Eur. J Biochem. 204,815-820 (1992) c” FEBS 1992

Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis Harry LAAN I , Jan KOK

’,Alfred J. HAANDRIKMAN ’,Gerard VENEMA2 and Wil N. KONINCS’

’ Department or Microbiology and of ’ Ccnetics, University of Croningcn, Haren, The Nctherlands (Received September S/Novembcr 19, 1991) - EJB 91 1184

Lactococcus lactis strains produce a n extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30 Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.

-

Growth of Lactococcus Iuctis. a predominant species in Dutch-cheese starter cultures. depends on the availability of small peptides or free amino acids [l. 21. These nutrients are present at low concentrations in milk, and growth of L. lactis in this medium is limited unless proteins are hydrolyzed. To obtain these nutrients, L. lactis produces a number of (extracellular) proteinases and peptidases which act in concert and hydrolyse the milk protein casein (for a review see [3]). Casein is first partially degraded by an extracellular proteinase and subsequently by peptidases which release peptides and amino acids that are taken up by the cells and used in biosynthesis [2 - 51. The characteristics of proteinases and peptidases are important for cheese ripening and flavour development. Proteinase specificity and activity can vary significantly between different Lactococcus strains [6, 71. Biochemical and genetical analysis revealed that the lactococcal proteinases are subtilisin-related serine proteinases of 100 -210 kDa. They are associated with the cell envelope and optimally active between pH 6 and 7 [3, 81. The nucleotide sequences of a number of proteinase genes of different L. luctis strains were very similar [9 - 111. The lactococcal proteinases show remarkable sequence similarity to the serine proteinases of the subtilisin family. The similarity is especially high around the active-site region. However, unlike the suhtilisin family, the lactococcal proteinases have a large C-terminal domain of about 1200 amino acid residues with unknown function. The 30 C-terminal amino acid residues serve as a membrane anchor [9, 1 1 -131. The lactococcal proteinases can be classified according to their specificity [7]. their activity [6] or their immunological characteristics [14] (see Table 1). Three groups of proteinases can be distinguished on the basis of their specificity; the HPtype ( P I ; degrading 8-casein), the AM1-type (P3;degrading

a,,-casein and p-casein, but with a specificity different from the HP-type) and a group possessing both types of specificities. Immunological analysis revealed that the proteinases of different L. lactis strains gave different precipitation patterns in crossed immunoelectrophoresis with their specific antibodies [ 141. Four immunologically distinct proteins, which originate from one native proteinase molecule [15], were detected and designated as A, B, C and A’ (Table 1). Component A is present in all strains and, in addition, in at least one of the components B, C or A‘. Components A and A‘ and also components B and C have common antigenic determinants [14, 161. Component A and component B do not share any antigenic determinants. Components A and B have been shown to be proteolytically active. How these immunologically different components can be formed has not yet been clarified, but a n autocatalytic process has been suggested [15]. The proteinase of L . luctis subspecies cremoris WG2 has also been characterized immunologically using monoclonal antibodies [16]. Analogous to the results obtained with the polyclonal antibodies, distinct proteinase components have been recognized using monoclonal antibodies. In this paper.

Table 1. Classification of the proteinases of different L. Iuctis strains. The four immunologically distinct proteinase components are A. B, C and A‘ and can be distinguished by specific antibodics [14]. P, (HPtype) specificity degrades only j-casein. P3 (AMl-type) specificity degrades z.,-casein and 8-casein (with a specificity different from P I ) [7]. n.d., not detcrrnined. L . lactis strain

Immunoreaction

Specificity

Subspecies cremoris

Wg2 A B E8 A C AM1 A C A‘ SKI1 A C A’ ML3 A B

PI PI

~~

C’orrcspondeerice to W. N. Konings, Department of Microbiology, University of Croningen, Kcrklaan 30, NL-9751 N N Haren, The Netherlands

Subspecies lucris

p3 p3 p3

PI

816 these monoclonal antibodies have been used to analyse the origin of the proteinase components A and B.

MATERIALS AND METHODS Bacterial strains, plasmids and cultivation L. lactis subspecies cremoris strains WG2, E8, SK11, AM1 and HP, and L . luctis subspecies lactis strains ML3 and MG1363 were maintained at -20°C in 10% skimmed milk [16, 171. The plasmid-free L . luctis MG1363 was used for the expression of proteinase derivatives encoded by the plasmids used in this study. The construction of plasmid pGKV5OO and its derivatives, as well as plasmids encoding hybrid proteinases of L. lactis subspecies cremoris strains WG2 and SK11, has been described elsewhere [15, 17- 191. A description of these constructs is given in Fig. 1. L. luctis MG1363-carrying pNZ521c was kindly provided by W. M. de Vos. Organisms were grown at 29°C in MRS broth [20] with modifications as described by Hugenholtz et al. [14]. The medium was supplemented with 15 mM CaCl, and 0.5% glucose (mass/vol.) and, for the recombinant strains, with 5 pg/ml erythromycin or chloramphenicol. The organisms were harvested at the beginning of the stationary phase of growth.

Proteinase isolation The proteinases of L. lactis subspecies cremoris strains and

of L. lactis subspecies lactis strains ML3, MG1363(pGKV552c) and MG1363(pNZ521c) were isolated as follows. Cells from a 100-mlculture were washed in 10 ml50 mM Tris/HCl pH 8.0 containing 15 mM CaCl, and suspended in 10 ml 50 mM Tris/HCI pH 8.0 containing 15 m M EGTA. After 20 min incubation at 29"C, the cells were removed by centrifugation. By this method, 80% of the total proteinase content of the cells could be released into the medium [13]. Proteinases produced by the MG1363 strains carrying one of the vectors pGKV5OO -pGKV512, which encode proteinases that lacked the membrane anchor region [12], were isolated by 10% perchloric acid precipitation of the culture medium (100-fold concentration of the medium).

I

I

I

I

I

Gel electrophoresis SDS/PAGE on 8% polyacrylamide gels was performed as described by Laemmli [27] using the Bio-Rad minigel system (Bio-Rad Laboratories, Richmond, Calif., USA). The protein samples were diluted fourfold with sample buffer (50 mM Tris/HCl, pH 6.8,10% SDS, 25% glycerol, 0.1% bromphenol blue and 10% 2-mercaptoethanol) and applied to the gels. After electrophoresis, the gels were stained with 0.1YO Coomassie brilliant blue or used for immunoblotting (see below). The molecular masses of the protein bands were estimated using the following reference proteins : myosin (200.0 kDa); /?-galactosidase (1 16.3 kDa); phosphorylase u (97.4 kDa); bovine serum albumin (66.2 kDa); ovalbumin (43.0 kDa). DNA analysis on 1YOagarose gels was performed according to standard procedures [22]. Immunoblotting After SDS/PAGE, protein was transferred to ImmobilonP poly(viny1idene difluoride) transfer membranes (Millipore Corp., Bedford, Mass., USA) with a semi-dry Electroblotter A (Ancos, Copenhagen, Denmark) as described by KyhseAndersen [23]. Proteinase detection using monoclonal antibodies was performed as described before [13]. Bound monoclonal antibodies were detected with alkaline-phosphataseconjugated antibodies raised against mouse antibodies (Sigma Chemical Co., St. Louis, Mo, USA) and the immunoblots were stained with 150 pg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma) and 300 pg/ml 2,2'-bis@-nitrophenyl)5, S'-diphenyl-3,3'-dimethoxy-4,4'-diphenylene-ditetrazolium chloride (Sigma) in 1 rnM MgC12 and 50 mM sodium carbonate, pH 9.8. Crossed immunoelectrophoresis Crossed immunoelectrophoresis was carried out as described previously [24]. The gels were run at 2.5 V/cm for 16 h in the second dimension. The second-dimension gel contained 250 pg/ml polyclonal antibodies raised in rabbits against the

I

I

I

pwvos

prtP I

pGKV5OO

I I

I

I

I I

pGKV502

I

pGKV503

I

pGKV512

I

pGKV509 I

pGKV1500 pGKV552c

I

I

pNZ521c

Fig. 1. Schematic representation of the constructs of the L. luctis subsp. cvemoris Wg2 proteinase genes. The sizes (in kb) of pGKV500, deletion derivatives and Wg2-SK11-hybrid proteinase genes are indicated. The bar represents 10 kbp. (M), Wg2 gene; ( O ) ,SKI1 gene; *, mutation of Asp30 4 Asn at the active site (pGKVI500) [lS].

817 proteinase of strain WG2. Crossed immunoelectrophoresis gels were stained with 0.5% Coomassie brilliant blue. Polymerase-chain reaction Amplification of DNA [25, 261 was carried out for 30 cycles in a DNA thermocycler. Each cycle included 60 s denaturation at 94"C, 120 s annealing of primers at 50°C and 60 s primer extension at 74°C. The reaction mixture, in a total volume of 100 pl, consisted of 10 mM Tris/HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCI2, 0.1% (massivol.) gelatin, 1 5 ng DNA, 500 pM deoxynucleoside triphosphates, 1 pM forward primer (5'-AGCGCGCTATCCCAGAC-3'), 1 pM reverse primer (5'-AGTTTGGATCCTTGGTA-3')and Thermus aquaticus DNA polymerase (25 U, Promega Corp., Madison, W1, USA). To prevent evaporation of the solution, 100 pl mineral oil was layered on top of the reaction mixture. Analysis of the amplification products was performed after electrophoresis of 2 pl reaction mixture on a 1YOagarose gel. Table 2. lmmunoreactivity on Western blots of different monoclonal antibodies (groups 1- VI) with deletion derivatives and hybrid proteinases of L. [actis. The proteinase dcrivatives are specified by the related plasmids. Monoclonal antibodies which recognized a given proteinase deletion mutant arc indicated as antibody that fails to recognize a given proteinase is indicated as -. A weak reaction is indicated as - n.d., not determined.

+,

+.

MG1363 clone

Reaction with monoclonal antibody group I

I1

111

IV

v

v1

+ + + + + +

+ + +

+ + + + +

+ + +-

+ + +

+ + +

-

-

f

-t n.d. n.d.

+

n.d. n.d.

-

+ +

-

n.d. n.d.

-

The regions in the lactococcal proteinase recognized by the monoclonal antibodies used in this study, were localized using a variety of C-terminally truncated L. luctis proteinases. These proteinases were synthesized by L. lactis MG1363 in which plasmids with a 3'-deleted proteinase gene had been introduced (Fig. 1). The reactions of the monoclonal antibodies of group I -VI with these proteinases were studied (Table 2). All monoclonal antibodies reacted with proteinases from all clones, except for the antibodies of groups I V VI. These antibodies did not react with the proteinases of MG1363(pGKV509) and MGl363(pGKV512). These observations indicate that there are two antigenic regions (Fig. 2); region A is the region upstream of the Ile604 (upstream of the EcoRI site in the gene; see Fig. I), to which the monoclonal antibodies of group I - 111 bind and region B is located between Thr816 and Leu1219 (encoded by the region NurISacI) to which the antibodies of groups IV-VI bind. The binding site for the monoclonal antibodies of group 1-111 could be further narrowed down to a region between Gln402 and Ile604, since the 58 kDa autoproteolytic fragment P58 of the WG2 proteinase, having Gln402 as the N-terminal amino acid, also reacted with these monoclonal antibodies [27]. A schematic representation of the binding-site regions A and B is shown in Fig. 2.

To investigate whether a relationship exists between the characteristics of the proteinase and binding of the monoclonal antibodies, the interaction of proteinases from different L. luctis strains with the monoclonal antibodies were tested. The breakdown patterns of the proteinases from the different strains were rather similar and a more detailed study of this phenomenon is described elsewhere [27]. The immunoblot reactions of the monoclonal antibodies of group I, 1V and V with the proteinases of the L. lactis subspecies cremoris strains

-

+

Localization of epitopes

Immunoreactivity with proteinases from other strains

-.

pGKV5OO pGKV502 pGKV503 pGKV.509 pGKV.512 pGKV552c pNZ521c

RESULTS

L . lvctis

pruteinaae

flN I

coo

/ -

region A

/

/

\

\

/

'\

\ \

\

/

/

2-

.

l.rEll

EroQ.1

tsoni

GCC ACC GAC GAA GAT GGC / / ATG TTG AAG AAT TCT GTG ACG TTC GAT CAA GGT GTG ACA TTT GGT GCC AAT

108')

pmtein:

\

/

WG2 DNA:

\

/

Lys Val T h r

Ala

T h r Asp G l u Asp G l y

Wet

AAT GCC I I,'

111'.

Leu Lys Asn Ser V a l T h c Phe Asp Gln Gly Val T b r Phe Gly A l e Assn Glu Pbe Asn Ale

SKI 1 DNA,

5- *

IlnrElI

Xpnl

AAG GTG ACC GCC ACC GAC AAA GAT GGC / / ATG TTG AAG AAG TCT GTG ACG TTC GAT CAA GGT GTG AAA TTT

protein. Lys V a l Thr A l e T h r Asp Lxf Asp GI7

AAT AM TTC AAT GCC

net Leu Lys LJlS Ser V a l T h c Pbe Asp Cln 617 Val Lxs Phe GIy Tht Asn Ly8 Phe As" M a

Fig. 2. Schematic representation of epitope-mapping data of the L. luctis proteinase. The proteinase IS rcpresented showing different epitopes rccognized by monoclonal antibodies (region A and region B). In the lower scheme, the DNA sequence and amino acid sequence of part of the proteinaws of L. /actis subsp. cremoris strain Wg2 (upper) and SKI 1 (lower) are shown. The protein fragments start at Lys1089 and end at Alal134.

81 8

Fig. 3. Lmmunoblot reactions of the monoclonal antibodies of group I, IV and V (indicated at the top of the figure as A, B and C, respectively) and the proteinases of the L. lacris subsp. cremoris strains. WG2 (lane l), E8 (lane 2), SKI 1 (lane 3), AM1 (lane 5) and subsp. luctis ML3 (lane 4). The molecular masses of the standard proteins arc indicated at the left (in kDa).

of group V recognize exclusively component B [I 61. Previously we have seen that both antibody groups bind to the region Thr816- Leu1219. To find out what differences in these binding regions are responsible for the different behaviour of the antibodies, antibodies of group IV and V were tested on two proteinases specified by pGKV552c and pNZ521c. In these constructs, the Sac11- BstEII gene fragments of the proteinases of the crernoris strains WG2 and S K l l were exchanged reciprocally, resulting in the synthesis of hybrid proteinases (Fig. 1) [19]. As expected, antibodies of group IV reacted with both hybrid enzymes, while those of group V reacted only with the proteinase specified by pGKV552c. These results indicate that the epitope for monoclonal antibodies of group V is located in a region specified by the nucleotide sequence downstream of the BstEII site, over Alal092-Leu1219 (see Fig. 2). Restriction-enzyme-site polymorphism Fig. 4. Restriction analyses of DNA fragments (0.912 kbp) of the L. factis strains WG2 (lanes 1, 6 and ll), E8 (lanes 2, 7 and 12), S K l l (lanes 3, 8 and 13), ML3 (lanes 4, 9 and 14), and A M 1 (lanes 5, 10 and 15). DNA was amplified via polymerase-chain reaction and the fragments (lanes 6 - 10) were incubated with restriction enzymes Kpnl (lanes 1-5) or EcoRI (lanes 11 - 15).

WG2, SK11, E8, AM1 and subspecies lactis ML3 are shown in Fig. 3. Antibodies of groups I (which recognize component A) and 1V (directed against components B and C) were found to react with all these proteinases. The monoclonal antibodies of group V (which recognize component B) react with the proteinases of strains WG2 and ML3 (lanes 1 and 4), but not with the proteinase of strains E8, SK11 and AM1 (Fig. 3, lanes 2, 3 and 5). These results demonstrate that the monoclonal antibodies from groups 1V and V can be used in immunoblot analysis to distinguish between proteinases of strains WG2 and ML3 and of strains SK11, E8 and AM1. lmmunoreactivity of hybrid proteinases Previously, it was found that the monoclonal antibodies of group IV recognize both components B and C, while those

The results obtained above indicate that amino acids in the region Ala1092- Leu1219 determine the binding of the monoclonal antibodies of group V. The nucleotide sequences of the proteinase genes of the cremoris strains WG2 and SKI 1 reveal that two EcoRI sites, present in thc WG2 gene, are lost in the S K l l gene due to mutations of nucleotides 5114 and 5157 (Fig. 2) [lo, 111. In the same region, around nucleotide 5142, a KpnI site is present in the proteinase gene of strain SK11, while it is absent in the WG2 gene. To determine the possible relationship between these mutations in the gene and the binding of the group V antibodies to the proteinase, the proteinase genes of several L. lactis strains were tested for the presence of these EcoRI and KpnI restriction-enzyme sites. Amplification of the DNA fragments was performed by polymerase-chain reaction. The fragments obtained as a result of the polymerase-chain reaction started at nucleotide 4555 and terminated at nucleotide 5467 of the proteinase gene of WG2 [lo], and had a length of 912 bp. The polymerase-chain reaction fragment of strain WG2 was not cleaved by KpnI, and digestion with EcoRI resulted in three fragments of 562, 31 1 and 40 bp, of which the latter is not detectable in the agarose gels (Fig. 4). The proteinase gene of L. lactis subspecies l a d y ML3 showed the same restriction-enzyme pattern in the

819

Fig. 5. Crossed immunoelectrophoresis of the proteinases produced by Wg2 and clone MG1363(pGKV1500). (A) pGKV1500; (B) tandcm crossed immunoelcctrophoresis of pGKVt 500 and WG2.

amplified region; both EcoRI sites were present and the fragment was insensitive to Kpnl. In contrast, the polymerasechain-reaction fragments obtained from the L. lactis subspecies cremoris strains SKI 1, E8, and AM1 were all sensitive to KpnI and two subfragments of 590 bp and 320 bp were formed. The DNA fragments of these strains were not cleaved by EcoRI. The fact that both fragments had the S K l l pattern, indicated that the proteinases of the three strains had the same amino acids at positions 1117, 1129 and 1131.

Crossed-immunoelectrophoresis pattern of inactive proteinase Proteinase was isolated from MG1363 carrying pGKVl500 [18]. This plasmid encodes a proteinase that is inactive due to the substitution ofAsp30 at the active site by an Asn residue via site-directed mutagenesis [18]. Its proteolytic activity is less than 0.1% of the activity of the proteinase synthesized by MG1363-carrying pGKV5OO. Crossed immunoelectrophoresis using polyclonal antibodies against the proteinase of WG2 [14], resulted in one precipitation line (Fig. 5), while the proteinase of strain WG2 (wild type) and the proteinase isolated from L. luctis MG1363(pGKV500) gave two precipitation lines, A and B (not shown). The precipitation line of the pGKV1500-specified proteinase fused with line A of the wild-type proteinase after tandem cross immunoelectrophoresis (Fig. 5), indicating that, in the inactive proteinase, only the epitopes of component A are accessible.

DISCUSSION The first part of this study presents the mapping of epitopes recognized by monoclonal antibodies against the L. lactis subspecies cremoris WG2 proteinase. Deletion mutants of the proteinase were used in Western-blot analysis to localize the binding regions. Lack of recognition of a deletion mutant of the proteinase defines the missing sequences as essential for antibody interaction with the proteinase, either directly or indirectly via protein conformation. Our finding that all antibodies can react with denatured proteinase (in a Western blot), strongly indicates that the missing sequences are part of the epitope. Two distinct regions were found to be involved in binding of antibodies directed against either proteinase component A (group 1 - 111) or component B (group IV VI). These two regions are, for component A, Gln215 - Ile604 and, for component B, Thr816- Leu1219 (Fig. 2). A previous study using polyclonal antibodies indicated that the proteinase

of L. lactis MG1363-carrying pGKV512 expressed only component B [I 51. Our results show that monoclonal antibodies directed against component A react, in Western blots, only with this truncated proteinase. Possibly other epitopes that represent component B are recognized by the polyclonal antibodies, but not by our set of monoclonal antibodies. The epitopes of component A of this proteinase derivative are not accessible for the monoclonal antibody under native conditions (crossed immunoelectrophoresis). After denaturation of the proteinase (Western blots) this A epitope becomes accessible for the binding of our set of monoclonal antibodies. Monoclonal antibodies of group IV (anti-B), but not of group V and VI (both anti-B) react with proteinases of L. luctis subspecies cremoris strains SK13, E8 and AM1 in ELISA [I61 as well as in immunoblots (Fig. 3). These strains express component C, a proteinase component which is not present in strain WG2, but they lack component B 1141. In contrast to the monoclonal antibodies of group V and VI, the antibodies of group IV cross-react with proteinase component C [16]. The observation that monoclonal antibodies of group V react with the hybrid proteinase specified by pGKV552c, but not with that encoded by pNZ521c, indicates that an antigenic determinant of component B is located between Ala1092 and Leu1219 of the mature proteinase (Fig. 2). The deduced amino acid sequences of the proteinases of L. lactis subspecies cremoris S K l l and WG2 reveal six amino acid differences between both proteins in this particular region [lo, 111. It is very likely that one or more of these six mutations are responsible for the lack of binding of group V monoclonal antibodies. The closely spaced amino acids Thr1126, Ala1129 and Glull31 of the WG2 proteinase that are changed in the S K l l proteinase into Lys, Thr and Lys, respectively, cause a change in the net charge in this small region. Possibly this charge difference leads to suppression of the binding of group V antibodies to the S K l l proteinase. Also, the mutation of Glu1095 + Lys, causing a change in two charges, could be responsible for the decreased binding of antibody. Comparison of the proteinase genes of the cremoris strains S K l l and WG2 reveals that, in this region, a number of nucleotide mutations result in the loss of two EcoRI restriction-enzyme sites and the formation of a KpnI site in S K l l (see Fig. 4) [lo, 1I]. DNA fragments of the strains AM1 and E8, comprizing this specific proteinases-gene region, were multiplied by polymerase-chain reaction. Both fragments had the SK11 restriction-enzyme pattern, indicating that the proteinases of these strains had the same amino acids as S K l l at positions 1117, 1129 and 1131. This correlates with the failure to bind antibodies of group V. In other words, differences in the proteinases of L. lactis strains can be detected using these monoclonal antibodies. The localisation of the binding regions of the proteinase components A, B and C of L. luctis raises the question as to how these proteinase components are formed. Components A and B originate from one native proteinase, but their precipitation lines in crossed immunoelectrophoresis do not fuse, suggesting that the respective epitopes are distinctly exposed [14, 151. In this report, we have shown that only one precipitation line was found in crossed immunoelectrophoresis with the inactive proteinase encoded by pGKV1500. This line fused with the precipitation line of component A. This indicates that exclusively the epitopes of component A are accessible for antibody binding in the inactive proteinase and that proteolytic activity is essential to exhibit other epitopes, such as the epitopes of B. It is therefore very likely that components A and B are different conformations of the L. luctis proteinase,

generated by autoproteolysis. This supports a previously presented model in which it was postulated that accessibility of different epitopes was the result of conformational changes [15].The results described in this paper indicate that in conformation A only, epitopes are exposed to which monoclonal antibodies of group I, 11 and I11 can bind and, in that conformation, the binding regions for the monoclonal antibodies of group IV, V and VI are not accessible. In conformation B only the epitopes of region B are exposed. We thank Dr Loe de Ley for valuable discussions and suggestions. We also thank Roe1 Haverkort for culturing of hybridomas and Judy Bun for immunizing rabbits for polyclonal antibodies.

REFERENCES 1. Thomas, T. D. & Mills, 0. E. (1981) Neth. Milk Dairy J . 35, 255 - 273. 2. Smid, E. J., Plapp, R. & Konings, W. N. (1989) J . Bacteriol. 171, 61 35 - 6140. 3. Laan, H., Smid, E. J., Tan, P. S. T. & Konings, W. N. (1989) Neth. Milk Dairy J. 43, 327 - 345. 4. Konings, W. N., Poolman, B. & Driessen, A. J. M. (1989) CRC Crit. Rev. Microbiol. 16, 419 -416. 5. Smid, E. J., Driessen, A. J. M. & Konings, W. N. (1989) J . Bacteriol. 171, 292 - 298. 6. Exterkate, F. A . (1976) Neth. Milk Dairy J . 30, 95-105. 7. Visser, S., Exterkate, F. A., Slangen, C. J. & de Veer, G. J. C. M. (1986) Appl. Environ. Microbid. 52, 1162- 1 166. 8. Kok, J . (1990) FEMS Microbiol. Rev. 87, 15-42. 9. Kiwaki, M., Ikemura, H., Shimizu-Kadota, M. & Hirashima, A. (1989) Mol. Microbiol. 3, 359-369. 10. Kok, J., Leenhouts, K. J., Haandrikman, A. J., Ledeboer, A. M. & Venema, G. (1988) Appl. Environ. Microbiol. 54, 231 238. 11. Vos, P., Simons, G., Siezen, R. J., de Vos & W. M . (1989) J . Mol. Biol. Chem. 264, 13579-13585.

12. Haandrikman, A. J., Kok, J., Laan, H., Soemitro, S., Ledeboer. A. M., Konings, W. N. & Venema, G. (1989) J . Bacteriol. f7l. 2789 -2794. 13. Laan, H. & Konings, W. N . (1989) Appl. Environ. Microhiol. 55, 3101 -3106. 14. Hugenholtz, H., Exterkate, F. A. & Konings, W. N . (1984) A p p l . Environ. Microbiol. 48, 1105-1110. 15. Kok, J., Hill, D., Haandrikman, A. J., de Reuver, M. J. B., Laan, H. & Venema, G. (1988) Appl. Enuiron. Microbiol. 54, 239244. 16. Laan, H., Smid, E. J., de Leij, L., Schwander, E. & Konings, W. N. (1988) Appl. Environ. Microbiol. 54, 2250-2256. 17. Kok, J., van Dijl, J. M., van der Vossen, J. M. B. M. & Venema, G. (1985) Appl. Environ. Microbiol. SO, 94-101. 18. Haandrikman, A. J., Meesters, R., Laan, H., Konings, W. N.. Kok, J. & Venema, G. (1990) Appl. Enuiron. Microbiol. 57, 1899- 1904. 19. Vos, P., Boerrigter, I. J., Buist, G., Haandrikman, A. J., Nijhuis, M., de Reuver, M. J. B., Siezen, R. J., Venema, G., de Vos, W. M. & Kok, J. (1991) Protein Eng. 4, 479-484. 20. De Man, J. C., Rogosa, M. & Sharpe, M. E. (1960) J . Appl. Bacteriol. 23, 130 - 135. 21. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685. 22. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 23. Kyhse-Andersen, J. (1984) J . Biochem. Biophys. Meth. 10, 203209. 24. Van der Plas, J., Hellingwerf, K. J . , Seijen, H. G., Guest, J. R., Weiner, J. H. & Konings, W. N. (1983)J. Bacteriol. 153,10271037. 25. Mullis, K. B. & Faloona, F. A. (1987) Methods Enzymol. 155, 335 - 350. 26. Saiki, R. K., Gelfmd, D. H., Stoffel, S., Scharf, S., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 23Y, 487 -49 1. 27. Laan, H. & Konings, W. N. (1991) Appl. Envirun. Microhiol. 57, 2586 - 2590.