FEMS Microbiolos~Reviews87 (1990) 15-42 Publishedby Elsevier

15

FEMSRE00157 Genetics

Genetics of the proteolytic system of lactic acid bacteria Jan Kok Department of Ce~'etics, University of Groningen, Haren, The Netherlands

Key words: Proteinase, Peptidase: Amino acid/peptide uptake; Plasmid, Gene cloning; DlqA sequence

1. SUMMARY The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellullar peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococci, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.

Correspondence to: J. Kok, Departmentof Genetics,KerHaan

30, 9751 Nlq Hatch, The Netherlands.

2. INTRODUCTION Lactic acid bacteria are important components of the microflora of a wide variety of fermented foods and feeds from milk, mea: and vegetable origin. Their main function is the rapid conversion of the available sugar to lactate resulting in the low pH which is necessary for good product quality. The proteolytic system of lactic acid bacteria is crucial for the growth of these organisms on protein-rich substrates as they are nutritiously very fastidious. In addition to nucleotides and vitarains, all species of lactic acid bacteria have an absolute requiremunt for exogenous suppfies of various amino acids [1-4]. In natural milk the concentrations of free amino acids and low molecular weight peptides initially present are only sufficient to give growth to cell densities corresponding to 8-16~ of the maximum found in coagulated milk and are thus well below the minimum necessary to support rapid acid production [5]. As the cell density increases, milk protein becomes the most important source of nitrogen and a functional set of proteinases and peptidases has to supply the cells with the required amino acids and small peptides. Amino acid and peptide uptake is the next crucial step in the utilization of milk protein by lactic acid bacteria and is mediated by. separate systems [6,7]. Lactococci have been shown to possess distinct dipeptide and oligopeptide transport systems [6,7]. A fortuitous but important side effect of the

0168-64A5/90/$03.50© 1990Federationof EuropeanMicrobiologicalSocieties

action of the proteolytic system is the generation of amino acids and peptides which give the fermentation products their characteristic organoleptic properties. The evident world-wide importance of fermentation processes using lactic acid bacteria has led to a rapi0 increase in the research devoted to these organisms during the last 20 years. The analyses of the proteolytic system has given new insights in the protein utilization of these organisms and will be reviewed here. The emphasis will be on the recent progress in the knowledge of the genetic basis of the proteolytic system as was largely gained after the second Symposium on Lactic Acid Bacteria, held in The Netherlands in 1987.

3. PROTEINASES The proteinases of lactic acid bacteria perform the first step in the cascade of reactions leading to the breakdown of casein. Because of their prime importance in fermentation processes, the enzymes are subject to intensive biochemical research for some 15 years now. In this paragraph the knowledge acquired from these studies on the lactococcal proteinases will be summarized and reference will be made to the corresponding enzymes in other species of lactic acid bacteria. As this paper will deal with the genetics of proteoly-

sis, it is beyond its scope to go into details of biochemical aspects of the proteolytic system of lactic acid bacteria. The reader's attention is drawn to recent excellent reviews specifically dealing with this subject [8-10]. 3.1. Classification o f proteinases

Biochemical studies on lactococcal proteinases show that the enzymes are high molecular weight proteins (the published molecular sizes ranging from 80-145 kilodalton (kDa)), having p H optima around 5.5-6.5 and isoelectric points of 4.40-4.55, and are either actiwAted or stabilized by Ca2+-ions. The enzymes are blocked by phenyl methylsulphonylfluoride (PMSF) or diisopropyl fluorophosphate (DFP) and are, therefore, serine-type proteinases, l, generally agreed that the proteinases are located primarily in the cell wall [8,11-17]; for a detailed review of this subject see Thomas and Pritchard [10]. Although the proteinases from the various lactococcal strains studied solar share these general properties, certain differences do exist and those have been extensively examined. Lactococci characteristically contain a number of different cell-associated proteolytic activities which vary between strains. Multiple cell wall proteinases have been shown to exist in Laetococcus lactls ssp. lactis [18] and several attempts to

Table 1 1. lactis up. cremoris strain classification Strain

Wg2, HP, C13 E8 TR FD27 AM1, SKII

Classificationbased on proteinaseactivity" specificityb Pl Pll Pl (Pill) Pl Pll Pill Pl Pll Pill Pill

HP HP AMI HP (AM1) HP AM1 AM1

immunology Polys © A B A C A B C A B C A A'(B) C

Mouos d I-VI I-IV I-VI l-Vl l-Vl

monm, polycional, and monoclonal antibodies against the purified prote~atso of L. lactls up. cremor/s Wg2 [22,23]. Data derived from [19], [20], [22] and [23], respectively.Plll-aetivity and AMl-specifidty have been added to E8 and TR, ~vely, oa the basis of the comidetation [20] that the proteinases from these strains cannot be isolated in a reproducible way. Comequentiy, E8, TR, and FD27 may be comidered as one group. Monoelonalantibody groups I, II, and III react with proteina~ component A; monodoual antibody groups IV, V, and VI react with protcinase component B. C13 was not included in the analysis rains the monodoual antibodies [23].

clarify the proteinase complex in a set of strains of the subspecies cremoris have been undertaken. These studies have resulted in the classification of the strains on the basis of differences in their proteinase activity, -specificity, and -immunology and are s - ~ T a b l e 1. The first classification was based on the enzyme activity of whole cells and discriminated between two acid activities differing in temperature optimum (30°C for the Pill-activity and 40°C for PI) and a neutral activity optimal at 30°C (PII) [19]. In a later study, the neutral activity at 30°C (Pll) was shown to be an artifact of the acid activity at this temperature (PI) [20]. The PI activity from /.. lactis ssp. cremoris Hp had the same temperature optimum irrespective of being cell wall-bound or soluble [19,20]. However, the cell wall-bound proteinas¢ of the cremoris strain ACI [13] had a temperature optimum of 30°C, characteristic of PlII, whereas the soluble, purified enzyme was most active at 4G°C (ffi. Pl). These results suggest that the same proteinase can have different proteolytic activities depending on the state of purification. Since it is difficult to characterize the proteinases by using intact cells, and, since highly purified proteinase preparations are very unstable, in later studies partially purified proteinases were used. A strain classification based on the difference in action of their proteinases on the various casein types present in milk was put forward by Visser et al. ([20]; see Table 1 and Fig. 1). Using the distinctive breakdown patterns of asl-, ~-, and s-casein two enzyme specificities could be detected, an HP-type (Pl) and an AMl-type (PlII). Twenty-three proteolyficaily active cremoris strains isolated from mixed strain starters and five strains of the subspecies lactis apparently belong to the PI-type because they only degraded t-casein [14,21]. Polyclonal antibodies against the proteinases of a number of cremoris strains have been isolated and with immunological techniques four proteins, A, A', B, and C, have been identified in the partially purified proteolytic systems of these strains. Proteins A and B have been shown to be proteolytically active. Component A is present in all strains tested and is always accompanied by at least one of the other three proteins. Proteins A and A' share common antigenic determinants as

~ - -

Po

6 Pn,

PI

Pol,

- -

Fig. 1. Cleavage of a- and ~-easein by P! and P i l l prot¢inas~ isolated from L. lactis ssp. cremor/s strains Wg2 and S K l l , respectively. The casein breakdown products were separated by

SDS-polyam'ylamide(20~$)gel electrophoresis.- , undigested controls. do B and C ([15,22]; see Table 1). Monoclonal antibodies have also been used to study the proteinases of the same set of strains. The monoclonal antibodies reacted either with protein A or with protein B but never with both components [23]. Monoclonals reacting with protein A reacted with all strains studied, confirming the conclusion reached with the polyclonals that component A is expressed in all s:ralns [22]. 3.2. Genetics of !actococcal proteinases

The genetic studies of the proteinases and the corresponding genes from the lactococci have led to a major breakthrough in our understanding of these enzymes and have led to a considerable clarification in the body of biochemical data gathered on the proteinases. In an earfier pubfication [24] we have dealt with the genetics of the proteinases of l a c t o c ~ i . The availability of the nucleotide sequences of the proteinase loci allowed the detailed analysis of specific mutations in this region and has led to a rapid furth~

18 i n c r e a s e in t h e k n o w l e d g e o f t h e l a c t ~ teinases.

pro-

3.2.1, Proteinase plasmids P r o d u c t i o n o f p r o t e i n a s e s b y lactococci is a n u n s t a b l e trait [25-28]. S p o n t a n e o u s l y a n d in s o m e c a s e s w i t h c o n s i d e r a b l e frequency, p r o t e i n a s e n e g a t i v e v a r i a n t s a p p e a r in c u l t u r e s o f w~ld-typc P r t + strains. T h e s e m u t a n t s s h o w a r e d u c e d ~c~d p r o d u c t i o n a n d a r e u n a b l e to g r o w in m i l k to t h e h i g h cell densities n e c e s s a r y for g o o d f e r m e n t a t i o n

practice ( " s l o w " p h e n o t y p e ) . Irrever~ibility o f this loss o f p r o t e i n a s e activity a n d t h e fact t h a t t h e m u t a t i o n f r e q u e n c y c o u l d b e e n h a n c e d b y treatm e n t o f cells w i t h a c r i d i n e d y e s a n d e t h i d i u m b r o m i d e , o r b y g r o w t h at e l e v a t e d t e m p e r a t u r e s suggested the involvement of plasmid DNA ([29,30]; see Fig. 2). T h i s g e n e t i c m e t h o d o l o g y to identify p r o t e i n a s e p l a s m i d s h a s b e e n r a p i d l y adopted and, by now, curing studies have prov i d e d e v i d e n c e for p l a s m i d l i n k a g e o f p r o t e o l y t i c activity in a n i n c r e a s i n g n u m b e r of lactococc. '.

Table 2 Localization of proteinase genes in iactococci Subspecies and strain

Size of (suspected) Prt+-plasmid in kb

Evidence apart from curing

Hybridization with Wg2 probe

Reference

/act/s C2 712

45 Ip, 27,19 56]p

Transduction Transduction Conjugation Cloning Conjugation Cloning

nd +

[148-150] [32,46,137]

nd nd nd nd -- © nd +

[31,151] [47] [151] [151] [48] [152] [155,156]

-t-d + + +

[44,153]

+ + + + nd + (93 100) * nd nd nd

[156] [1551

ML3 763 C10 M18 SSL135 DRC1 UC317 cremor/s HP MLI Wg2 E8 SKll UC205 UC411 P8/2/47 H2 SD8, SD9, SDI1 VI6 VI8 1200 M12R

49.5Ip 55tp 60 Ip 67.5tp - " 46.5 Ip 69Ip 13.5 3.3 26

n~ 78 34.5 30 14 63]p 93 (93 100)tp 93¥ 21 19.5

Cloning b Conjugation Cloning Cloning Cloning Cloning Conjugation Cloning Conjugation Conjugation Conjugation Conjugation

[1531 [43,44] A.J. Haandrikman t [45,154]

A. Geis, B. Kiefer f [158] H. Neve, A. Geis t H. Neve, A. G©is f H. Neve, A. Gels t [31] [1571

• A plasmid-frce derivative was still Prt+, suggesting chromosomal linkage of this trait in SSL135 [48]. see refe~nce [24] for a discussion of these r~ults. d No hybridization with the 3.3-kb plasmid was observed. Instead, hybridization was found with a high molecular weight plasmid (A.M. Ledcbocr, personal communication), from which the scnc was cloned (P. Vos and W.M. de Vos, personal communication). • Hybridization was observed but no discrimination could be made as to which of the two plasmids hybridized (A. Ocis and B. Kidcr, pasonal communication). t Pasoaal communication. ~' h ~ c / p r o t e i n a s e p~unid. rid, not dctamincd.

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I Fig. 2. Agarose (0.8%) gel electrophoresis of plasmid DIqA isolated from acriflavine.inducedPrt" variants of I_ lactis ssp. cremoris Wg2 ($2, S3, $8, $9, $23, S32). The plasmid complement of a spontaneous Pn" variant (Prt') and of the wild-type Wg2 strain (Prt + ) are also shown. Only the covalently closed circular DNA of the six distinct plasmids of W82 are indicated. pWV01 (2.2 kb) was used to construct a series of broad host range lactococcal cloning-, promotor- screening- and expression vectors [39,142,146]. pWV05 is the 26-kb proteinase plasmid of Wg2. Adapted from [43].

strains. Plasmids ranging in size from 13.5 to 100 kilobasepairs (kb) have been implicated in proteinase production. In some strains the genes for proteolytic ability are located on a plasmid ~vhich is also involved in lactose metabolism. A summing-u p of strains and their proteinase plasmids is given in Table 2. Structural instability of proteinase plasmids and loss of plasmids which are not involved in proteinase production during curing experiments make interpretation of plasmid curing data difficult [31-33]. Several research groups, therefore, have started to use gene transfer systems to physically rink proteolytic ability with certain plasmids. Natural gene transfer systems viz. transduction and conjugation as well as prot.oplast transformation and electrotransformation have been used to provide evidence for plasmid linkage of proteinase determinants in strains of lactococci (see Table 2). 3.2.2. Proteinase gene cloning

The recently developed cloning vectors and techniques to genetically manipulate lactic acid bacteria ([34-39]; for reviews see [40-42]) have, in a number of instances, been used to localize pro-

teinase genes. In I~ lactis ssp. cremoris W g 2 loss cf the 26-kb plasmid pWV05 was accompanied by loss of proteolytic ability ([43]; see Fig. 2). A 6.5-kb H i n d l I I fragment of this plasmid was inserted in a pWV01-based cloning vector [39] and transferred to a proteinase-deficient strain of L. lactis by protoplast transformation. The strain thus obtained expressed a functional proteolytic activity and was able to grow normally in milk 044]; see Fig. 3). With immunological techniques it was show~ that in both Bacillus subtilis a n d L lactis carrying the recombinant plasmid the fragment specified the two proteins A and B of the proteolyric system of Wg2 ([22]; see 3.2.1). The fact that the proteinase antigen could be detected in the heterologous host B. subtilis proved that the structural gene(s) for a proteolytic enzyme(s) had been cloned. By now, a number of proteinase genes from different lactococcal strains have been cloned and analysed. De Vos et al. identified, in a lambda-phage b'ddk of the 78-kb proteinase plasmid p S K l l l of the cremoris strain S K l l , plaques in which proteinase was detectable with

Fig. 3. Left: ~owth in milk and in a transparant citratcd m/Ik-based agar plate (containing bromocresolpurple as a pH indicator) of L lact~ ssp. ~¢t~ MG1363, a plasmid.frec Pn" variant of L lact~ ~sp. ~¢t~ 712 [32]. The strain forms only tiny coloniesand fails to comsulatemilk within 18 h. Right: the same strain, carrying a rec~mbimmt plasmid with the proteinase gene region cloned from the proteinase plasmid pWV05 of L. lactis ssp. cremoris Wg2. The DNA fragment restores the strain's ability to grow rapidly in milk with good acid .~roduc,tion, resulting in milk coagulation and the formation of large colonies surrounded by a white precipitate of casein in the milk-agar plate.

the genes are ldghly conserved ([24,42,47]; see Fig. 4). The recently cloned genes of the cremoris strains E8, ML1 and UC317 also show the same conservation of restriction enzyme sites (A.J. Haandrikman, J. Law, P. Vos, W.M. de Vos and G.F. Fitzgerald, personal communication). Therefore, it appears that many (if not all) lactococci carry essentially the same gene for a cell wall-associated proteinase which has only slightly been subject to change during evolution. From the genes of three proteinases, those of the cremoris strains Wg2 and SK11 and that of the L. lactis ssp. lactls strain NCDO763, the nucleotide sequences have been elucidated completely [47,51,52]. Three additional genes have been sequenced partially (that from strain E8, in our group, and those from strains ML1 and UC317 (P. Vos, J. Law, W.M. de Vos and G.F. Fitzgerald, personal communication). The conclusions derived from the comparison of restriction enzyme sites of the cloned protcinase genes were fully confirmed by the nucleotide sequences. The genes from the two cremoris strains appeared to be almost identical with an overall homology of 98~ on the nucleotide and amino acid level (44 amino acid substitutions). Although some amino acid substitutions seem to be clustered, most of them are more or less randomly distributed [52]. The differences with L lactis ssp. lactis NCDO763 are

immunological methods. Subcloning of the specific DNA fragment on a pSH71 derivative allowed the complementation of the proteinase defect of L. lactis MG1363 [41,45]. The lactose/proteinase plasmid pLP712 of L lactis 712 has been mapped in detail using transductionally shortened derivatives or deleted forms which arose frequently in a strain carrying pLP712 as the single plasmid [32]. In this way the proteinase gene(s) could be localized on a 4.8-kb BglII fragment. This fragment was cloned in a pSH71 derivative and partly restored, in strain L. lactis MG1363, the ability to grow in milk [46l. The protcinase genes of L. lactis ssp. lactis 763 [47], of the cremoris strains E8 and ML1, and the lactis strain UC317 have also been cloned and functionally expressed in L lactis (A.J. Haandrikman, J. Law, P. Vos, W.M. de Vos and G.F. Fitzgerald, personal communication). Until now, it is not clear whether the chromosomal fragment from L. lactis SSL135, which encodes the ability to grow rapidly in milk, carries a prc~ teinase gene a n d / o r other genes involved in proteolysis [48-50]. 3.2.3. Proteinase gene sequences and homology From a comparison of the restriction enzyme maps of the cloned proteinase genes of the L lactis ssp. lactis strains 712 and 763 and the cremoris strains Wg2 and S K l l it is obvious that

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Fig. 4. Phydcal maps of the proteinaseplasmidsof L. lactb ssp. lactis 712 (pLP712, 56 kb), and the cremor/s strains SK11 (pSK11, 78 kb) and Wg2 (pWV05,26 kb). The plasmidsare alisned such that maximaloverlapis obtainedin the regionwhere the proteinLse genes are located, as deduced from the nucleotidesequencesof the W82 and SKll genes[51,52].Thick lines indicate the restriction fragmentsinitiallycloned in L/act/s specifyingproteinaseactivity.The bar represents 1 kb of DNA. (For details: see text), prtP, proteinesesene; prtM, 8ene for proteinasematurationprotdn; IS, insertionsequence;B, Bglll; A, BamHl; C, Clal; E, £coRl; H, Hindlll; X, XkoI.(W.M.de Vos end M3. Gasson,personalcommunication).

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MATURE PROTEINASE SS PRO Fig. 5. Comparison of the amino acid sequences of the proteinases from L~ lactis ssp. cremods swains Wg2 and S K l l , and L./actis ssp. lactis NCDO 763 (763), as derived from the nucleotide sequences of the respective genes [47,51,52]. The hotizoaud bar represents the amino acid sequence of the W82 proteinase. Differences with the amino acid sequences of the S K l l and 763 protcinases are shown as vertical lines above and below the Wg2 sequence, respectively. Identical amino acid substitutions in the S K l l and 763 proteinases are indicated by an uninterrupted vertical line. The 60 amino acid dupfication in the C-terminus of the S K l l enzyme is shown by the double arrow above the bar. The membrane anchor is indicated as a small horizontally striped region. The bar at the top-left of the figure represents 100 amino acids. The three regions of homology of the proteinasas with subtilisin are indicated by the black boxes. The amino acids constituting the active site are shown: D, Asp.30; H, tlis.94; S, SerA33. Double headed m o w s : SS (stippled area), signal sequence; PRO (hatched box), pro-sequence. Adapted from [24].

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-187

:

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I 1715

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o."..', -107 ",'1

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® and su0tili~dnq~.;e~,:be~&DY,

amyloliquefaciens and subtilis. Subtilisin sequences are from the National Biomedical Research Foundation (NBRF) ~l~t¢~n bank, Octobar 1986. Only those amino acids differing from the residues in the Carlsberg enzyme are s'-o'~, identical res} ~u~s are boxed. The active site amino acids Asp, His, and Set are indicated by the vertical dotted lines. B. "rtae h ~ l o 3 o u s regions ~rtym A (black boxes) are drawn to scale on a linear map of the whole proteina~ and compared with a lin~tr map of sub "tdhin. Dc.tted box, signal sequence; hawated box, pro-~quence; s ~ p e d region, membrane anchor. Numbers refer to amino acid residue~ -;-i, N.tenninal amino acid of the mature proteinase; D, Asp32/30; H, His64/94; Ser221/4~3. Adapte2 from [2~,].

even smaller: the Wg2 proteinase and that of strain 763 differ in only 18 amino acids [47]. Some of the amino acid differences involve conservative amino acid replacements, while others result in functionally different amino acids. There is a strong bias for more basic amino acid substitutions in the S K l l proteinase, especially in the first 1200 amino acids. The most striking difference between the S K l l proteinase and the other two is the presence in the former of a duplication of 60 amino acids near the C-terminus. The differences in the amino acid sequences between the Wg2 proteinase (PI-type) and the S K l l enzyme (PIIItype) are responsible for the different caseinolytic properties of both enzymes (see sections 3.1. and 3.2.8.). A summary of the homology comparison is given in Fig. 5. 3.2.4. Homology with subtilisins The nucleotide sequences of the proteinase genes have revealed a number of interesting features of the proteinases they specify [47,51,52]. First, protein homology comparisons indicated three regions in the lactococcal proteinases which have extensive homology with serine proteinases of the subtilisin family (see Fig. 6). Especially, amino acids involved in the formation of the active center (viz. Asp.32, His.64, and Ser.221 of the subtilisins) are well conserved in the lactococcal proteinases. The homologous sequences are separated by stretches of amino acids not found in the subtilisins, most notably, a sequence of approximately 175 amino acids between the His and Ser residues of the active site. Another marked structural feature of the lactococcal proteinases is the presence of a long C-terminal extension not present Jn the subtilisins. The coding regions of the Wg2 and 763 proteinase genes contain 1902 codons which could specify proteins with molecular weights ( M w) of 200 000. The S K l l proteinase is somewhat larger due to the duplication of 60 amino acids in the C-terminal part of the enzyme. The proteinase genes are flanked by transcription- and translation regulatory sequences which closely resemble those reported for B. subtilis and Escherichia coli and are in good agreement with the regulatory sequences found in lactococci [42,53]. Like the sub-

tilisins, the lactococcal proteinases are synthesized as pre-pro-proteins. At the N-terminus a typical signal peptide-like sequence of 33 amino acids is present. The N-terminal amino acid sequences of the isolated proteinases have been determined and indicate that in all three cases the mature enzyme starts with the aspartic acid residue at position 188 of the predicted amino acid sequence [47,52,54]. 3.2.5. Proteinase localization A clear difference between the subtilisins and the lactococcal proteinases is their localization. While subtilisins are fully secreted and found in the culture medium of producing cells [55,56], the proteinase activity of lactococci is associated with the cells ([11-13,15,18,19,22]; for reviews [8-10]). At the extreme C-terminal end of the lactococcal proteinases a region was identified which shows homology with a number of membrane attachment sites of proteins of Gram-positive bacteria ([47,52,57]; see Fig. 7). A very hydrophobic a-helix of 18 amino acids is flanked by an N.terminal profine residue and a C-terminal hydrophylic region containing three basic amino acids followed by two acidic residues. Upstream of the a-helix a proline-rich region with a number of conserved amino acids is located. It follows that the lactococcal proteinases are attached to the cells by anchoring in the cell membrane while the prolinerich region directly upstream of the membrane anchor most probably provides for contacts with the cell wall. Therefore, it is more accurate to refer to the proteinases as cell envelope-attached enzymes. Removal of the C-terminal domain from the proteinases results in the release of the proreins from the cells: all initial proteinase gene clones were truncated at the Y-end and the Cterminally truncated proteinases (lacking 130 or 403 amino acids) were fully secreted [45,47,57]. Moreover, these results show that C-terminal truncation is possible without abolishing proteinase activity (see also section 3.2.7.). 3.2.6. Proteinase maturation A second open reading frame (ORF) is present immediately upstream of the proteinase gene and directed in opposite orientation [47,51,52]. It is

found in all the proteinase gene regions sequenced so far and is completely conserved. O n the basis of the similarities in the restriction enzyme maps of the proteinase plasmids of L, lactis 712 and of other lactococcai str:~ins, this O R F also seems to b e present in these cases [24]. Deletion analysis has shown that the O R F is essential for proteolyric activity: it is necessary for the maturation of the proteinase a n d was consequently (re)named p r t M ([57,58]; see Fig. 4). In the same two papers it was proposed to use the notation prtP for the proteinase gene. I n the original clones, p r t M was truncated a n d lacked four amino acids at the C-terminus, apparently without interfering with its functioning. The entire gene specifies a protein of I ~

33 kDa. Cells carrying an intact proteinase gene but lacking p r t M synthesize a n d correctly locate the protcinase antigen b u t they are phenotypically proteolytically negative (see Fig,. 7). Apparently, the proteinase produced is inactive. The inactive proteinase formed in the absence of p r t M was larger than that made when p r t M was present, suggesting a proteolytic step in proteinase activation, effected by the prtM-gene product. P r t M can exert its effect in trams [58,59]. T o examine the roles of PrtM and the proteinase itself in the activation process, a mutation in the proteinase was made to destroy the active center of the enzyme. By changing the aspartic acid residue, which is part of the active center of the lactococcal

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II

Fig. 7. Effect on proteolytie activity and cell attachment of the proteinase specified by plasmids carrying mutations in the proteinase locus o f / , lacti$ ssp. cremoris Wg2. Piasmid designations a~'cgiven on the left. The top line shows the prt locus as present on plasmid pWV05 of strain Wg2. PrtP: proteinase gene; prtM: gene for the proteinase maturation protein. Whether the plasmids specify the maturation protein or (incomplete) proteinase is indicated by the small and long open bar, respectively.The small hatched area at the extreme C-terminus of the proteinase represents the membrane anchor. Asp.30, His.94 and Set.433 of the active site ave shown as is the Asp.30 to *Asn.30 mutation. SS, signal sequence; PRO, pro-sequence. B, H, N, C, M: sites for restriction BamHl, Hindlll, Nrul, ClaI, and Mlul, respectively. Fat arrows: promotors for prtM and prtP. For details: see text. (Taken from AJ. Haandrikman et aL, unpublished).

24 proteinase (at position 30 of the matured enzyme), into asparagine using site directed mutagenesis (SDM) techniques, an inactive proteinase was obtained (Fig. 7; A.J. Haandrikman, personal communication). The SDM-inactivated proteinase had a M w which was higher than the active enzyme. The elucidation of the role of PrtM in the maturation process, either d i r ~ t , b y performing the activating cleavage step, or indirect, b y inducing the pro-proteinase to perform a n autocatalytic conversion into the mature active form awaits the determination of the N-terminal amino acid'sequences of the inactive enlarged forms of the

proteinase. In a subsequent study, H a a n d r i k m a n a n d coworkers overexpressed the p r i M gene in E. coil and isolated PrtM-specific antibodies. Using these antibodies in immunogold labelling experiments, it was shown that PrtM is located in the cell envelope of lactococci, while cell fractionation studies showed that the protein was enriched in the m e m b r a n e fraction. The observation from the nucleotide sequence that P r t M might be a lipoprotein [57,58] was confirmed in experiments with 3H-labelled palmitic acid. A 32-kDa 3H-labelled protein specifically precipitated with the P r t M di-

s~gual sequence and Cys.24 of the maturation protein (PriM) C

slSnal sequence, pro-re~oa and Asp.188 of the proteiaese C-terminus of the pro(e'mu~ with membrs,,,e ahchor

N

~-

Cysteine residue with lipomodificetion

):~

putative sctfdlgestlon ~e(s) N, C: N- tud C-terminus, respcctlveiy. M, W: call membrene and .wall, respectlvuly.

w :.:.~ii

~:~.~.~.~.:

:::::: ~>

~ :::::::::::::::::::.::::::i:~i

FiB. 8. The proteinese model. (1) Precursor forms of the maturation protein and the proteinase are synthesized from a proteinase plasmid carrym8 the prtM and prtP 8enes. (2) Lipomodificationat position Cys.24 of pre-maturation protein and subsequent cutting by the lactocoocalequivalent of E. coil SPase If (prolipoproteln signal peptidase) results in membrane attachment of the maturation protein. Pre-pro-proteinase is transloeated across the cell membrane, cleaved by the equivalent of SPare I (signal peptidase) and remains attached to the cell membrane through the membrane anchor in the extreme C-terminus of the enzyme. (3) The maturation protein effects, in a way not yet undel'stood, the removal of the pro-sequence by cleavage between Thr.187-Asp.188 to sire a mature active proteinasc (numbers refer to amino acid positions in the pre-pro enzyme). (4) Seifdigestion results in the release of a C-terminally truncated protelnase (which can still be active). Incubation in Ca2÷-frec buffer is thought to stimulate selfdigestlon by enhancing proteinase activity or by unmasking of selfdigestionsites in the proteinase molecule.

25 rected antibodies only in cells carrying the prtM gene (A.J. Haandrikman, personal communication).

3.2. 7. The proteinase model The available genetic data presented above on the lactococc~ proteinases now allow a reinterpretation of the biochemical and immunological data collected in recent years and their integration in a model for lactococcal proteinase. Fig. 8 schematically depicts the salient features of this model. The first important conclusion from the genetic information is that the caseinases, the extracellular serine proteinases of lactococci, are all very similar enzymes which differ in only relatively few emino acids. This holds true for the enzymes of both subspecies. The amino acid differences, although few in number, do divide the proteinases in two specificity classes: enzymes which can rapidly degrade a- and t-casein (Pill-type) and those which primarily act on ~-casein but with a specificity differing from that of the PIII enzymes (PI-type). Enzymes with a chimeric specificity are found in certain strains and have been produced by genetic manipulation (see Table 1 and section 3.2.8.). The second new concept is that the proteinases are attached to the cells via a membrane anchor which is present at the extreme C-terminal end of the molecule. The proline-rich region in the Cterminus directly upstream of the membrane anchor might make contacts with cell wall components and serve as a "spacer" between the Cterminus and the N-terminal proteinase domain. Removal of the anchor sequence results in the release of the proteinase, as was the situation with the initial proteinase gene clones, all of which specified C-terminally truncated secreted proteins. The third aspect of the model concerns the fact that a second gene, prtM, is essential to obtain a functional proteinase. The priM gene product is a membrane located lipoprotein which is, in a way not yet understood, involved in the activation of the proteinase. In this respect, the lactococcal proteinases differ from the subtilisins which perform autocatalytic activation without the need for an additional cellular function [56]. ,Although no direct proof could be obtained due to the fact that the N-termini of both inactive proteinases, the one

produced in the absence of PrtM and the active site mutant, could not be determined ([58]; AJ. Haandrikman, personal communication) it is tempting to speculate that the maturation step involves the removal of the pro-region from the inactive pro-proteinase, a step effected by PrtM. We originally postulated a proteinase degradation model to reconcile biochemical and immunological work with genetic data indicating that a discrepancy existed between the Mw of the proteinase isolated from L lactis ssp. cremoris Wg2 and the size of the product predicted from the proteinase gene of this strain [24,60]. The principals of this model still hold true and, actually, have been substantiated by the new data. In the new model shown in Fig. 8 the data on PrtM have been incorporated. As is the case for subtilisin, the lactococcal proteinase is synthesized as a pre-proprotein. The size of the proteinase of strain Wg2 as deduced from the nucleotide sequence of its gene is 200 kDa [51]. The putative signal peptide contains 33 amino acids [51,52], while the pro-region consists of 154 amino acids [47,51,54]. Removal of this N-terminal stretch of amino acids would result in a mature enzyme of 1715 amino acids with a molecular size of 187 kDa. Additional proteolytic cleavage was postulated to account for the difference between this calculated size and that of the isolated enzyme (140-165 kDa) [22,51,54]. As the purified enzyme starts with Asp.188 [47,51,54], the cleavage occurs at the Cterminus of the proteinase. Apparently, large Cterminal deletions can occur without abolishing proteinase activity. This observation was confirmed by in vitro deletion analysis of the proteinase gene: large 3"-end deletions resulted in the production of smaller but still active proteinase molecules ([60], M.J. Gasson, personal communication). Protcolytically active degradation products of the proteinase have been isolated which were approximately 60-80 kDa ([17,61]; J. Erkelens, personal communication). The 80-kDa proteinase initially isolated from the lactis strain 763 [16] must be considered to be such a degradation product as in a later study a 145-kDa proteinase was purified from the same strain. Interestingly, the 80-kDa proteinase fragment was not only active, but also showed the same specificity towards

casein as the larger enzyme ([62-64]; see below). Recently, it was shown that an 87-kDa degradation product of the Wg2 proteinase still had the same N-terminal amino acid (Asp.188) as the largest proteinase band of 165 kDa present in this preparation, indicating that extensive C-terminal degradation had taken place [54]. In the model presented in Fig. 8, Ca 2+ is thought to play an important role in proteinase degradation as it can reduce proteolytic activity at concentrations higher than 10 mM or enhance activity at lower concentrations. The preferred method of proteinase isolation, the "release" of the enzyme from the cells in a Ca2+-free buffer, is considered to result in autocatalytic degradation. This can either be envisaged by an increase in proteinase activity or by assuming that at low Ca 2+ concentrations a putative cleavage site in the C-terminus of the proteinase becomes accessible to proteolytic cleavage. In both cases the result is the fiberation of the mature cell envelope-bound proteinase as a large C-terminally truncated prorein of 140-165 kDa. This large proteinase fragment can then digest itself to proteolytically active products of lower molecular weight. Several putative "selfdigestion sites" with similarities to d~gestion sites of lactococcal proteinases in/3-casein have been identified in the C-terminus of the Wg2 proteinase [60,64]. Whether one of these sites is actually used for the release of the proteinase from the cell wall remains to be estabfished. The postulated selfdigestion step predicts that proteinase activity is necessary to release the enzyme from the cell envelope. It has already been shown that the inactive proteinase, produced in the absence of PrtM, can not be releas~ from the cells ([58]; A J . Haandrikman, personal communication). One possibility could be that PrtM performs the Cterminal cleavage step. It appears, however, that the active center of the proteinase itself is involved in proteinase release. The active center mutant of the proteinase (Asp.30 to Asn.30) was normally produced and could be detected on the outside of the cells with monoclonal antibodies. The inactive proteinase, howe~'er, cou'~ not be released from the cells by incubation in Ca2+-free buffer (AJ. Haandrikman, personal communication). Evi¢tenee in favour of the selfdigestion model was

obtained in proteinase inhibition studies [54]. Cells producing proteinase were incubated in the presence of the serine proteinase inhibitor PMSF prior to the incubation step in Ca2+-free buffer. By using monoclonal antibodies it was shown that the proteinase antigen could not be released from these cells. The inhibited proteinase could only be released by the addition of other proteinases. These observations make a model in which the proteinase is thought to be C-terminally processed, after which the enzyme stays attached to the membrane-bound C-terminus via Ca~+-bridges, less likely [58,61]. The proteinase degradation model was also used to explain the occurrence of four immunologically distinct proteins, A, A', B and C, in the proteolytic system of several L. lactis ssp. cremoris strains ([15,22,24,60]; see section 3.1). Only one proteinase is isolated from all strains tested. The released proteinase is found in two immunologically distinct and proteolytically active conformations A and B. Autoprotcolytic cleavage is assumed to lead to an irreversible conformational change resulting in the exposure of the antigenic sites of conformation B and, at the same time, in the masking or loss of the antigenic sites for conformation A. With the aid of monoclonal antibodies it has been shown that component A can be converted to component B, but not vice versa [65]. B can be further degraded to give smaller products, one of which may be protein C found in some Cremoris strains [22]. 3.2.8. Engineering of the proteinase specificity As discussed in section 3.1., two main proteinase substrate specificity classes exist: PI enzymes mainly hydrnlyse /]-casein whereas PIII proteinases degrade ~8-casein with" a different specificity and, moreover, ,,-casein. The genes of a representative of each class have been cloned and sequenced: that of the cremoris strain Wg2 (PI; [44,51]) and that of cremoris strain SKII (PIll; [45,52]). Both proteinases had the original specificity in the heterologous host L. lactis ssp. lactis. Moreover, they retained their specificity despite the fact that their genes had been cloned only partially, resulting in C-terminally truncated enzymes. Three L. lactis ssp. cremoris strains seem

to have a mixed Pl- and PIII-type specificity: TR, FD27 and E8 [20]. To examine whether this was due to the presence of two proteinases with different specificities in one cell, or to a proteinase with a mixed specificity, the proteinase gene of strain E8 was cloned (A.J. Haandrikman et al., unpubfished). L. lactis ssp. lactis MG1363 carrying the E8 gene produced a proteinase with the original E8 specificity, indicating that the mixed specificity of strain E8 is caused by a single proteinase. The knowledge of the sequences of the two protcinase genes of the different specificity classes was the basis for a joint Dutch proiect in which hybrid P I / P I I I proteinase genes were constructed to investigate which of the 44 amino acid substituo tions contributed to the differences in casein cleavage specificity (P. Vos et al., unpubfished). DNA fragments were reciprocally exchanged between the two proteinase genes. The proteinases produced in L. lactis host strains carrying the various hybrid genes thus constructed were isolated and their caseinolytic properties were examined. In this way, two regions in the lactococcal ~roteinase were identified which are involved in ,~teavage specificity. One region showed homology to the substrate binding region of subtilisin and comprises seven amino acid differences. The other region is located in the C-terminal part of the lactococcal proteinase which is not present in subtilisin. Two amino acids in this part of the p r o teinase, residues 747 and 748 of the mature enzyme, appeared to be important for cleavage specificity. Interestingly, some of the hybrids had a specificity of casein degradation which was different from either of the wild-type enzymes. With simple genetic techniques it is therefore possible to create proteina~s with new caseinolytic specificities. Seeh "new" proteinases may also be generated by single amino acid substitutions using SDM techniques, when the two regions essential for cleavage specificity have been further delineated. 3.2.9. Proteinases in lactic acid bacteria other than lactococci Only few data are available on the presence of proteinases in species of lactic acid bacteria other .hall lactococci (lactobacilli, leuconostoes, pediococci) and information on the genetics of these

enzymes is lacking completely. Proteinases have been detected in Lactobacillus helveticus, Lb. lactis, Lb. bulgaricus, Lb. casei, Lb. acidophilus and Lb. plantarum [66-71]. The enzymes from Lb. acidophilus and Lb. helveticus have been isolated and characterized [70,71]. Both proteinases could be liberated from whole cells by repeated washing in a Ca2+-free buffer. In the case of the Lb. helveticus enzyme the level of LDH activity in the crude extract was 35 indicating that fiberatioa of intracellular enzymes was limited. Using polyacrylantide gel electrophoresis, the molecular size of the protein~ ~ of Lb. acidophilus was estimated to be approximately 145 kDa and is therefore in the range of that of the lactococcal proteinases. Both lactobacillus enzymes were severely inhibited by DFP and PMSF and not by ethylenediaminetetraacetic acid (EDTA), indicating that they are serine proteinases. The Lb. acidophilus proteinase resembles the lactococcal enzymes in having a rather acid pH-optimum (PHop, around 5.7) while the optimum pH of the helveticus enzyme was 7.5-8.0. The digestion sites of the proteinase from Lb. helveticus on a s l - and ~ have been determined and show that the enzyme has a broad cleavage specificity. Leuconostoc species show a limited ability to grow in milk, which, in part, is caused by the lack of proteolytic activity in these organisms. Via conjugation, lactococcal proteinas¢ plasmids have been introduced in Leuconostoc and lactose/proteinase deficient wild strains of L. lactis ([72]; C. Daiy, G.F. Fitzgerald and M J . Gasson, personal communication). The resulting transconjugants were weakly proteolytically active. Transfer of the proteinase plasmid from the transconjugants back into the original I, lactis strain resulted in full proteolytic activity in this recipient. Apparently, the weak proteolytic activity was not caused by alterations in the proteinase gene but was the result of inferior gene expression or poor utilization of the products resulting from initial p r o t e ~ lysis. 4. PEPTIDASES The second step in casei~l utilization involves the degradation of the relatively large peptide

28

"

fragments p r o d u c e d b y the proteinases to smaller p r o d u c t s b y peptidases. O w i n g to their importance, research o n this set o f enzymes has intensified since the early 70s, which is reflected in the publication o f several reviews o n the subject in recent years [8-10,65,/3,74]. T h e study o f peptidases has involved the isolation a n d biochemical characterization o f the (partially) purified e n z y m e s a n d b y n o w a n u m b e r o f different p e p t i d a s e activities has b e e n described. A m i n o p e p t i d a s e s , di- a n d tripeptidases, a n aryl-peptidyl amidase, a m i n o p e p t i d a s e P, proline iminopeptidase, prolinase a n d prolidase, X-prolyl-dipeptidyl a m i n o p e p t i d a s e s , endopeptidases, a n d c a r b o x y p e p t i d a s e s have all

b e e n f o u n d in various species o f lactic acid bacteria. A s u m m a r y o f the available d a t a is p r e sented below. A s the first genetic d a t a o n peptidases are emerging, the i m p o r t a n c e o f this develo p m e n t in relation to the b i o c h e m i c a l analyses will b e discussed. 4.1. P e p t i d a s e isolation a n d c h a r a c t e r i z a t i o n

A n u m b e r o f factors c o m p l i c a t e the analysis o f p e p t i d a s e s in lactic acid bacteria. T h e s e h a v e b e e n recognized a n d thoroughly discussed in recent literature [8-10]. T h e r e s e e m to b e a large n u m b e r o f different p e p t i d a s e s p r e s e n t in a n y o n e strain,

Table 3 Peptidases purified from lactic acid bacteria Peptidase

Strain

Mw ( x 103) mono/multimer

Type a

Reference

General AP General AP General AP General AP General AP General AP General AP

Lb. acidophilus L, cremoris AC1 Lb. lactis 1183 L. cremoris Wg2 L. lactis 267 Lb. bulgaricus L. cremoris AM2

38+/156 * 36 + 78+/81 * 95 + 85 * 95 * 50+/300 *

metallo metallo metallo metallo metallo metallo -

[81] [13] [82] [85] [83] [114] [84]

Ap A

L. cremoris HP

43 +/130 *

metailo

[86]

X-PDAP X-PDAP X-PDAP X-PDAP X-PDAP X-PDAP

Lb. lactis S. thermophilus L. cremorls 138/2/47 Lb. bulgaricus 397 L. lactis 763 Lb. helvetlcus 32

90+/165 * 80+/165 * 90+/160-180 * 82 * 85+/190 * 72 b

serine serine serine serine serine serine

[90] [90] [91] [93] [92] [94]

Prolidase

L, cremoris H61

43 *

metailo

[88]

Proline imino peptidase Dipeptidase Dipeptidase

/, cremoris HP L cremoris H61 I.. cremoris W82

45+/130 * 100 * 49 +

metallo metallo metallo

[95] [96] [98]

Tripeptidase Tripeptidase

L, lactis 267 L ¢remoris Wg2

75 * 52+/103 *

metallo metallo

[83] [104]

Endopeptidese Endopepfidese

!,, cremoris H61 L, cremoris H61

98 ÷ 40+/80 *

metallo metaUo

[108] [109]

Aryl peptidyl fmidase

Lb. easei 151

ND

-

[105]

• metalIo, serine: enzymes are inhibited by EDTA or PMSF, respectively. b determined on both native- and SDS-polyacrylamide gels. + de~'~mined by SDS-polyacrylamide gel electrophoresis. * determined by gel fdtration. AP, aminopeptidese; X-PDAP, X-prolyi dipeptidyl aminopeptidase. ND, not determined.

which, of course, might reflect the complexity of the peptidase system itself. However, part of this complexity is caused by the choice of substrates used to characterize the enzymes while the differences in the substrates used by the various authors make it difficult to compare the peptidases under study. Furthermore, the use of ceil-free extracts, even when they are fractionated, may result in the failure to resolve distinct peptidase activities. Fractionated cell extracts have been used in many studies to estimate the number of different peptidase activities present in one or only a few strains [75-80]. In a recent study [78], I I s~rains of lactococci were classified into three groups on the basis of peptidase a3tivity profiles towards a wide range of substrates (including amino acyl paranitroanilides (-pNAs), di-, tri-, and tetrapeptides and carbobenzoxypeptides). After DEAE cellulose fractionafion of cell-free extracts, three to four different peptidases were detected and characterized on the basis of substrate specificity, pH dependency and effect of inhibitors and metal ions. This and other work strengthens the view [10] that the peptidase complex of lactic acid bacteria consists of only a relatively small number of exopeptidases. Recent literature shows that the emphasis in this field of research is changing towards the isolation and purification of single peptidases to allow a detailed analysis of the enzymes. A summary of the peptidases purified so far is given in Table 3. 4.1.1. General aminopeptidases An aminopeptidase with a broad substrate specificity has been detected in L. lactis ssp. cremoris nTR [78]. Apart from amino acyl-pNAs, the enzyme was able to hydrolyse certain di-, triand tetrapeptides. In this respect, the enzyme is similar to the purified aminopeptidases from Lb. acidophilus R26 [81], Lb. lactis [82], the AP I enzyme from L. lactis ssp. lactis CNRZ 267 [83] and the aminopeptidases from the L. lactis ssp. cremoris strains AM2 [84] and Wg2 [85] (see Table 3). The aminopeptidase from strain Wg2 does. not hydrolyse dipeptides containing N-terminal alanine or phenylalanine, while the enzyme from Lb. lactis is able to degrade such dipeptides, pro-

vided that the second amino acid is basic or aromatic. Ala-Ala (Ala2) is not hydrolysed by the Wg2 aminopepti~se whereas Ala 3 to Ala 6 are substrates, suggesting that the enzyme preferentially hydrolyses peptides with more than two amino acids. The aminopeptidase from cremoris AC1 possibly falls in the same class of broad substrate specificity general aminopeptidases as it reacts not only with amino acyl-pNAs and dipeptides but also with the larger peptides pro. duced from ~-casein by the action of lactococcal proteinase [13,70]. All the aminopeptidases mentioned above, with the exception of that from strain AM2, are metalloenzymes as they are inhibited by metal-complexing agents such as EDTA. The molecular size of the AM2 aminopeptidase is different from that of the Wg2 enzyme (50 kDa versus 95 kDa) and both enzymes have different N-terminal amino acid sequences [84,85]. All enzymes, including the one from strain AM2, are inhibited by sulfhydryl-blocking reagents and do not show carboxypeptidase or endopeptidase activity. It appears that aminopeptidases with a broad substrate specificity are common in lactic acid bacteria, and it is of interest to see how many different enzymes constitute this class of general aminopeptidases. In L. lactis ssp. lactis 527 an aminopeptidase activity of much more confined specificity has been detected. It only hydrolyses amino acyl-pNAs and Leu 3 but none of the other di-, tri- and tetrapeptides tested [78]. This enzyme, therefore, does not belong to the broad-substratespecificity-class. A completely different aminopeptidase was purified from L. lactis ssp. cremoris HP. This enzyme appears to be highly specific for glutamyl- and aspartyl peptides and thus resembles mammalian aminopeptidase A. It is a trimeric metalloenzyme with identical subunits and has a native molecular size of 130 kDa [86]. 4.1.2. Proline-specific peptidases Proline-containing dipeptides and ofigopepfides containing an N-terminal proline residue are not hydrolysed by the general amlnopeptidases nor by the broad specificity di- and tripeptidases (see below). In order to be able to degrade the proline-rich oligopeptides liberated from casein by

3o the action of the proteinase, lactic acid bacteria depend on profine-specific peptidases. Aminopeptidase P, proline iminopeptidase, iminodipeptidase (prolinase), imidodipeptidase (prc~ lidase) and X-prolyl dipeptidyl aminopeptidase (X-PDAP) have all been detected in the various species of lactic acid bacteria [70,75,78,87-95]. Several of these enzymes have been purified to homogeneity and characterized in detail ([88,9092,94,95]; see Table 3). All X-PDAPs specifically degrade peptides of the structure X-Pro-Y... (X and Y can be any amino acid) but the rate of hydrolysis depends on the type of amino acids surrounding proline. Some differences exist between the X-PDAPs with respect to the substrate which is hydrolysed most efficiently. Slight activity wA'th AIa-Ala-pNA is detected but no aminopeptidase activity is observed. The lactococcal enzymes have been shown to split ~8-casomorphin (Tyr-Pro-Phe-Pro-Gly-Pro-!!e) starting from the amino terminus into the successive X-Pro dipeptides and lie [91,92]. The native enzymes are dimers of identical subunits and have molecular sizes of 160-180 kDa. The published monomeric sizes range from 80-90 kDa [90-93]. The enzymes are inhibited by the serine proteinase inhibitors PMSF and DFP and not by EDTA. The X-PD from Lb. helveticus is somewhat different in having a native monomeric size of 72 kDa [94]. The prolidase of L. lactis ssp.cremoris H61 is perfectly suited to degrade the products of X-PDP activity as it is highly specific for X-Pro dipeptides. The prolidase is completely inhibited by the metal-chelators EDTA and 1,10-phenantroline, and has a Mw of 43 000 ([88]; see Table 3). Recently, a proline iminopeptidase f r c ~ L. lactis ssp. cremoris HP has been purified to homc~ geniety. The metalloenzyme is a 130-kDa multimer of 45 kDa subunits with a preference for tripeptides containing proline as the amino terminal residue ([95]; see Table 3). 4.L3. Dipeptidases Dipeptidases of broad substrate specificity have been purified and characterized from two strains of L, lactis ssp. cremoris ([96-98]; see Table 3). Both enzymes are metallopeptidases as they are severely inhibited by EDTA and 1,10-phenantro-

line. The dipeptidase from strain H61 has a Mw which is markedly higher than the enzyme from strain Wg2 (100000 versus 49000, respectively). Both enzymes hydrolyse various dipeptides but not tri- and tetrapeptides. Both enzymes fail to hydrolysc dipeptides having glycine or proline as the N-terminal amino acid. Differences in Mw, turnover numbers, metal dependencies and substrate specificities indicate that both enzymes are distinctly different. Dipeptidase activities showing a broad substrate specificity have been detected in several other lactic acid bacteria [78,80,99-103]. The genera! dipeptidase peak detected in Lactococcus [78] is contaminated with a prolinase as, most probably, are the dipeptidase peaks observed by Kolstad and Law [80]. Even when the substrate specificity profiles are reinterpreted on the basis of this assumption, the remaining dipeptidase activity in the peaks is of the broad-substratespecificity-type, indicating that these types of dipeptidases may be widely distributed among lactic acid bacteria. 4.1.4. Tripeptidases Various authors have described the existence of tripeptidase activity in lactic acid bacteria [75,77,78,83,95,103,104]. To date, there are three reports on the purification of a trlpeptidase. One concerns the proline-specific tripeptidase described above [95]. The specificity of this enzyme clearly differs from the partially purified tripeptidase (AP II) from L. lactis ssp. lactis CNRZ 267 and a tripeptidase which has been purified to homogeniety from the cremoris strain Wg2 ([83,104]; see Table 3). All three enzymes are inhibited by EDTA. EDTA-inactivated AP II can be reactivated by Co2+-ions while the Wg2 enzyme can be reactivated by Zn 2+, Mn 2+ and to a lesser extent by Co~ +. The Wg2 tripeptidase is inhibited by reducing agents such as dithiothreitol and /~. mercaptoethanol and has a broad specificity for tripeptides only. It does not attack tripeptides containing proline in the second position. Like the Wg2 enzyme, AP II is active on a number of different tripeptides. Both tripeptidases differ in their molecular size which is 103-105 kDa for the Wg2 enzyme and 75 kDa for AP II. Both similarities (EDTA inactivation and reactivation) and dif-

ferences ( M w and sensitivity to /]-mercaptoethanol) exist with the intracellular tripeptidase activities analysed by Law [77] in cell-free extracts of different lactococcal strains. These results suggest that a broad substrate specificity tripeptidase may be a common enzyme in the peptidase complex of lactococci. As yet, no data are available on the possible existence of such broad substrate specificity tripeptidases in other species of lactic acid bacteria. 4.1.5. Carboxypeptidases The peptidases described above hydrolyse their substrates starting from the N-terminus. The presence in lactic acid bacteria of carboxypeptidases, attacking substrates from the C-terminal end, has been examined in a number of studies. The conclusion from this work seems to be that this activity is not present in lactococci, S. thermophilus, Lb. helveticus and Lb. bulgaricus [78,87,105,106]. Apparently, casein derived peptides are degraded by these organisms from the N-terminus only. Two studies, however, show that Lb. casei and Lb. plantarum do contain an activity on N-terminally blocked dipeptides: a carboxypeptidase which was highly specific for carbobenzoxyslycyl arginine was found in Lb. casei 151 [101,107]. This is in contrast with a later study in which it was shown that the same strain contained a carboxypeptidase activity w;th a broad substrate specificity. A similar actiwty was found in Lb. plantarmn [103]. Lb. lactis appears to produce a carboxypeptidase hydrolysing the substrates N-benzoylGly-Arg and N-benzoyl-Gly-Phe [82]. 4.1.6. Endopeptidases The peptidases described so far are exopeptidases, enzymes which remove one or, in the case of the X-prolyl dipeptidyl aminopeptidase, two amino acids from a peptide. Recently, two studies describe the purification and characterization of two endopepfidases, LEP I and LEP II ([108,109]; see Table 3). Both enzymes were purified from L. iactis ssp. cremoris H61 and are metallopeptidases with unique substrate specificities. They were isolated using asl-CN(1-23) as the substrate. This peptide, fiberated from asl-casein by chymosin, and asl-CN(91-100) are hydrolysed by both

peptidases (but at different peptide bonds), t - or ~-casein arc not degraded, no more than a s l casein, asl-CN(1-54) and asl-CN(61-123) although the latter three substrates do contain the susceptible peptide bonds. Apparently, the spatial conformation of the substrates plays a role in enzyme specificity [108,109]. LEP II has a broader substrate specificity than LEP I and hydrolyses v ~ o u s small polypeptides (up to a size of 3.5 kDa) at peptide bonds involving the amino groups of hydrophobic amino acids. In the other reports on endopeptidolytic activity, either N-glntaryl- or succinyl-phenylalanine-pNA or N-acetyl-alanine~ pNA were used as the substrate [78,79,101,105107,110]. As shown by El Soda and Desmazeaud [105], interpretation of results using this kind of substrates should be done with great care as hydrolysis does not necessarily mean that the enzyme involved is an endopeptidase. The activity "in Lb. casei on succinyl-Phe-pNA appeared to result from a ribosomal aryl-peptidyl amidase (E.C.3.5.1). Recently, an intracellular endopeptidase was partially purified from an L lactis ssp. lactis 763 mutant which lacked the cell envelopebound proteinase (caseinase) [111]. The enzyme had a Mw of 93000 by gel filtration, was not inhibited by scrine or thiol enzyme inhibitors but was affected by EDTA. The enzyme only slowly degraded t-casein but readily cleaved oxidized insulin B-chain with a specificity similar to other intracellular metalloproteinases, isolated from L. lactis and S. thermophilus [109,112,113]. 4.2. Peptidase localization Despite many attempts none of the above-meno tioned peptidases has been unequivocally assigned a cellular location. Determination of the true cellular location of peptidascs using fractionated cell extracts is hampered by cross contamination of the fractions. Although both intracellular and extracellular peptidase activities have been described, conclusive evidence on their in vivo location is lacking. As many of the primary breakdown products of casein are too large to enter the cell (the size exclusion being approximately six amino acid residues [2,6,7], it follows that the cells need (an)

32 extracellular peptidase(s) to reduce the peptides liberated by the protcinases to a manageable size. To assign an extraceUular status to peptidases in the presence of highly active intracellular peptidases requires careful estimation of contamination of the "extracellnlar" fraction with cytoplasmic enzymes such as aldolase or lactate dehydrogenase. When applying the rigorous criteria needed evidence for the existence of extracellnlar enzymes in lactic acid bacteria is scanty. Several of the general aminopeptidases are thought to be extracellular [13,82,85,114]. Only for the crernoris Wg2 and the Lb. bulgaricus enzymes intraeellnlar marker enzymes have been used to assess cell lysis [85,114]. The aminopeptidase from cremoris AM2 appears to be intracellular [84] while for the aminopeptidase AP I from L. lactis CNRZ 267 an intracellular location was also claimed [83]. With respect to dipeptidases both intracellular [77,80,98] and extracellular activities have been described. Lact~occal protoplast supernatants devoid of intracellular marker enzymes contained an apparently cell wall-located dipeptidase distinct from the intracellular dipeptidase liberated upon osmotic lysis of the protoplasts [77]. The observation that most of the LEP I endopeptidase activity was present in the cell wall fraction was not substantiated [108]. Except for one report [91], all publications concerning X-prolyl dipeptidyl aminopeptidase agree on an intracellular location for this enzyme ([90,92,93,94]; see below). The location of the prolidase from L. lactis ssp. cremoris H61 has not been determined [88]. The fact that proline is an essential amino acid for cremoris [1] and the observation that proline is not taken up by L. lactis as a free amino acid but, instead, is very efficiently transported as a dipeptide ([115]; see section 5.2.) justify the speculation that this enzyme (and, in fact, all peptidases liberating free proline from a peptide) is intracellnlar in L lactis ssp. cremoris. 4.3. Peptidase mutants The elucidation of the complexity of the peptidase system, of the number of different peptidases involved and the way peptidase synthesis is regulated, awaits the isolation of peptidase

mutants. The first examples of such mutants have now become available. Atlan et al. [114] isolated peptidase mutants from Lb. bulgaricus by classical mutagenesis techniques: cells were treated with nitrosoguanidine and mutants were screened for their inability to hydrolyse chromogenic substrates by a direct enzymatic assay on bacterial colonies. In this way, mutants were obtained which lack a lysyl-aminopeptidase (AP If). Arginyl- and leucylaminopeptidase activities are reduced in t!~e mutants and the remaining activity was attributed to the presence of three other aminopeptidases. In a subsequent study [93], mutants for the X-prolyldipeptidyl aminopeptidase of the same strain of Lb. bulgaricus were obtained. Both types of mutants are proteinase positive and have the wild-type amino acid requirements and sugar fermentation patterns. Both peptidases were shown to be non-essential for growth in milk. The mutants have the wild-type doubling time in rich (MRS) medium but are characterized by a shorter exponential growth phase leading to a significantly lower biomass. Lack of X-prolyl-dipeptidyl aminopeptidase results in an enhancement of prote~ lyric activity while mutants devoid of AP II dis.. play an increase in X-PDAP activity when cells are grown in milk. These data are indicative of the presence of a common regulatory mechanism controlling the biosynthesis of the three enzymes [93,114]. Mutants in X-PDAP activity have already proved useful for the cloning of X-PDAP structural genes. 4.4. Cloning and sequence of X-prolyl dipeptidyi aminopeptidase To date, the only peptidase genes cloned and characterized at the nucleotide level are those for X-prolyl dipeptidyl aminopeptidase from two strains of Lactococcus. The X-PDAP gene of L. lactis ssp. cremoris P 8 / 2 / 4 7 was isolated in our laboratory by screening an E. coli plasmid bank of chromosomal DNA of P 8 / 2 / 4 7 for X-PDAP activity using a chromogenic substrate (B. Mayo et al., unpublished). Expression of the gene in this heterologuus host and in B. subtilis proved that the structural gene for X-PDAP had been cloned. The chromosomal piece of D N A was subeloned

33 on a pWV01-derived cloning vector [39] and with the resulting recombinant plasmid the X-PDAP deficiency of an L. lactis mutant obtained after methylmethane sulphonate treatment could be restored. In an independent study, the X-PDAP gene of L. lactis ssp. lactis 763 was cloned by constructing a chromosomal DNA bank from strain 763 in an isogenic X-PDAP-negative mutant (obtained by nitrosognanidine mutagenesis). Subsequently, this bank was screened using an enzymatic plate assay and a chromogenic substrate (M. Nardi et al., personal communication). In this way, a plasmid was obtained which not only restored the X-PDAP deficiency in the lactococcal host, but also conferred the X-PDAP phenotype to B. subtilis, indicating that the entire structural gene had been cloned. In both cases, the plasmids carrying the X-PDAP structural genes had copy numbers of approximately 5 to !0 per chromosome equivalent. However, the increase of the number of copies of the X-PDAP gene in L. lactis did not result in a significant increase in X-PDAP activity, suggesting regulation of (expression of) the X-PDAP gene. Both genes have been sequenced completely and contain 763 codons. An amino acid comparison indicated an almost complete homology between the two peptidases. The size of the protein deduced from the inferred amino acid sequence (88 kDa) corresponds well with that of the purified enzyme (approximately 90 kDa; [91,92]). The N-terminus of the predicted protein does not contain a signal peptide-like sequence. Rather, the N-terminal amino acid sequence of purified X-PDAP of cremoris P 8 / 2 / 4 7 (W. Bockelmann, personal communication) was identical to the N-terminal stretch of amino acids deduced from its gene. These results indicate that X-PDAP is not processed at the N-terminus and that the cellular location of the enzyme awaits careful reexamination of the localization studies using specific antibodies.

5. AMINO ACID AND PEPTIDE UTILIZATION During the first stages of multiplication in milk and, subsequently through the combined action of

proteinases and peptidases on milk proteins, lactic acid bacteria are confronted with small peptides and free amino acids which serve as substrates for growth. Many amino acids are either essential or stimulatory for growth of these multiple auxotrophic organisms. Most lactic acid bacteria can utilize ~ i n o acids and peptides to satisfy their growth requirements [6-8,116]. Lactococ~ possess separate transport systems for amino ~ d ~ , dio (and tri-), and oligopeptides [6,7,115-117]. Until recently, only limited information was available on the mechanisms by which the amino acid and peptide transport systems operate. The recent development of a procedure for the incorporation of functional proton-motive-force (pmf)-generating systems into bacterial membranes has led to a rapid increase in the knowledge of the bioenergetics of amino acid and peptide transport by lactococci. The intention of this review allows only a brief summary of these data. Excellent reviews on the bioenergetics of amino acid and peptide trae~ port have appeared recency [118-120]. 5.1. Amino acid transport

Three types of amino acid transport systems have been identified in lactococci: (A) pmf-driven transport, in which the uptake is primarily coupled to the proton motive force. The uptake of L-methiouine and the branched amino acids L-lencine, L-isolencine and L-valine, of the neutral amino acids L-serine, L-threonine, L-alanine and glycine, and of the basic amino acid L-lysine is pmfodriven [121-126]. The uptake of L-histidine, L-cysteine, L-tyrosine and L-phenylalanine might also be coupled to the pmf [120,125,127]. L-proline is not taken up in a free form ([115]; see below); (B) phosphate-bond-linked transport, which is driveit by the high-energy phosphate bond of ATP or an ATP-derived metabolite. The uptake of glutamate, glatamine, asparaglne and, possibly, aspartate have been shown to be phosphatebond-driven in lactococci [120,128,129]; (C) exchange transport, which is exemplified by the arglnine/ornithine antiporter present in / . lactis ssp. lactis. The driving force for argivine uptake and oruithine excretion is supplied by the

concentration gradients of the two amino acids. The arginine taken up is metabolized via the arginine deiminase pathway [130-131].

5.2. Peptide transport Different mechanisms of utilization of peptides with up to six amino acid residues can be envisaged [132,133]. The intact peptide c a n b e transported into the cell where it is subsequently hydrolysed, the peptide uptake can be directly coupled to peptide hydrolysis by a transmembrane peptidase which releases the liberated amino acids inside the cell, or the peptide is first hydrolysed on the outside of the cell, after which the free amino acids are taken up. The latter mechanism has been ruled out for most of the various peptides studied since high extracellular concentrations of the amino acids constituting the peptides did not inhibit the uptake of the peptides [6,7,134]. In a study to discriminate between the first two mechanisms, evidence was presented that peptide transport is not directly coupled to peptide hydrolysis [117]. Dipeptide transport was studied in L lactis membrane vesicles which were fused with liposomes containing beef heart cytochrome c oxidase as a pmf-generating system. In this way, peptide transport could be studied in the absence of peptidase activity. Accumulation of dipeptides was only observed upon addition of an electron donor to the system and was thus shown to be driven by the pmf. Therefore, dipeptide utilization takes place by a two-step process: first, the peptide is translocated across the cytoplasmic membrane via a specific peptide transport system, while in the second step the peptide is hydrolysed by an intraceUular peptidase. The dipeptide transport system has a broad substrate specificity [117]. Slow uptake of dipepfides by de-energized cells via the secondary peptide transport system is driven by the inwardly directed chemical gradient of the peptide which is maintaL~ed l~y intracellular peptidase activity. The latter mechanism of dipeptide utilization is thought to play an important role during cheese ripening [117]. In a subsequent study, Smid et al. isolated spontaneous mutants of L / a c t / s ML3 deficient in dipeptide transport, by selecting for resistance to the toxic chlorated di-

peptide L-alanyl-/~-chloro-L-alanine. With this mutant they showed that the dipeptide transport system can also transport tripeptides [115]. The mutant has full protcinase- and peptidase activity and the uptake of amino acids and tetra-, penta-, and hexapeptidcs is unaffected. Nevertheless, the mutant is unable to grow on media containing casein as the sole source of nitrogen, indicating that during casein degradation one or more essential or stimulatory amino acids mainly become available as di- or tripeptides. Because L lactis can not take up L-proline, this amino acid, most likely, enters the cell in a peptide-bound configuration. Apart from a functional set of proteolytic enzymes, dipeptide transport is therefore also an essential component in the process of casein utilization by lactococci. Interestingly, the uptake rate of various amino acids is siguificantly increased in the dipeptide transport mutant, suggesting that dipeptide transport plays a role in the regulation of some of the amino acid transport systems. The possible involvement of peptides in the expression of amino acid transport systems was noted earfier [135]. The 10-fold reduced expression of the branched-chain amino acid carrier in cells grown in complex broth, as compared to cells cultivated in a chemically defined medium supplemented with amino acids, was postulated to be attributable to a (partially) repressed synthesis of the carrier by the presence of peptides.

6. CONCLUSIONS AND PECTS

FUTURE

PROS-

The knowledge of the genetic basis for the proteotytic activity of lactococci has increased considerably since clear proof was furnished in the 70s that plasmid DNA governs this trait in many strains. Now, some 10 years after the first report on proteinase plasmids, proteinase genes of several strains have been cloned and three of these have been sequenced. The sequences have convincingly demonstrated that the lactocoecal proteinases belong to the serine proteinases. By combining biochemical, immunological, and genetic data a substantial simplification of the proteinase complex

35 was obtained. Only one cell-envelope-bound proteinase is produced by lactococci from a "basic" proteinase gene. Because the enzyme is extremely unstable, smaller active degradation products may be formed and it is this instability which has caused the confusion about the apparent complexity of the proteinase system. The proteinase synthesized may be slightly different in the various lactococcal strains as a result of a small number of mutations in the proteinase gene. These mutations may lead to amino acid substitutions and to the production of a proteinase with an altered cascinolytic specificity. With molecular genetic techniques two regions in the proteinase have been identified which are involved in enzyme specificity. This knowledge was used to manipulate the specificity of the proteinase and has led to the production of proteinases with new caseinolytic specificities. Ultimately, site directed mutagenesis strategies might result in the construction of proteinases with improved properties such as a higher stability or an altered casein breakdown specificity such that the liberation of bitter peptides from casein is reduced or even abolished. In the future, by usin8 the homolosy with subtilisin as a 8uide-line, knowledse-based alterations in the proteinase will lead to the unravellin$ of the function of the various regionsin the enzyme. One such alteration, the change of the active site aspartate (residue 30 of the mature enzyme) into asparasine has led to the production of an inactive form of the proteinase. This mutation has already helped in ~nining more insight in the process of proteinase maturation. Maturation of the inactive precursor-form of the lactococcal proteinase into the active mature enzyme involves a digestion step for which a second protein is indispensible. This maturation process is unique for prokaryotes and the next goal will be the elucidation of the precise mechanism. As DNA probes of the proteinase system are available now, an important field of study in the future will be the regulation of expression of the genes for proteinase and the proteinase maturation protein, an at present underexposed aspect of proteinase synthesis. Furthermore, the proteinase gene probes are used at the moment to investigate

wheth~ similar genes are present in other lactic acid bacteria. The availability of synthetic DNA primers for the proteinase gene opens the possibility to use the Polymerase Chain Reaction to speed up the cloning of such genes. An explanation for the similarity, in the organization and nucleotide sequences of the progeinase loci in lactococci is provided by the occurrence of insertion elements on proteinase plasmids [136141]. Immediately downstgeam of prtM an insertion sequence is present in the proteinase plasmids of strains Wg2 and S K l l (see Fig. 4). The IS elements have been sequenced [141] and both are similar to the two IS elements found on the lactose/proteinase plasmid pSK08 (which is probably identical to pLPT12 shown in Fig. 4). It is conceivable that through cointegratc formation with conjugative plasmids the genes have been rapidly disseminated within strains of lactococci. Compared to the genetic knowledge of the proteinase system, that of the peptidases of lactic acid bacteria is lagging far behind. This is partly due to the lack of mutants in peptidase activity. The genes for the various peptidases are, most probably, located on the chromosome, which makes their analysis more complicated. Using classical mutagenesis techniques, the first peptidase mutants have been isolated and characterized. With these mutants the first data on regulation of peptidase synthesis have been obtained. Moreover, they enabled the detection of peptidase activities which were partly masked in the wild-type strain. The availability of X-prolyl-dipeptidyl aminopeptidase mutants facilitated the cloning and analysis of the structural gene of this peptldase. These studies are the first to illustrate the way to tackle the next important challenge: the elucidation of the complexity of the peptidase system of lactic acid bacteria. In particular, the important issue of peptidase localization needs attention. For this, the isolation to homoganiety of the various enzymes is a prerequisite and is presently undertaken by several groups. Undoubtedly, this will lead in the (near) future to the availability of specific antibodies so that the question of the cellular distribution can be properly addressed with immunological techniques. In this respect, the availability of antibodies against a strictly

membrane-bound protein (the proteinase maturation protein PrtM) may prove of great help as they could be used to determine the degree of contamination of cell extracts with membranes. The know-how on the mechanisms and energetits of amino acid and peptide transport in lactococci is rapidly increasing. This is largely due to the developngnt of a procedure for the functional incorporation of protun-motive-force-generating systems into bacterial membranes. Three distinct mechanisms of amino acid transport are operative in lactococci, while a pmf-dependent dipeptide transport system has recently been identified. The isolation of a mutant in di- and tripeptide transport enabled the detailed analysis of peptide uptake and indicated that it is essential for growth of Lactococcus lactis in milk. Again, the isolation and characterization of mutants and of genes involved in amino acid and peptide uptake will be inevitable for a complete understanding of these uptake systems. The development during the 80s of techniques which enabled the application of recombinant DNA methodology to lactic acid bacteria stood at the basis of the current understanding of the proteinase system of lactococci. Continued fundamental research devoted to the development of genetic engineering techniques to effectively manipulate lactic acid bacteria is a guarantee that new insights will be rapidly gained. Such new developments which have already proved useful are the construction of expression vectors and vectors which can integrate in the chromosome of lactic acid bacteria [42,142-147]. With the first type of vectors it is now possible to express heterologons genes and to increase the expression of homologous genes in lactic acid bacteria. By using an expression vector the neutral proteinase of B. subtilis has been expressed in L, iactis, resulting in an increased proteolytic ability of the new strain (M. van de Gachte, personal communication). The integration vectors have been helpful in two ways. First, they have been used to stably integrate one or multiple copies of the proteinase gene of cremoris strain W82 into the chromosome of L, /act/s resulting in new strains of which the properties are under investigation (K.J. Lecnhouts, personal communication). Second, one of the vectors

was used to make a strain in which the chromosomal X-prolyl dipeptidyl aminopeptidase gene had been inactivated (K.J. Leenhouts, personal communication). The fact that by using the integration vectors we can now make specific mutations in the chromosome will be of major importance for the analysis of genetics and regulation of proteolysis in the lactic acid bacteria. In conclusion, we can be confident that we will see a rapid increase in knowledge of the genetic basis of proteolysis in lactic acid bacteria and in our understanding of the functioning of this trait which is of such eminent importance in food fermentations.

ACKNOWLEDGEMENTS I would like to thank Dr. Gerard Venema for critical reading of the manuscript, Alfred Haandrikman, Tjakko Abee, Harry l.aan and Eddy Smid for helpful discussions throughout this work and Henk Mulder for preparation of the figures. I am grateful to my colleagues for communicating their results prior to publication.

REFERENCES [1] Reitcr, B. and Oram, J.D. (1962)Nutritionalstudies on cheese starters. 1. Vitaminand aminoacid requirements of singlestrainstarters. J. Dairy Res. 29, 63-77. [2] Law, B.A., Sezgin, E. and Sharpe, M.E. (1976) Amino acid nutrition of some commercial cheese starters in relation to their growth in peptone-supplementedwhey media. J. DairyRes. 43, 291-300. [3] Bracquart, P. and 1.orient,D. (1979) Effect des amides amin~ et peptides sur la croissance de Streptococcus thennophilus, 3. Peptides comportant Gin, His et Met. Milchwisscnschaft34, 676-679. [4] Morishita,T., Deguchi,Y., Yajima,M., Sakurai,T. and Yura, T. (1981) Multiple nutritionalrequirements of lactobacilli:geneticlesionsaffectingaminoacid biosynthetic pathways.J. Bacteriol.148,64-71. [5] Mills, O.E. and Thomas,T.D. (1981) Nitrogen sources for growth of lactic streptococci in milk. N.ZJ. Dairy Sci. Teclmol.15, 43-55. [6] Law, B.A. (1978) Peptide utilization by group N streptococci. J. Gen. Microbinl.105,113-118. [7] Rice,G.H., Stewart, F.H.C., Hillier,A.J. and Jago, G.R. (1978) The uptake of amino a.:ids and peptides by Streptococcuslactis.J. DairyRes. 45, 93-107.

[8] Thomas, T.D. and Mills, O.E. (1981) Protanlytic enzymes of starter bacteria. Neth. Milk Dairy J. 35, 255-273. [9] Law, B.A. and Kolstad, J. (1983) Protanlytic systems in lactic acid bacteria. Antonie van Lceuwenhoek 49, 225245. [10] Thomas, T.D. and Pritchard, G.G. (1987) Proteolytic enzymes of dairy starter cultures. FEMS Microbiol. Rev. 46, 245-268. [11] Thomas, T.D, Jarvis, B.D.W. and Skipper, lq.A. (1974) Localization of proteinase(s) near the cell surface of Streptococcus lactis. J. Bacteriol. 118, 329-333. [12] Exterkate, F.A. and de Veer, G.J.C.M. (1985) Partial isolation and degradation of caseins by cell wall proteinase(s) of Streptococcus cremoris HP. Appl. Environ. Microbiol. 49, 328-332. [13] Gels, A., Bockelmann, W. and Teuber, M. (1985) Simultaneous extraction and purification of a cell wall-associated pepfida~ and ~umein specific proteinase from Streptococcus cremocis AC1. Appl. Micsobiol. Biotechnol. 23, 79-84. [14] Geis, A., Kicfer, B. and Textber, M. (1986) Proteolytic activities of lactic acid streptococci isolated from dairy starter cultures. Chem. Mikrobiol. Technol. Lebensm. 10, 93-95. [15] Hugenboltz, J., van Sinderen, D., Kok, J. and Konings, W.N. (1987) The cell-wall-associated proteinases of Streptoco¢c~ cremor/s Wg2. Appl. Environ. Microbiol. 53, 853-859. [16] Monnet, V., Le Bars, D. and Gripon, J.-C. (1987) Purification and characterization of a cell wall proteinaso from Streptococoas lactis NCDO 763. J. Dairy Res. 54, 247255. [17] Exterkate, F.A. and de Veer, GJ.C.M. (1987) Complexity of the cell wall proteinase of Lactococcus/act/s ~p. cremor/s HP and purification of the enzyme. Syst. Appl. Microbiol. 9,183-191. [18] Cliffe, AM. and Law, B.A. (1984) Discontinuous polyacrylamlde gel electrophoresis of call wall proteinase from vmiants of Streptococcus kact/s. J. Appl. Bacteriol. 58, 245-250. [19] Exterkate, F.A. (1976) Comparison of strains of Streptococcus cremom for pmteolytic activities associated with the cell wall. Neth. Milk Dairy J. 30, 95-105. [20] Visser, S., Exterkate, F.A., Slangen, CJ. and de Veer, GJ.C.M. (1986) Comparative action of cell wall proteillases from various strains of Streptococcus cremoris on bovine asl-, p-, and g-casein. Appl. Environ. Microbiol. 52,1162-1166. [21] Monnet, V., Le Bars, D., Neviani, E. and Gripon, J.C. (1987) Partial characterization and comparison of cell wall pmteinases from 5 strains of Streptococcus lactis. Le Lait 67, 51-61. [22] Hugcmholtz,J., Exterkate, F. and Koning& W.N. (1984) The proteolyfic system of Streptococcus crenmris--An immunological analysis. Appl. Environ. Microbiol. 48, 1105-1110. [23] l.un, H., Staid, E.J., de Le/j, L., Schwander, E. and Konings, W.N. (1988) Monoclonal antibodies to the

cell-wall-associated proteinase of Le~ococcus/act/s sap. cremor~ Wg2. Appl. Environ. Micsobiol. 54, 2250-2256. 124] Kok, J. and Venema, O. (19SS) Genetics of prmeina~ of lactic acid bacteria. Biochimie 70, 475-488. [251 Harriman, L.A. and Hammer, B.W. (1931) Vmiation in the coagulation and proteol~s of n ~ k by Strq~ococcus lactis. J. Dairy Sci. 14, 40-49. [26] Oarvie, E.I. and Mabbitt, L.A. (1956) Acid production in milk by starter culturm. J. Daix~ Rm. 23, 305-314. [27] Cirri, J.F-, Sandlnc, W.E. and Elliker, P.R. (1965) Comparison of slow and fast acid-produclng Streptococe~ Ioct~s. J. Dairy Sci. 48,14-18. [28] Westhoff, D.C., Cowman, R.A. and Speck, M.L (1971) Isolation and partial characterization of a particulate proteinase from a slow acid producins mutant of Streptococcus/act/s. J. Dairy ~ 54,1253-1258. [29] Davies, F.L. and ~ MJ. (1981) Reviews of the progress of dairy science: genetics of lactic acid bacteria. J. Dairy Res. 48, 363-376. [30] McKay, L.L. (1983) Functional propmies of p ~ in lactic streptococci. Anionic van Lceuwenhock 49, 259-274. |311 Oasson, MJ. and Davies, FJ... (1984) The geaetica of dairy lactic acid bacteria, in: Advances in the Microbiology and Biochemistry of Cheese and Fermeated Milk (Davies, F.L. and Law, B.A., eds.), Elsevier Applied Science Publishers, London, New York, pp. 99-126. [32] Gasson. MJ. (1983) Plasmid complements of Streptocaccus/act/s NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154,1-9. [33] Ramos, P., Novel, M., Lemosquet, M. and Novel, O. (1983) Fmgmantation du plasmide l a c t o s e - ~ chez des d&riv,~slactose n~gatifs de Streptococc~ lactis et de $. lactis ssp. diacetylactis. Ann. MierobioL (Inst. Pasteur) 1MB, 387-399. [34] Kondo, J.F. and M¢Kay, L.L (1982) Tmnsfmmation of Streptococcus lactis protoplasts by plamaid DNA. Appl. Environ. Microbiol. 43,1213-1215. [35] Kondo, J.F. and McKay, LL. (1984) Plnsmid trandormarion of Streptococcus lactis protoplasts: optlmlzatloa and use in molecular cloning. Appl. Environ. M/crobioL 48, 252-259. [36] Simon, D., Rouan]t, A. and Chopin, M.-C. (1986) Highefficiency transformation of Streptococcus lactis protoplasts by plasmld DNA. AppL Envircm. MicrobioL 52, 394-395. [37] Wright, A., Talmisto, AM. and Sivela, S. (1985) Effect of Ca2+ ions on plasmid transformation of Streptococcus /act/s protoplasts. AppL Environ. MicrobioL 50, 11001102. [38] Gasson, MJ. and Anderson, P.H. (1985) High copy number plasmid vectors for use in lactic streptococci. FEMS Microbiol. Lctt. 30,193-196. [39] Kok, J., van dv'r Vcamm, J.M.B.M. and Vcmcma, (3. (1984) Construction of plasmld cloning vectors for lactic streptococci which also replicate in Bad//us mbd//s and Escherichia coll. Appi. Environ. Microbiol. 48, 726-731. [40] Kondo, J.F. and McKay, L.L. (1985) Gane lrandef

systems and molecular cloning in group N streptococci: a review. J. Dairy Sci. 68, 2143-2159. [41] De Vm, W.M. (1986) Gone cloning in lactic streptococci. Neth. Milk Dairy J. 40,141-154. [42] De Vce, W.M. (1987) Gone clonin8 and expression in lactic streptococci. FEMS Microbiol. Rev. 46, 281-295. [43] Otto, R., de Vos, W.M. and Gavricli, J. (1982) Plasmid DNA in Streptococcus cremoris Wg2: influence of pH on selection in chemostats of a variant lacking a protease plasmid. AppL Environ. Microbinl. 43,1272-1277. [44] Kok, J., van Dijl, J.M., van der Vossen, J.M.B.M. and Venema, G. (1985) Cloning and expres~on of a Streptococcus cremor~ protcinase in Bacillus subtilis and Streptococcus lactis. Appl. Environ. Mieroblol. 50, 94101. [45] De Vos, W.M., Vo$, ~P.,De Haard, H. and Boerrigter, I. (1989) Cloning and expression of the Lactococ~.v lactis ssp. cremoris SKll 8one encoding an extraeellular serine proteinase. Gone 85,169-176. [46] Gasson, M3. (1986) Identification and cloning of genes which encode enzymes for improved dairy fermentation processes, in: Biomolecular Ensineering in the European Community (Masnien, E., ed.) Final Report, Maninus Nijhoff Publishers, Dordrecht, Boston, Lancaster, pp. 489-496. [47] Kiwaki, M., Ikemura, H., Shimizu-Kadota, M. and Hirashima, A. (1989) Molecular characterization of a cell wall.associated proteinase gone from Streptococcus lactis lqCDO763. Mol. Microbiol. 3, 359-369. [48] Von Wright, A., Tynkloynzn, S. and Suominen, M. (1987) Cloning of a Streptococcus lactis ssp. loctis chromosomal fragment associated with the abifity to grow in milk. Appl. Environ. Microbiol. 53,1584-1588. [49] Tynkkynen, S. and yon Wright, A. (1988) Characterization of a cloned chromosomal fragment affecting the protcinue activity of Streptococcus lactis ssp. lactis. Biochimie 70, 531-534. . [50] Tynkkynen, S., yon Wright, A. and Syvlioja, E-L. (1989) Peptide utilization encoded by Laetococeus la¢gv ssp. /aetLv SSL135 chromosomal DNA. Appl. Environ. Mierobiol. 55, 2690-2695. [51] Kok, J., Leenhonts, KJ., Haandrikman, AJ., Ledeboer, A.M. and Venom& G. (1988) Nucientide sequence of the wall protcinase gem: of $trepto¢ocem cremoris Wg2. AppL Environ. Microbiol. 54, 231-238. [52] Vm, P., Simon~ ¢3., Siexen, R.J. and de Vm, W.M. (1989) Primary structure and organization of the gone for a prokaryotic, cell envelope-located serine protcinase. J. Biol. Chem. 264,13579-13585. [53] Van da- Voesen, J.M.B.M., van der Lelle, D. and Venema, ~.3.(1987) lsohttion and characterization of Streptococcus Wg2-specific promoters. Appl. Environ. Microbiol. 53, 2452-2457. [54] Laan, H. and KoninSs, W.N. (1989) Mechanism of proreletwe from Lacwcoc¢~/act/s up. c r ~ - / s Wg2. AppL Environ. Microbiol. 55, 3101-3106. [55] Markland, F.S. and Smith, E.L. (19"/1) Subtilisins: primary structure, chemical and physical properties, in:

]'he enzymes, (Boyer, P.D., ed.) Academic, New York, voi. 3, pp. 561-608. [56] Power, S.D., Adams, R.M. and Wells, J.A. (1986) Secretion and autoproteolytic maturation of subtifisin. Proc. Natl. Acad. Sfi. USA 83, 3096-3100. [57] Ha&ndrikman, A.J., Kok, J., Laan, H., Soemitro, S., Ledeboer, A.M., Koninss, W.N. and Venema, G. (1989) Identification of a gone required for the maturation of an extracellular serine proteinase. J. Bacteriol. 171, 27892794. [58] Vos, P., van Asseldonk, M., van Jeveren, F., Siezen, R., Simons, G. and do Vos, W.M. (1989) A maturation protein is essential for the production of active forms of Lactococcus lactis SKll serine proteinase located in or secreted from the cell envelope. J. Bacteriol. 171, 27952802. [59] Van der Vossen, J.M.B.M. (1988) Development and use of host vector systems for the characterization of lactococcal expression signals. PhD thesis, University of Groningen, The Netherlands. [60] Kok, J., Hill, D., Haandrikman, A.J., de Renver, MJ.B., Laan, H. and venema, G. Deletion analysis of the prc~ tcinase sene of Streptococcus cremoci$ W82. Appl. Environ. Microbiol., 54, 239-244. [61] Exterkate, F.A. and de Veer, GJ.C.M. (1989) Characterization of the cell wall proteinase Pill of Lacwcoccus la~,l/s ssp. cremor~ AM1, and its relatioship with the c~talytically different cell wall proteinase Pl/Pll of strain HP. Syst. Appl. Microbiol. 11,108-115. [62] Monnet, V., D. Le Bars, and Gripon, J.C. (1986) Specificity of a cell wall protcinase from Streptococcus lactis NCDO763 towards bovine B-cascin. FEMS Microbiol. Lett. 36,127-131. [63] Bockelmann, W., Monnet, V., GCIS, A., Teuber, M. and Gripon, J.C. (1989) Comparison of cell wall protcinases from Lactococcus ~actis ssp. cremoris AC1 and Loctocoo cus lacti~ ssp. lactis NCDO 763. I. Purification and action of the enzymes on caseins. Appl. Microbiol. Biotechnol. 31, 278-282. [64] Monnet, V., Bockeimann, W., Grlpon, J.C. and Tanber, M. (1989) Comparison of cell wall pmteinases from Lactococcus lactis ssp. cremoris ACI and Lactococcus lactis ssp. lactis NCDO 763. !I. Specificity towards bovine ~,Jscin. Appl. Microbiol. Biotechnol. 31, 112118. [65] Laan, H., Staid, E.J., Tan, P.S.T. and Koninp, W.H. (1989) Enzymes involve~ in the degradation and utilization of ca,q~ in Lactoceccus lactis. Neth. Milk Dairy J. 43, 327-345. [66] Ar~lle, PJ., Mathison, G,E. and Chandran, R.C. (1979) Production of cell-bound protcinase by Loctobocill~ bu/gar/cus and its location in the bactmrial cell. J. Appl. B~teriol. 41,175-186. [67] Ezzat, N., [] Soda, M. Bouillanne, C. Zevaco, C. and Blanchurd, P. (1985) Ccll wall asso~ated protcinascs in Loctobacillus helveticus, Lactobacillus bldgaricus and Loctobocillus/act/s. Milchwisse~chaft 40,140-143. [681 El Soda, M., Desmazcaud, MJ., Le Bars, D., and Zevaco,

C. (1986) Cell-wall-associated protcineses in Lactobacillus casei and L plantarum. J. Food Protect. 49, 361-365. [69] Ezzat, N., Zevaco, C., E1 Soda, M. and Gripon, J.C. (198~ Partial purification and characterization of a cell wall associated proteinase from Loctobocillus bulgaricus. Milchwissenschaft 42, 95-97. [70] Bockelmann, W. (1987) Das proteolytische system yon milchsiurebakterien. PhD thesis, University of Kicl, Kid. [71] Zevaco, C. and Gripon, J.-C. (1988) Properties and specifwity of a cell.wall proteinase from Lactobacillus hel~eticus. Le Lalt 68, 393-408. [72] Caprice, E., Fitzserald, G.F. and Daly, C. (1987) Conjugafive transfer of a recombinant protcinase plasmid from Streptococcus loctis to Leuconostoc mesenteroides. FEMS Microbiol. Rev. 46, P35. [73] Marshall, V.M.E. and Law, B.A. (1984) The physiology and growth of dairy lactic acid bacteria. In: Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk (Davies, F.L. and Law, B.A., ~ds.), pp. 67-98. Elsevier Appfied Science Pubfishers, London. [74] Van Boven, A. and Konings, W.N. (1986) Uptake of peptides by microorganisms. Neth. Milk Dairy J. 40, 117-127. [75] Mou, L., Sullivan, J.J. and Ja8o, G.R. (1975) Peptidase activities in group N streptococci. J. Dairy Res. 42, 147-155. [76] Schmidt, R.H., Morris, H.A. and McKay, L.L. (1977) Cellular location and characteristics of peptidase enzymes in lactic streptococci. J. Dairy Sci. 60, 710-717. [77] Law, B.A. (1979) Extracellolar peptidases in group N streptococci used as cheese starters. J. Appl. Bacteriol. 46, 455-463. [78] Kaminosawa, S., Ninomiya, T. and Yamauchi, K. (1984) Aminopeptidase profiles of lactic streptococci. J. Dairy Sci. 67, 2483-2492. [79] Exterkate, F.A. (1984) Location of peptidases outside and inside the membrane of Streptococcus cremoris. Appl. Environ. Microbiol. 47,177-183. [80] Kolstad, J. and Law, B.A. (1985) Comparative specificity of cell wall, membrane and intracallolar peptidases of group FI streptococci. J. Appl. Bacteriol. 58, 449-456. [81] Machuga, EJ. and Ires, D.H. (1984) Isolation and characterization of an aminopeptidase from Loctobacillus acidophilus R-26. Biochem. Biophys. Acta 789, 26-36. [82] E881mann, B. and Bachmann, H. (1980) Purification and partial characterization of an aminopeptidase from Loctobacillus lactis. AppL Environ. Microbiol. 40, 876882. [83] D e s ~ u d , M.J. and Zevaco, C. (1979) Isolation and general properties of two intracellular amino peptidases of Streptococcus diacetylactis. Milchwissenschaft 34, 606610. [84] Nevlani, E., Boquien, C.Y., Monnet, V., Phan Thanh, L. and Gripon, J.-C. (1989) Purification and characterization of an aminopeptidase from Lactococcus lactis ssp. cremoris AM2. Appi. Environ. Microbiol. 55, 2308-2314. [85] Tan, P.S.T. and Koninss, W.N. (1990) Purification and characterization of an aminopeptidase from Lactococca~

lactis ssp. cremoris Wg2. Appl. Environ. Mierobiol., 56,

526-532. [86] Exterkate, F.A. and de Veer, GJ.C.M. (1987) Purifw.afion and some properties of a membrane-boond aminopeptidase A from Streptococcus ~ . Appl. Environ. Microbiol. 53, 577-583. [87] Hickey, M.W., Hillier, AJ. ami Jago, G.R. (1983) Peptidase activities in lactobacilli. Aust. J. Dairy Tocb nol. 38,118-123. [88] Kaminogawa, S., Azuma, N., Hwang, I.K., Suzuki. Y. and Yamanchi, IL (1984) Isolationand characterization of a prolidase from Streptococcus ¢remoris H61. Alp'it. Biol. Chem. 48, 3035-3040. [89] Casey, M.G. and Meyer, J. (1985) Presence of X-prolyldipeptidyl-peptidase in lactic acid bacteria. J. Dairy SCI. 68, 3212,-3215. [90] Meyer, J. and Jordi, R. (1987) Pu.,Ww~ationand characterization of X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus lactis and from Streptococc~ thermophilus. J. Dairy Sci. 70, 738-745. [91] Kiefer-Partsch, B., Bockelmann, W., Geis, A. and Tenber, M. (1989) Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Loctococcus lactis ssp. cremoris. Appl. MicrobinL Biotechnol. 31, 75-78. [92] Zevaco, C., Monnet, V. and Gripon, J.-C. (1990) lntracellularX-prolyl dipeptidyl peptidase from Lactococcus Ioctis ssp. lactis: purification and properties. Appl. Bacteriol. 68, 357-366. [93] Arian, D., Laloi, P. and Portalier, R. (1990) X-prolyl-dlpeptidyl aminopeptidase of Lactobacillus d~lbrueckii ssp. bu/gor/cus: characterization of the evzyme and isolation of deficient mutants. Appl. Environ. MicrobioL 56, 2174--2179. [94] Khalid, N.M. and Marth, E.M. (1990) Purification and partial characterization of a prolyl-dipeptidyl aminopeptidase from Lactobocillus itelveticua CIqRZ 32. Appl. Environ. Microbioi. 56, 381-388. [95] Baankreis, It. and Exterkate, F.A. (1990) Purification and characterisation of a prollne-specilic peptidase from Lactococcus lactis ssp. cremorts HP. Proc. 3rd Nesherlands Bioteclmology Congress 1990, p. 336. {96] Hwang, I.IC, Kaminogawa, S. and Yamauchi, K. (1981) Purification and properties of a dipeptidase from Streptococcus cremoris. Agric. Biol. Chem. 45,159-165. [97] Hwans, LK., Kaminogawa, S. and Yamamr.hi, IC (1982) Kinetic properties of a dipeptidase from Streptococcus cremoris. Agric. Chem. Biol. 46, 3049-3053. [98] Van Boven, A., Tan, P.S.T. and Konings, W.N. (1988) Purification and characterization of a dipeptidase from Streptococcus cremoris Wg2. Appl. Environ. MicrobioL 54, 43-49. [99] Rabier, ]13.and Desmazeaud. MJ. (1973) Invantaire des diff&entes activit~s peptidariques intracellulalres des Streptococcus thermophilm. Purification et propriet~s

d'une dipeptide-hydrolese et dune aminopeptidase. Biochimie 55, 389-404. [lOO1 Desmazcaud, MJ. and Zevaco, C. (1977) General prop-

erties and substrate speclf'~-ityof an intracellular soluble dipeptid~se from Streptococcus diucetylactis. Ann. Biol. Anita. Bioch. Biophys. 17, 723-736. [101] [] Soda, M., Desmazcaud, M.J. and Berg~re, J.-L. (1978) Peptide hydrolase$ of l.octobacillus casei: isolation and ganetai properties of various peptidase activities. J. Dairy Res. 48, 445-455. [102] Szrhang, T. and Solberg, P. (1973) Fractionation ~f dipeptidese activities of Streptococcus lactis and dipeptidase specificity of some lactic acid bacteria. Appl. Mierobiol. 25, 388-395. [103] Abo-Elnaga, I.G. and Plapp, R. (1987) Peptidases of Lactobacillus casei and L. plantarum. J. Basic Microbiol. 27,123-130. [104] Bosman, B.W., Tan, P.S.T. and Konings, W.N. (1990) Purification and characterization of a tripeptidase from Lactococcus lactis ssp. cremoris Wg2. Appl. Environ. Microbiol. 56,1839-1843. [105] El Soda, M. and Desmazeaud, M.J. (1981) General properties of a new aryl.peptidyl amidase in Lactobacillus casei. Agric. Biol. Chem. 45,1693-1700. [106] Cliffe, A.J. and Law, B.A. (1979) An elactrophoretic study of peptidases in starter streptococci and in cheddar cheese. J. Appi. Bacteriol. 47, 65-73. [107] [] Soda, M., Ikrg~re, J.-L. and Desmazeaud, M.J. (1978) Detection and localization of peptide hydrolases in Lactobocillus casei. J. Dairy Res. 45, 519-524. [108] Yan, T-R., Azuma, N., Kaminogawa, S. and Yamauchi, IL (1987) Purification and characterization of a substrate-size-recognizing metalloendopeptidase from Streptococcus cremoris H61. AppL Environ. Micsobiol. 53, 2296-2302. [109] Yah, T-R., Azuma, N., Kamlnogawa, S. and Yamanchi, K. (1987) Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. Eur. J. Biochem. 163, 259-265. [110] Exterkate, F. (1975) An introductory study of the proteolyric system of Streptococcus cremoris HP. Neth. Milk Dairy J. 29, 303-318. [111] Muset, G., Monnet, V. and Gripon, J.-C. (1989) Intracellular proteinese of Lactococcus lactis ssp. lactis NCDO 763. J. Dairy Res. 56, 765-778. [112] Desmazcaud, M.J. (1974) General properties and specificity of an intraceilular neutral endopeptidase from Streptococcus thermophilus. Biochimie 56,1173-1181. [113] Desmazeaud, M.J. and Zevaco, C. (1976) General prop. erfies and substrate specificity of an intracellular neutral protease from Streptococcus diacetylactis. Ann. Biol. Anlm. Bioch. Biophys. 16, 851-868. [114] Atlan, D., Laloi, P. and Portalier, R. (1989) Isolation and characterization of amlnopeptidase-deficient Lactobaeil. /us bu/gar/cus mutants. Appl. Environ. Microbiol. 55, 1717-1723. [115] Smld, EJ., Plapp, R. and Koninss, W.N. (1989) Peptide uptake is essential for 8rowth of Lactococcus iactis on the milk protein casein. J. BacterioL 171, 6135-6140. [116] Van Boven, A. and Konings, W.N. (1988) Utilization of

dipept~des by Lactococ cus lactis ssp. cremori~'. Biochimie 70, 535-542. [11'7] Smid, E.J., Driessan, A.J.M. and Konings, W.N. (1989) Mechanism and energetiesof dipeptide transport in membrane vesicles of Loctococcus lactis. J. Bacteriol. 171, 292-298. [118] Driessen, AJ.M., Helfingwerf, K.J. and Konings, W.N. (1987) Membrane systems in which foreign protein pumps are incorporated. Microb. SCI. 4,173-180. [119] Driessen, A.J.M. (1989) Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria. Antonle van Leeuwenhoek 56,139-160. [120] Konings, W.N., Poohnan, B. and Driessen, A.J.M. (1989) Bioenergefics and solute transport in lactococei. CRC Crit. Rev. MierobioL 16, 419-476. [121] Crieiaard, W., Driessen, AJ.M., Molenaar, D., Hellingweft, K.J. and Konings, W.N. (1988) Light-induced amino acid uptake in membrane vesicles of Streptococcus cremoris and Clostridium acetobutylicum fused with reaction center containing proteoliposomes. J. Bacteriol. 170, 1820-1824 [122] Driessen, A.J.M., Kodde, J., de Jon8, S. and Konings, W.N. (1987) Neutral amino acid transport by membrane vesicles of Streptococcus cremoris is subjected to regulation by internal pH. J. Bacteriol. 169, 2748-2754. [123] Driessen, A.J.M., Hellingwerf, K.J. and Konings, W.N. (1987) Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris. J. Biol. Chem. 262, 12438-12443. [124] Driessen, A.J.M., de Jong, S. and Konings, W.N. (1987) Transport of branched chain amino acids in membrane vesicles of Streptococcus cremoris. J. Bacteriol. 169, 5193-5200. [125] Driessen, A.J.M. (1987) Amino acid transport in lactic streptococci. PhD thesis, University of Groningen,

Oroningen. [126] Driessen, AJ.M., van Lecuwen, C. and Konings, W.N. (1989) Transport of basic amino acids by membrane vesicles of Lactococcus lactis. J. Bacteriol. 171,1453-1458. [127] Otto, R., Lageveen, R.G., Veldkamp, H. and Konings, W.N. (1982) Lactate efflux induced electrical potential in membrane vesicles of Streptococcus cremoris. J. Bacteriol. 149, 733-738. [128] Poolman, B., Helfingwerf, K.J. and Konings, W.M. (1987) Regulation of the glutamate-81utamine transport system by intraceUular pH in Streptococcus Ioctis. J. Bacteriol. 169, 2272-2276. [129] Poolman, B., Staid, E.J. and Koninss, W.M. (1987) Kinetic properties of a phosphate-bond-driven 81utamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris, J. Bacteriol. 169, 2755-2761. [130] Driessen, AJ.M., Poolman, B., Kiewiet, R. and Konings, W.N. (1987) Arsinine transport in Streptococcus Ioctis is catalyzed by a cationic exchanser. Pro¢. Natl. Acad. SCI. USA 84, 6093-6097.

[131] Thompson, J. (1987) Ornithine transport and exchange in Streptococcus loctis. J. Bacteriol. 169, 4147. [132] Payne, J.W. (1980) Transport and utilization of peptides by bacteria, in: Microorganisms and NitroSen Sources (,I.W. Payne, ed.), pp. 211-~56. John Wiley & Sons Ltd., Chichester, U.K. [133] Payne, J.W. (1980) in: Microorganisms and Nitrogen Sources (J.W. Payne, ed.), pp. 359-377. John Wiley & Sons Ltd., Chichester, U.K. [134] Van Boven, A. and Konings, W.N. (1987) A phosphatebond-driven dipeptide transport system in Streptococcus cremoris is rogulated by the internal pH. Appl. Environ. Microbiol. 53, 2897-2902. [135] Poolman, B, and Konings, W.~I. (1988) Growth of Streptococcus cremoris in relation to amino acid transport. J. ILacteriol. 170, 700-707. [136] Walsh, P.M. and MeKay, L.L. (1982) Restriction endonuclease analysis of the lactose plasmid of Streptococcus ioctis ML3 and two recombinant lactose plasmids. Appl. Environ. Mi~obiol. 43,1006-1010. [137] Gas.~n, MJ. (1983) Genetic transfer systems in lactic acid bacteria. Antonie van Leeuwanhoek 49, 275-282. [138] Anderson, D.G. and MeKay, L.L. (1984) Genetic and physical characterization of recombinant plasmids associated with cell asgresa!ion and hish-frequency conjugal transfer in Streptococcus lactis ML3. J. Bacteriol. 158, 954-962. [139] Polzin, K.M. and Shimizu-Kadota, M. (1987) Identification of a new insertion element, similar to Gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3. J. BacterioL 169, 5481-5488. [140] Novel, M., Huang, D.C. and Novel, G. (1988) Transposition of the Streptococcus lactis ssp. Ioctis Z270 lactose plasmid to pVA797: demonstration of an insertion sequence and its relationship to an inverted ~peat sequence isolated by self-annealins. Biochimie 70, 543-551. [141] Haandrikman, A.3., van Lecuwen, C., Kok, J., Vos, P., de Vex, W.M. and Venema, G. (1990) IS elements on lacto-coceal proteinese plasmids. Appl. Environ. Microbiol. 56,1890-1896. [142] Van de Guchte, M., van der Vossen, J.M.B.M., Kok, J. and Venema, G. (1989) Construction of a lactococcal expression vector: expr~sion of henn e88 white lysozyme in Lactococcus lactis ssp. lactis. Appl. Environ. Microbiol. 55, 224-228. [143] Leenhouts, K.J., Kok, J. and Venema, G. (1989) Camp bell-like intearation of heterolosous plasmid DNA into the chromoson~ of Lactococcas Ioctis ssp. lactis. Appl. Environ. Microbinl. 55, 394-400. [144] Chopin, M.-C., Chopin, A., Rouault' A. and Galleron, N, (1989) Insertion and amplification of foreign genes in the Lactococcus loctis ssp. lactis chromosome. Appl. Environ. Microbiol. 55,1769-1774. [145] .Scheirfinck, T., Mahillon, J., Joos, H., Dhaese, P. and Michiels, F. (1989) Integration and expression of aamylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl. Environ. Microbiol. 55, 2130-2137.

[146] Van der Vossen, J.M.B.bL. Kok, J. and Vanema, G. (1985) Construction of cloning, promotog-screcainf,, and ternlinatog-screening shuttle vectors fog Bac///w .vu~///.v and Streptococcus Iocti3. Appl. Environ. Miesobiol. 50,

540-542. [147l Simons, G., Rutten, G., Homes, M. and de Vo~ W.M. (1988) Production of bovine prochymosin by lactic acid bacteria. Proceedings 2nd Netherlands Bioteclumle~ Congress 1988, pp. 183-187. [148] Klaenhammer, T.R., MeKay, I...L. and Baldwin, K.A. (1978) Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxydbonacleic acid. Appl. Environ. MicrobioL 35, 592600. [149] McKay, L.L., Baldwin, K.A. and Efstathinu, ,I.D. (1976) Transductional evidence for plasmid linkage of lactose metabolism in Streptococcus lactLv C2. AppL Environ. Mierobiol. 32, 45-52. [150] McKay, L.L. and Baldwin, K.A. (1974) Simultaneous loss of proteinase, and ~ u t i l i z i n g enzyme activities in Streptococcus Iocti$ and reversal of loss by transdtgtion. Appl. Microbiol. 28, 342-346. [151] Kuhl, S.A.. Larsen, L.D. and McKay, LL. (1979) Plasmid profiles of luctose(171 }negative and protcinase-deficient mutants of Streptococcus lactis C10, ML3, and MI8. Appl. Environ. Microbiol. 37,1193-1195. [152] Kempler, G.M. and McKay, L.L. (1979) Characterization of p|~.-v.~d deox~'ibonuclcic acid in Streptococcus Ioctis ssp. diacetyioctis: evidence for plasmidJinked citrate utilization. Appl. Environ. Microbiol. 37, 316-323. [153] Larsen, L.D. and McKay, L.L. (1978) Isolation and characterization of plasmid DNA in Streptococcm cremoris. Appl. Environ. MicrobioL 36, 944-952. [154] De Vos, W.M. and Davies, F.L. (1984) Plasmid DNA in lactic streptococci: bacteriophage resistance and proteinase plasmids in S. cremor/s SKIL in: Third European Congress on Biotechnolosy Vol. I!!, Vcrlag Chemic, Munich, pp. 201-206. [155] Daly, C. (1986) Exploitation of recombinant DNA techo nology to provide improved cultures for dairy fermentations, in: Biomolecular Engineering in the European Community (Magnien, E., ed.), Final Report, Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster, pp. 453-462. [156] Hayes, F., Caprice, E., McSweeney, A., Fitzgerald, (3.1:. and Daly, C (1987) pAM~l-associased mobilization of proteinase plasmids from Loctococcus /oct/s ssp. /oct/.v UC317 and L Ioctis ssp. c r e n u ~ UC205. Appl. Environ. Microbiol. 56,195-201. [157] Steenson, L.R. and Klaenhammer, T.R. (1986) lqasmid heterogeneity in Streptococcus cremoris MI2R: effects on proteolytic activity and host-dependent phage replication, J. Dairy Sci. 69, 2227-2236. [158] Yu, P.L., Appleby, R.D., Pritchard, G.G. and Limsowtin, G.K.Y. (1989) Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris pDI-21. Appl. Microbiol. Bioteclmol. 30, 71-74.