E), EcoRV (R), Hind111 (H) and PstI (P) on the enlarged proteinase region (sites in parentheses are not unique in this region). The bar indicates a DNA size of 1 kb.

was. constructed in IEMBL3, which was subsequently screened for the expression of the SK11 proteinase gene using peroxidase-labeled antibodies directed against the SK1 1 proteinase. Phages which reacted with the antibodies were obtained with a frequency of approx. 5 % . The DNA present in six of these recombinant phages was analyzed by restriction enzyme mapping. On the basis of the alignment of the insert DNA with the physical map of pSKll1, these phages could be grouped in two classes. Representatives of each class are the phages 1NZ501 and ANZ243, which have in common a region of approx. 4.5 kb located to the left of the unique Sal1 site of pSKll1 (Fig. 1). Immunoblot experiments showed that, after infection of E. coli by these phages, high-& proteins were produced, which reacted with proteinase antibodies (results not shown). Since DNA inserted in the IEMBL3 derivatives is flanked by Sal1 sites as a consequence of the design of this vector (Frischauf et al., 1983), Sal1 digestion of the pSKll1 DNA in JNZ501 resulted in two Sal1 fragments of 4.5 and 8 kb. These were subcloned in A47.1 and used to infect E. coli. It appeared that only phages with the 4.5-kb Sal1 fragment resulted in the synthesis of proteins that reacted with the antiproteinase antibodies. These results indicate that the structural proteinase gene (prtP) is located on the 4.5-kb Sal1 fragment from 1NZ501 which is also contained in phages of the lNZ243 class. (b) Subcloning of the SK11 prtP gene on plasmid vectors in Escherichia coli and Lactococcus lactis

The 4.5-kb Sal1 fragment from ANZ501 was cloned in the proteinase-deficient, plasmid-free L. lactis subsp. lactis derivative MG1363 with the use of the high-copy-number, heterogrammic vector pNZ12. Plasmids, designated pNZ54 and pNZ55, containing both orientations of the insert DNA were readily obtained (Fig. 1). Unexpectedly, no expression of the SK11 prtP gene could be measured immunologically or via complementation of the proteinase deficiency of the L. luctis subsp. lactis host. Similarly, when pNZ54 or pNZ55 were introduced in E. coli no expression of the proteinase could be measured using anti-proteinase antibodies. However, in contrast to L. lactis subsp. lactis where both plasmids were stably maintained, a high frequency ($0 pNZ55; */lo pNZ54) of structural deletions of

173

the pSKll1 DNA was observed when plasmid DNA was extracted from E. cdi transformants, indicating the ~s~b~ity of the proteinase gene in this heterologous host. The observation that the 4.5kb Sal1 fragment of IN2501 only resulted in the synthesis of proteinase when cloned in a Avector suggested that expression of the SK1 1 prtP gene depends on signals that are present on the phage vectors and hence are absent from the cloned fragment. Preliminary sequence analysis (not shown) of the DNA region in LNZ243 upstream from the PstI site showed the presence of a promoter, ~bosome-bind~g site and the start of a long ORF extending to the righthand side (Fig. l), indicating the orientation and initiation of the prtP gene. Therefore, a 5’ extension of the prtP gene was constructed by isolating a 6-kb ~i~dIII-S~~I fragment from LNZ243. This DNA fragment was subsequently cloned into L. lactk subsp. lactis MG1363 using either pNZ 12 or its lower-copy-number derivative pNZ122 (Fig. 1). Cm-resistant transformants were readily obtained but appeared to contain recombinant plasmids only when pNZ 122 was used as vector. Two orientations of the insert DNA in pNZ122, designated pNZS11 (Fig. 1) and pNZ512, were further analyzed. In the proteinase-deficient L. lasts subsp. lactis strain MG1363 these plasmids directed the synthesis of a functional proteinase (see below). In addition, the presence of pNZ511 or pNZ512 in MC1363 complemented the proteinase deficiency of this host and resulted in wt growth in milk (not shown). These results indicate that the 6-kb HindIII-Sal1 fragment from lNZ243 contains the prtP gene including its expression signals which are located upstream from the P&I site (Fig. 1). Therefore, synthesis of the proteinase in llphages of the 1NZ501 type is most likely a result of a transcriptional and translational fusion. (c) Specificity of the SK11 proteinase in la&cocci Because of the unique caseinolytic specificity of the SK1 1 proteinase it was of interest to determine if its PIII-type specificity is determined by the prtP gene and not by the lactic streptococcal host. To this purpose use was made of the L. la&is subsp. Iact& strain MG1363, since previous studies had indicated that proteinases with a PI-type specificity are pro-

A

B

Fig. 2. Caseinolytic specificity of protemase encoded by the wt or cloned prtP gene. Proteinase was isolated from milk-grown lactococcal cells (release fraction) or supematant (medium fraction) following centritugation (15 mm at 6000 X g) of neutraliied (pH 6.5) and clarified (by the addition of 1% citrate) cultures. The supematant was adjusted topH 5.0 and the precipitated caseins were removed by low-speed centrifugation. Proteinase was released from the cells by resuspending cells in Ca2 + -free buffer with a pH of 6.5 at 30°C (Exterkate and de Veer, 1985). After dialysis against water the proteinase was concentrated by freeze-drying. The proteinase preparations (release fraction for SKI 12 or medium &action for MG1363 carrying pNZ511) was used to digest as,, /I- and a-casein (panels A, B and C, respectively) as described by Visser et al. (1986). Degradation products were separated on a 0.1 y0 SDS-15% PA gel (Laemmli et al., 1970) and subsequently stained with Coomassie blue. Lanes: 1, incubation with proteinase from strain SKI 12; 2, incubation with proteinase from strain MG1363 containing pNZ511; 3, control with undigested casein.

duced in this strain when containing the native proteinase-encoding plasmid pLP712 (De Vos, 1987; W.M. de V., ~pub~shed results) or plasmids containing the cloned L. iactis subsp. cremoris Wg2 proteinase-encoding gene (Kok et al., 1988b). Therefore, proteinase was isolated from MG 1363 containing the plasmid pNZ511 and its caseinolytic specificity was compared to that of proteinase isolated from the wt L. luctis subsp. cremoris strain SK112. The results (Fig, 2) show that both proteinase preparations possess the same, PI&type specificity towards ~si-, jI- and rc-casein. These results clearly indicate that the caseinolytic specificity of the proteinase is determined by the prtP gene and not by the lactic streptococcal host.

174

(d) Complete secretion of and overproduction of the SK11 proteinase in lactococci In the course of analyzing the caseinolytic specifcity it was observed that when strain MG1363 carrying pNZ5 11 was grown in milk the proteinase activity could not be isolated from the cell-envelope but was found exclusively in the growth medium. This is not due to the medium composition or growth phase (Fig. 3), which shows the fermentation in whey-broth and concomitant extracelhtlar proteinase production of the lactose-proficient L. tactis subsp. lactLs strain MG1820 carrying pNZ511. To determine the factors involved in the cellular location of the lactococcal proteinase, various proteinase plasmids were introduced into the proteinasedeficient L. luctis strains MG1363 and SK112 and their expression products were compared with those

(A60

extracellular proteal~ic activitv (uni mil

10.0

10

encoded by the natural proteinase plasmids in these hosts (Fig. 4). Complete secretion of proteinase was observed in strains MG1363 or SK1128 carrying pNZ5 11 but not in the parental wt strains. These results demonstrate that the inability of the pNZ5 1lencoded proteinase to become attached to the cellenvelope is not a result of the absence of a host factor involved in this process. Furthermore, pNZ521 containing a 1.8-kb 3’ extension of the prtP gene

CELLS 1234567

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1.0

1.0

0.1

0.1

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2

4

6

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8

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12

Fig. 3. Secretion of proteinase during fermentation. Strain L. facris subsp. la& MGI 820 containing pNZ5l I wasgrownin a pEI-controlled fermentor on a medium based on ultrafhrated whey cont~n~g 0.1% casiton (M. Timmer and W.M. de V., unpublished) pH6.5. Samples, taken at regular intervals, were used to determine the absorbance at 600 nm (&J and, after centrimgation, assayed for proteolytic activity using [methylr4C]j-casein. One unit is arbritarily defined as the amount of proteinase activity which is required to render 10% of total radioactivity soluble in 6% trichloroacetic acid in 10 min. Only trace amounts of proteinase activity (between 0.001 and 0.05 units/ml) were found to be associated with the cell fractions during the entire fermentation period (results not shown).

Fig. 4. SDS-PA gels of cell-envelope released or secreted proteinases. Cultures were grown to late logarithmic phase and proteinase was recovered from the cells or supernatant as described in the legend to Fig. 2. Medium and release fractions from equal amounts of cells (approx. 2 ml) were separated on a 0.1% SDS-lo% PA gel (Laemmli et al., 1970), which was subsequently stained with Coomassie blue (only top half of the stained gel is shown). Iw, markers (in kDa) are indicated. P indicates the SK11 proteinase and an asterisk indicates a degradation product of the proteinase (Vos et al., 1989). Lanes: l-7, Preparations from (1)L. la&r subsp. la& MG1363 (Prt- ); 2, L. la& subsp. cremorir SK1128 (Prt-); 3, L. Ia& subsp. cremorir SK1 12 (wt, Prt ‘); 4, L. la&s subsp. la& NCDO 4109 (wt, Prt +); 5, L. la& subsp. lactis MG1363 containing pNZ521 (Prt’ ); 6, L. lack subsp. Iacrir MG1363 containing pNZ511 (Prt + ); 7, t. la& subsp. cremuris SK1 128 containing pNZ.511 (Prt +).

175

(Fig. 1) appeared to specify a proteinase that was found predominantly in the cell-envelope release fraction (Fig. 4). This suggests that the prtF gene cloned on pNZ5 11 contains a 3’ deletion and specifies an active, C-terminally truncated proteinase that lacks a topogenic sequence(s) which is present on the proteinase encoded by pNZ521 and wt plasmid pSKll1. Support for the existence of a topogenic sequence in the SK1 1 proteinase has recently been obtained from the nt sequence determination of the SK1 1 prtP gene. The deduced aa sequence of the SK11 proteinase showed a cell-membrane anchor sequence at the C-terminal end which is homologous to similar sequences in cell-envelope-located proteins from other Gr~-~sitive cocci (Vos et al., 1989). Interestingly, cells of L. Iactis subsp. lactis MG1363 carrying pNZ521 contain at least threefold more proteinase in the cell-envelope release fraction than lactococcal cells carrying low copy-number, wt proteinase plasmids (Fig. 4). Most likely this overproduction may be att~but~ to a gene dosage effect due to the cloning of the SK1 1 prtP gene on the vector pNZ 122. (e) Conclusions

(1) The L. la& subsp. cremoti SK1 1 prtP gene encoding a proteinase that specifically degrades as,-, @- and Ic-casein (PIII-type proteinase) has been cloned and expressed in E. coli and L. /actis subsp. lactis and cremoris using phage and plasmid vectors, respectively. The location and orientation of the SK1 1 prtP gene on the 78”kb plasmid pSKll1 has been determined by analyzing the expression of various deletion derivatives. (2) The SK1 1 prtP gene can be stably maintained in E. coii using the R-derived vectors ilEMBL3 and 147.1. In contrast, a high degree of structural instability of the SK1 1 prtP gene cloned on plasmid vectors has been observed in E. coli. This is analogous with the reported inability to clone other genes specifying lethal gene products, including the Wg2 proteinase-encoding gene (Kok et al., 1985). In contrast, the SK1 1 prtP gene has been readily cloned, expressed and stably maintained in L. lactis subsp. lactrkand cremoris with the use of the plasmid vector pNZ122. (3) The cloned SK1 1 prtP gene complements all

investigated prote~ase-de~cient lactococcal strains to wt levels. It has been found that the unique caseinolytic specificity of the SK11 proteinase is determined by the cloned SK1 1 prtP gene and is not dependent on the host used to express this gene. More than threefold overproduction of the SK11 proteinase in L. iactis subsp. lactis has been realized with the plasmid pNZ521, which contains the complete proteinase gene. (4) A topogenic sequence(s) located in the C-terminal region of the proteinase most likely determines the location of the SK11 proteinase at the cell envelope in lactococci. Deletion of this sequence(s), as in a 3’ deletion of the SK1 1 prtP gene in pNZ511, results in a proteinase that retains its PI&type specificity but is completely and continuously secreted into the growth medium. Evidence for the existence of at least one such topogenic sequence, a membrane anchor sequence, has recently been obtained by analyzing the C-terminal aa sequence of the proteinase deduced from the SK1 1 prtP gene sequence (Vos et al., 1989). These topogenic signal(s) may have potential in targeting homologous or heterologous proteins to the cell envelope of lactic acid bacteria to include such proteins into products made with these food-grade bacteria.

This research was partly funded by contracts GBI-2-084-NL and BAP-OOll-NL of the Biomolecular Engineering and Biotechnology Action Programmes (BEP and BAP) of the Commission of European Counties. We are grateful to Fred A. Exterkate and Ser Visser for gifts of labeled or purified caseins, Liz Muhalan and Jan Peter Nap for phage samples, Joop Mondria for art work and to Roland J. Siezen for stimulating discussions and critically reading this manuscript.

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