Plasmid-derived type II restriction-modification systems from Lactococcits laclis

This invention relates to plasmid-derived type II restriction-modification (R-M) systems from Ixic/ococcus lactis, DNA fragments coding for individual methylases and restriction endonucleases thereof, DNA cassettes for increasing phage resistance in lactic acid bacte¬ ria, cloning and expression vectors including the R-M systems, a method of conferring in¬ creased phage resistance on a lactic acid bacterium, lactic acid bacteria and Ixtdococcus laclis strains carrying the expression vectors, as well as methylases and restriction endo¬ nucleases encoded by the R-M systems

BACKGROUND OF THE INVENTION

Lactococcits strains are used as starter cultures for the production of cheeses and fermen¬ ted milks Bacteriophage infection of the starter culture remains a serious problem for the cheese industry and can result in a slow or dead cheese vat Several mechanisms of phage defense have been identified in lactococci These include adsorption blocking, abortive in¬ fection, and R-M systems (26) A report has shown the beneficial effect of using different phage resistance mechanisms in rotation (42) By cloning phage resistance mechanisms from lactococci it would be possible to construct a "cassette" like system consisting of different phage resistance mechanisms

Restriction-modification (R-M) systems have been found in a wide range of bacteria At least three different types of R-M systems, type I, II and III, have been found and charac¬ terized with respect to their requirement of Mg ²⁺ , ATP and S-adenosyl-methionine (2) The type II R-M system is by far the most simple and best understood of the R-M systems, containing a separate methylase (MTase) which uses S-adenosyl-methionine as the methyl donor and an endo uclease (ENase), both recognizing the same sequence Today more than 200 different type II R-M systems have been identified and more than 100 of them have been cloned, mainly because of their importance as tools in molecular biology and the important knowledge which is achieved of protein-DNA interactions The genetic charac¬ terization of type II R-M systems shows that the genes for the ENase and the MTase are

closely located, although not always in the same orientation The MTase and the ENase from the same type II R-M system normally do not show any homology to each other at the amino acid level despite the fact that they recognize the same DNA-sequence Com¬ parisons between type II MTases have shown that strong similarities exist within the group consisting of 5-methylcytosine MTases (m ⁵ C MTases) and within the group consisting of N4-methylcytosine (m ⁴ C) and N6-methyladenosine (m ⁶ A) MTases (29) The m ⁵ C MTases have about ten common amino acid sequence motifs, whereas the ni'C and m ^f, A MTases share two major common amino acid sequence motifs In contrast, the ENases have gene¬ rally very little homology in common, and no strong sequence motifs have been found

Several plasmids encoding R-M systems have been found in lactococci However they ha¬ ve not been characterized at the molecular level, but only in vivo by their efficiency in re¬ stricting phages (38) Fitzgerald et al (1 1) examined eight different strains and found only one type II endonuclease activity, R,ScτFI, in one strain, L. laclis UC503 (originally designated Streptococcus cre oris F) This first type II R-M system, ScvFI, isolated and characterized from a L. lactis strain, has been found to be cliromosomally en¬ coded (11, 8) Later Daly and Fitzgerald (6) examined seven strains more from diffe¬ rent starter cultures and found that six of the strains had ENase activity similar to R,ScvFI No other type II ENase activities were found The ENase from ScvFI recog- nized 5'-CClNGG-3' TWO ScvFI MTase-encoding genes have been cloned and characte¬ rized, but neither of the two isolated M,ScvFI-carrying clones exhibited any ScvFI ENase activity (8) The nucleotide sequences of the two MTase-encoding genes have been de¬ termined (8, 45) Both contained all ten of the predictive motifs normally found in m ⁵ C MTases Mayo et al (32) have reported type II activity, Ua\ recognizing the sequence CC WGG from /,. lactis NCDO497, but they did not determine whether it was chromo- somally or plasmid encoded

A type IIS system, Ll l, has been identified on the lactococcal plasmid pTR2030, which also codes for an arbortive infection mechanism (17) The nucleotide sequence of a type IIS MTase, M.LUΛ, from the plasmid pTR2030 has been identified and determined (18)

TK5 is a Danish starter culture, which has been used for the production of Cheddar cheese since 1982 (33) This starter culture has a marked resistance to phages as the dairy during 11 years of continuous production did not observe any delay in acidification due to a pha¬ ge infection, even though phages were isolated from the whey The starter originates from an old traditional dairy starter culture, which consists of an unknown number of L. lactis strains Sixty-two bacterial isolates were purified from the TK5 starter 33 of the isolates were arranged according to their plasmid profiles into six groups of identical or nearly identical profiles, 27 of the isolates showed unique plasmid profiles (22) All isolates have between 5 and 10 plasmids

In order to identify plasmid-encoded phage resistance in Ixictococcus a cotransformation procedure was used Total plasmid DNA from L lactis strain W56 isolated from the TK5 starter culture (33) was transformed together with the vector pVS2 (53) into actococcus. lactis subsp demons MG1614 We selected for the Cm ^R marker on pVS2 Transfor- mants were tested for increased phage resistance In this way we identified three plasmids (pJW563, pJW565, and pJW566) which coded for R-M systems in W56 (23) These plasmids ranged in size from 1 1 to 25 kb The efficiency of plating (EOP) for the isome¬ tric-headed phage p2 (16) varied from 10 ^"2 to 10 ^' for different plasmids The existence of multiple R-M encoding plasmids in Ixictococcus strains has previously been reported by Chopin's group in France (5, 13)

That multiple R-M-encoding plasmids can increase phage resistance was confirmed by stacking two-three plasmids (23, 24) The data in the following Table A show the efficien¬ cy of plating (EOP) of phage p2 on the various transformants, the numbers in brackets show the EOP of phage p2 with the R-M plasmid alone in L. lad is MG 1614

Table A. Plasmid encoding R-M systems assembled in L. lactis and their efTects on the EOP of phage p2 or jj50\

Transformant s ^h Plasmid encoding R-M EOP

MG1614 none 1

T128 pJW563 (10 ^" ') + pJW565 (10 ^"2 ) 10 ^'5

T46 pJW563 (10 ^" + pJW566 (2xl0 ^"2 ) 4xl0 ^"6

T45 pJW565 ( 10 ^"2 ) + pJW566 (2x 10 ^"2 ) 1 O^

T8 pJW563 ( 10-') + pJW565 ( 10 ^"2 )+ pJW566 3x 10 ^"6 (2xl 0 ^-2 )

J96 pJW563 (10-') + pFV1001 (10 ¹ ) 10 ^"5

J92 pJW563 (10-') + pFV1201 (10 ^" ') 10 ^"5

J75 pJW563 (10 ^", ) + pFV1001 (10 ^', ) + pTRK12 4\W ^η do-')

' Phages p2 and jj50 were propagated on L. lactis MG1614[pVS2] L. lactis MG1614 is a sm ^R , Opp ^d derivative of L. laclis MG1363 (46) ^h All transformants also harbored pVS2

As shown in Table A, the effect of assembling R-M plasmids were additive in most cases This supports the importance of R-M systems in the phage resistance of the TK5 starter We did not obtain completely phage resistant strains, however, Sing and Klaenhammer (41) showed that in combination with other phage resistance mechanisms, e g , abortive infection, R-M systems are powerful tools

Transformant Tl 1 (L. laclis MG1614 + pJW563) and a transfor ant harbouring a plasmid pFW094 from /,. laclis W9 (34) exhibited type II endonuclease activity showing that type II R-M systems can be plasmid encoded in Lacfococats laclis.

The ENases R LlaAA and R Z./«BI from W9 and W56, respectively, were partially purified, and the recognition sequences for both ENases were identified by digesting well known DNA (pBR322/328, λ DNA) with the respective ENases, treating the fragments with either the Klenow fragment of DNA polymerase or mung bean nuclea- se and ligating the resulting fragments into pBluescriptIISK+ (Stratagene, La Jolla, CA, USA) digested with EcoKV By sequencing the junction fragments of the obtai¬ ned clones, the recognition sequence of the respective ENases could be determined (34)

We found that R LlaM and R ZVαBI recognized 5' GATC-3' and 5'-CiTRYAG-3', respectively, digesting as indicated by the arrows ENase R L/aAl is therefore an isoschizomer ofMbol from Moraxella bovts and Dp/ill from Streptococcus pnevmo- ae R LlaBl is an isoschizomer of Sfcl from Enlerococciis faecinm Identical ENase cleavage patterns were obtained in digests of pBluescriptIISK+, pBR322 and Ml 3mp20 with R L/aAJ and Mbo\, and with R ZVαBI and Sfcl, respectively

SUMMARY OF THE INVENTION

Attempts to clone L/ AJ or LlaBl in Escherichia colt by screening the transformants for increased phage resistance to λvir were unsuccessful However, it was possible to clone and subclone the LlaM, LlaBl and LlάDll R-M systems directly in Lactococcits (see the following examples) When fragments from L/aAl and L/aDll were later transferred to E. coli, only the MTase activity was expressed, while ENase activity could not be detected It was not possible to clone the entire ZVαBI system or the gene encoding the methylase in E. coli

From the nucleotide sequence of LlaAl we could identify three ORFs, transcribed in the same direction and coding for putative proteins of 284, 269 (or 257) and 304 ami¬ no acids By comparison of the deduced amino acid sequences with data in EMBL and GenBank we found that two of the proteins had about 80% homology to the two MTases from the Dpnll R-M system, while the third had about 30% homology to the

corresponding ENase This indicates that the LlaAl R-M system consisted of two putative MTases and one putative ENase Based on the observation that the two ENases, R Llakl and Dpiήl, recognize the same nucleotide sequence, 5'-GATC-3', that both have two MTases, that the two MTases of Dpn l methylate adenine (9), and that the LlaM ENase is sensitive to methylated DNA from dam ^* E.coli strain, we conclude that the LlaM MTases most probably methylate adenine It has been sugge¬ sted that the previously sequenced lactococcal MTases, MJJal and M,ScvFI, methy¬ late adenine and cytosine, respectively ( 18, 8)

From the nucleotide sequence of LlaBl we could identify two ORFs with predicted proteins of 580 and 299 amino acids They are transcribed divergently We did not find any very strong homology to other ENases or MTases in the EMBL and Gen¬ Bank

Accordingly, in a first aspect the present invention provides a plasmid-derived type II restriction-modification (R-M) system, termed LlaM, from Lactococcits lactis subsp cremorts W9, said system encoding at least one methylase and a restriction endonu- clease with the recognition sequence 5 ^, -4 _' GATC-3', characterized in that the system comprises i) an open reading frame, termed ORFl, from nucleotide 769 to nucleotide 1620 in the enclosed SEQ ID No 1, coding for a methylase, termed M LlaM A, having the amino acid sequence shown in the enclosed SEQ ID No 2, ii) an open reading frame, termed ORF2, from nucleotide 1613 to nucleotide 2419 in the enclosed SEQ ID No 3 (same as SEQ ID No 1) or from nucleotide 1649 to nu- cleotide 2419 in the enclosed SEQ ID No 5 (same as SEQ ID No 1 ), coding for a methylase, termed M LI a MB, having the amino acid sequence shown in the enclosed SEQ ID No 4 or SEQ ID No 6, respectively, and iii) an open reading frame, termed ORF3, from nucleotide 2412 to nucleotide 3323 in the enclosed SEQ ID No 7 (same as SEQ ID No 1 ), coding for a restriction endo- nuclease, termed LlaM, having the amino acid sequence shown in the enclosed SEQ ID No 8

This aspect of the invention also includes DNA fragments comprising each of the ORFs in the R-M system LlaM, i e

a) A DNA fragment coding for a methylase, termed M Lla AIA, said fragment com- prising the DNA sequence from nucleotide 769 to nucleotide 1620 in the enclosed

SEQ ID No 1

b) A DNA fragment coding for a methylase, termed M LlaMB, said fragment com¬ prising the DNA sequence from nucleotide 1613 to nucleotide 2419 or from nucleoti- de 1649 to nucleotide 2419 in the enclosed SEQ ID No 1

c) A DNA fragment coding for a restriction endonuclease, termed R LlaAl, said fragment comprising the DNA sequence from nucleotide 2412 to nucleotide 3323 in the enclosed SEQ ID No 1

In a second aspect the present invention provides a plasmid-derived type II restricti¬ on-modification (R-M) system, termed LlaBl, from Lactococcits lactis subsp demo¬ ns W56, said system encoding at least one methylase and a restriction endonuclease with the recognition sequence 5'-CiTRYAG-3', characterized in that the system comprises i) an open reading frame, termed ORFl, from nucleotide 422 to nucleotide 2161 in the enclosed SEQ ID No 9, coding in the complementary strand for a methylase, ter¬ med M LlaBl, having the amino acid sequence shown in the enclosed SEQ ID No 10, and ii) an open reading frame, termed ORF2, from nucleotide 2464 to nucleotide 3360 in the enclosed SEQ ID No 9, coding for a restriction endonuclease, termed R LlaBl, having the amino acid sequence shown in the enclosed SEQ ID No 1 1

This aspect of the invention also includes DNA fragments comprising each of the ORFs in the R-M system LlaBl, i.e

d) A DNA fragment coding in the complementary strand for a methylase, termed M LlaBl, said fragment comprising the DNA sequence from nucleotide 422 to nu¬ cleotide 2161 in the enclosed SEQ ID No. 9

e) A DNA fragment coding for a restriction endonuclease, termed R LIaBl, said fragment comprising the DNA sequence from nucleotide 2464 to nucleotide 3360 in the enclosed SEQ ID No. 9

In a third aspect the present invention provides a plasmid-derived type II restriction- modification (R-M) system, termed LlάDll, from Lactococcits laclis subsp demons

W39, said system encoding at least one methylase and a restriction endonuclease, characterized in that the system comprises i) an open reading frame, termed ORFl , from about nucleotide 743 to nucleotide

1282 in the enclosed SEQ ID No 12, coding for a restriction endonuclease, termed RA/oDII, having the amino acid sequence essentially as shown in the enclosed SEQ

ID No 13 and with the recognition sequence 5'-GCiNGC-3\ and ii) an open reading frame, termed ORF2, from nucleotide 1391 to nucleotide 2341 in the enclosed SEQ ID No 12, coding for a methylase, termed M /JαDII, having the amino acid sequence shown in the enclosed SEQ ID No. 14

This aspect of the invention also includes DNA fragments comprising each of the

ORFs in the R-M system LlaOll, i e.

f) A DNA fragment coding for a restriction endonuclease, termed R ZVαDII, said fragment comprising the DNA sequence from about nucleotide 743 to nucleotide

1282 in the enclosed SEQ ID No.12.

g) A DNA fragment coding for a methylase, termed M LlaDll, said fragment comprising the DNA sequence from nucleotide 1391 to nucleotide 2341 in the enclosed SEQ ID No. 12

Further, the invention includes a DNA cassette comprising one or more of the R-M systems and DNA fragments according to the invention in combination with DNA en¬ coding other phage resistance mechanisms selected from the group consisting of ad¬ sorption blocking, abortive infection and R-M systems.

The invention also provides a cloning vector including DNA selected from the group consisting of R-M systems, DNA fragments and DNA cassettes according to the invention, and more specifically the plasmid pSNAl introduced in Laclococciis lactis MG1614 and deposited under the accession number LMG P- 15720, the plasmid pAG55 introduced in Laclococciis laclis MG1614 and deposited under the accession number LMG P- 15719, and the plasmid pCADl introduced in Lactococcus laclis subsp demons LM2301 and deposited under the accession number LMG P- 16901

The invention also provides an expression vector including DNA selected from the group consisting of R-M systems, DNA fragments and DNA cassettes according to the invention under the control of a promoter capable of providing expression thereof in a host cell, particularly a Gram-positive bacterium, and more particularly a lactic a- cid bacterium, especially Laclococciis lactis.

Further, the invention provides a method of conferring increased virus resistance on a cell wherein said cell is transformed with an expression vector according to the inven¬ tion. In particular the invention provides a method of conferring phage resistance on a Gram-positive bacterium, more particularly a lactic acid bacterium, and especially a Laclococciis laclis strain, wherein said bacterium is transformed with an expression vector according to the invention The invention also comprises a cell, particularly a Gram-positive bacterium, more particularly a lactic acid bacterium, and especially a Lactococcus lactis strain, which carries an expression vector according to the inventi¬ on.

In addition, the invention provides

- a methylase, termed M L/aAlA, having the amino acid sequence shown in the enclo¬ sed SEQ ID No 2,

- a methylase, termed M LfaAlB, having the amino acid sequence shown in the enclo¬ sed SEQ ID No 4 or SEQ ID No 6,

- a restriction endonuclease, termed R LlaM, with the recognition sequence 5'-iGATC-3', said endonuclease having the amino acid sequence shown in the enclo¬ sed SEQ ID No 8,

- a methylase, termed M LlaBl. having the amino acid sequence shown in the enclo¬ sed SEQ ID No 10,

- a restriction endonuclease, termed R LlaBl, with the recognition sequence 5'-C-lTRYAG-3'. said endonuclease having the amino acid sequence shown in the enclosed SEQ ID No 1 1 ,

a restriction endonuclease, termed R LlaDll, with the recognition sequence

5'-GC-iNGC-3', said endonuclease having the amino acid sequence essentially as shown in the enclosed SEQ ID No 13, and

a methylase, termed M-LlaOll, having the amino acid sequence shown in the enclosed SEQ ID No 14

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Maps, restriction and modification activities of pFW094 and subclone fragments thereof and the products of the ZVαAI genes pFW094 is a wild-type plasmid and is shown linearised by cleavage at a XJiol site Arrows indicate the position of the putative open re-

ading frames and the direction of transcription The putative sizes in amino acids are indi¬ cated for the open reading frames

Figure 2 Maps, restriction and modification activities of pJW563, its derivatives and the subclone fragment in pSNB 1 as well as the products of the LlaBl genes pJW563 is a wild-type plasmid Plasmids are shown linearised by cleavage at a XJiol site Arrows indi¬ cate the position of the putative open reading frames and the direction of transcription The putative sizes in amino acids are indicated for the open reading frames The figure il¬ lustrates the activity measured for the different plasmids constructed A restriction map of pJW563, B effect on R-M activity by introducing a chloramphenicol resistance cassette in the Clal site at position 1 1 kbp, C effect on R-M activity by deletion of the 1 2 kbp Bell fragment, D effect on R-M activity by introducing an erythromycin resistance cassette in the BglH site at position 9 2 kbp, and E cloning of the 4 0 kbp Hindlll fragment Abbre¬ viations Be, B ll, Bg, BgH , E, EcoRl, H, Hwdlll, X, Xhol

Figure 3 Restriction map of plasmid pΗW393

Figure 4 Agarose gel (0,8%) showing the restriction patterns obtained by cleaving different DNA with B oFl (lane 1 and 3) and L/aOll (lane 2 and 4) Lanes 1 and 2 pS A3, Lanes 2 and 4 pBluescript IISK+

DETAILED DESCRIPTION OF THE INVENTION

In order to determine the biological diversity of the R-M systems we compared six plas- mids isolated in our laboratory with three isolated in Chopin's laboratory and one from Klaenhammer's laboratory by use of the prolate headed phage c2 (25) Phage c2 was pro¬ pagated on each plasmid-containing strain and tested for restriction by all strains The re¬ sults are assembled in the following Table 1

Table 1. The EOP of phage c2 on L lactis strains carrying diflerent R-M encoding plasmids

Phage c2 propagated on strain

MG1614 Tl l ^a T21 5 ^a T46 22 ^a T3442 T T912 2 ^a T2235 5 ^a TB 123 ^b IL1420 ^C IL1530 ^C IL

Plasmid none pJW563 pJW565 pJW566 pFV0802 pFVlOOl pFV1201 pTRK 12 pBL6 pIL7 pI

from isolate W56 W56 W56 T29W5 V32 2 KH NCK40 IL594 IL594 I

EOP ^d

MG1614 1 1 1 1 1 1 1 1 Tl 1 3 x 10 ^" ' 1 3 x 1 -' 3 x 10 ^" ' lO ^'1 lO ^" ' 2 x 10 ' 4 x 10 ^' ' 2 x 10 ' 4 T21 5 8 x 10 ^"4 4 x 10 ^'3 1 7 x 10° lO ^"3 lO ³ 7 x 10 ³ 4 x 10 ² 2 x 10 ^'3 5 x 10 ^-4 2 T46 22 5 x 10 ³ 9 x 10 ³ 5 x 10 ^"3 1 3 x 10 ^"3 3 x lO ^-1 7 x 10 ^"3 4 x 10 ^"3 5 x 10 ^'3 T3442.7 10 ^"2 8 x 10 ^"3 6 x 10 ^"3 1 1 lO ^"2 6 x 10 ^"3 10 ² 7 x 10 ^"3 6 x 10 ^'3 T912 2 9 x 10 ^-2 3 x 10 ' 2 x lO-' 4 \ 10 ^" ' 4 x lO ^" ' 1 2 x lO ^-1 5 x 10 ^"2 4 x 10 ² lO ^" ' T2235 5 6 x 10 ^"2 8 x 10 ^"2 2 x 10 ^' ' 9 x 10 ^'2 lO ^" ' 8 x 10 ^'2 1 3 x 10 ^" ' 8 x 10 ^"2 7 x 10 ^"2 TB123 4 x 10 ^"2 3 x 10° 10 ' 9 x 10 ^"2 l ^" ' 4 x 10 ^'2 10 ^": 1 lO ^"1 5 x 10 ^'2 IL1420 7 x 10 ^" ' 2 X 10 ^"4 2 x 10 ^"5 5 x 10 ^"4 2 x 10 ^"4 Iff ⁴ 7 x 10 ^-2 3 x 10 ⁶ 7 x 10 ² 2 x 10 ^'5 4 IL1530 6 x l(ϊ ³ 6 x 10 ³ 2 x 10 ^"3 6 x 10 ^"3 lϋ ³ 9 x 10 ^"4 l ^"3 8 x 10 ^"3 lO ^"1 10 ' 4 IL1813 3 x IP ⁵ lo- 4 x 10 ^'5 4 x 1Q-" 10 ^" ' 2 x 1 -' 3 x 10-' 7 x 10 2 x IP ^'5 3 x 10 ⁶

' The plasmids were cotransformed into L lactis MG1614 together with pVS2 and afterwards cured for pVS2 by growing the cells in the presence of novobiocin b A gift from T Klaenhammer The plasmid is in L /ac//.s MG1363 ^c AA ggiifftt ffrroomm MM..--CC CChhooppiinn.. TThhee ppllaassmmiiddss aarree iinn LL llaaccttiiss IILL11440033 ((55)

^J EEOOPP wwaass ccaallccuullaatteedd aass tthhee pphhaaggee ttiitteerr oonn tthhee tteesstt ssttrraaiinn ddiivviiddeedd bbyy the titer on L. lactis MG1614 The EOP is an average of at least four expenments Type-II ENase activit was carried out as written in Fig 1 +. indicate activit and -. no activity

The results in Table 1 demonstrate that phages propagated on T46 22 [pJW566] and T3442 7 [pFV0802] were not restricted by each other This showed that pJW566 and pFV0802 probably code for identical R-M systems Tl 1 did not restrict phages pro¬ pagated on TB123, but TB123 restricted Tl 1 propagated phages This indicated either that two R-M systems could be encoded on pTRK12, one of which is identical to the one encoded by pJW563, or that the MTase encoded by pTRK12 cross- protects against the pJW563-encoded R-M system We also observed that phage c2 propagated on IL1420 or IL1530 gave a lower titer on the propagating host, than on the plasmid-free strain L. lactis MG1614 This is not due to differences in strain back- ground, since this effect was not found in L. lactis IL1813 These data imply that in addition to a R-M system, pIL6 and plL7 also code for an additional phage resistance mechanism, not previously identified The remaining MTases were not able to protect the phage from restriction by the other R-M systems, indicating that they are different from each other This shows that L. laclis strains have a large diversity of plasmid- encoded R-M systems

The diversity of R-M-encoding plasmids identified in Laclococciis made us screen all isolates from the TK5 starter for the presence of type II ENase activity Five different type II ENase activities, R LlaM, R LlaBl, R L/aCl, R L/aOl and K /JaEl, were identified Table 2 depicts the distribution of ENase activity among the TK5 isolates

Table 2. Type-H ENase activity in L. lactis isolates from the TK5 starter culture.

Group" Type II ENase activity ¹¹

R LlaM R LlaBl R LlaC R LlaOl R L/aEl

W39 W12

" The isolates were grouped according to their plasmid profile (22) Numbers in parenthe¬ ses show the number of isolates belonging to the group

It is seen from Table 2 that none of the screened isolates expressed more than one ty¬ pe II ENase activity The most common ENase was the R LlaAl, which was found in 12 (19 %) of the isolates R LlaBl was found in five (8 %), R laCl in two (3 %), with R ZVoDI and R LlaEl in one (2 %) of the isolates Thus, approximately 1/3 of the strains in the TK.5 starter culture contained a type II R-M system

From two of the strains that showed type II ENase activity, W9 (Group 3) and W56 (Group 4), we had previously isolated R-M-encoding plasmids This enabled us to test if the activity was chromosomally or plasmid encoded The transformants Tl l [pJW563], T21 5[pJW565], T46 22[pJW566] (Table 1) carried plasmids from W56, TW093[pFW093] and TW094[pFW094] carried plasmids from W9 These we¬ re examined and we found that only Tl 1 and TW094 exhibited type II ENase activi¬ ty The two systems identified in the TK5 isolates W12 and W15 quite likely are

plasmid encoded In order to determine whether the LldDl endonuclease was plasmid encoded, the cotransfor ation procedure into L. lactis MG1614 was made with total plasmid DNA from W39 We found that only transformant L. laclis MG1614[pHW393] expressed ZVαDI endonuclease activity which shows that also the LldDl R-M system is plasmid encoded The other transformants from Table 1 were also examined None of these strains exhibited type II ENase activity This suggests that other ENase activity than type II exists in Laclococciis Additional experiments are required to determine the type of R-M systems coded for on these plasmids

The recognition sequences for LlaCl, LlaOl and LlaEl have not yet been determined It is interesting that we have found a much wider diversity of type II ENases in Lacto¬ coccus. than previously reported (27) Also the two ENases characterized by us have recognition sequences with at least 50% A+T, in contrast to the 5 bp-recognizing ENases (5'-CCNGG-3' or 5'-CCWGG-3') reported for Lactococcus and Sfreptococ- ens Ihermophiliis (27) This may have practical implications, as Lactococcus and lac- tococcal phages have approximately 60% A+T in their genomes (40)

When we tried to clone the LlaOl R-M system we discovered that the plasmid pHW393 also coded for another type II R-M system which we designated L/aOll

The cloning and sequencing of the R-M systems LlaAl, LlaBl and L/aDll is described in the following Examples 1, 2 and 3, respectively

EXAMPLE 1

In the following we describe the identification, cloning and sequence of the plasmid- derived LlaM R-M system from Lactococcus lactis subsp demons W9 We show that the plasmid-free Lactococcus strain MG1614 obtains a higher degree of phage resistance with the plasmid pFW094 or the plasmid pSNAl than without it The cloning and sequen- ce of the LlaM R-M system is shown, and the putative ORFs and orientation of two MTases and one ENase are shown The deduced amino acid sequences were compared

with known type II R-M systems and, surprisingly, strong homology was found to the isoschizomer Dpn l R-M system from Diplococciis pneii omae and to Mbo from Mo- rexella bovis

Materials and Methods-

Strains, phages, plasmids and growth condition. Ixictococcus laclis subsp demons (L. laclis) W9 obtained from E Waagner Nielsen (22), and Lactococcus laclis subsp demons MG1614 (12) (previously designated L. laclis subsp lactis MG1614 (14)), ob- tained from Atte von Wright, was grown at 30 °C in Ml 7 medium (44) supplemented with 0,5 % glucose (GM17) When required, 10 μg/ml chloramphenicol or 5 μg/ml ery- thromycin was added The Eschericlva coli strains, XL 1 -Blue (Stratagene) and TCI 685. obtained from Tove Atlung, were grown at 37 °C in LB supplemented with chloramphe¬ nicol, erythromycin, tetracycline or ampicillin at the concentrations 10, 100, 12,5 and 100 μg/ml, respectively Plasmid pVS2 was cured with 1 μg/ml novobioein The isometric headed phages jj50 (23) and c2 (25) were propagated and plaque assayed as decribed by Terzaghi and Sa dine (44) Phage sensitivity was performed by plaque assay and cross streaking with phage jj 50 as described previously (23) Phage λ Λ2 was propagated as de¬ scribed by Sambrook el al (37) The plasmids used in this study are shown in Table 3

Table 3. Plasmids.

Preparation of cell extracts. A 500 ml fresh over-night culture of L. laclis or E.coli was harvested by centrifugation at 8 000 x g, washed twice in ice-cold lysis buffer (50 mM Tris-HCl pH 7 6, 10 mM MgCl ₂ , 25 mM NaCl, 7 mM mercaptoethanol) Cell were sus¬ pended in 10 ml cold lysis buffer and disrubted using a French press (Aminco, USA) at 1 000 psi (6,9 MPa) The crude extract was centrifiigated for 30 min at 40 000 x g before glycerol was added to a final concentration of 20% Aliquots were stored at -20 °C Small scale preparation of E coli extract was carried out by centrifugation of a 10 ml overnight culture at 15 000 x g, washing twice with lysis buffer The cells were resuspended in 0 5 ml lysis buffer before sonical disruption After centrifugation at 15 000 x g the supernatant was stored at -20 °C in 20% glycerol

Determination of endonuclease activity. Type II endonuclease activity in vitro was de¬ termined by incubating cell extract or purified enzyme with pBluescriptIISK+ or phage λ DNA in 50 mM Tris-HCl pH 7 6, 80 mM NaCl, 10 mM MgCl ₂ , 0 001 M DTT After 2 hrs at 37 °C the reaction mixtures were analyzed by horizontal electrophoresis in agarose gel with TEA buffer (37) //; vivo endonuclease activity was determined as an average of three independent determinations of the efficiency of plating (EOP) performed as descri¬ bed earlier (23)

Determination of methylase activity Phage jj50, or λ Λ2 was purified three times over single plaques and propagated on the selected strains Phage DNA was isolated by stan¬ dard procedure (37) Phage DNA was incubated with purified LlaM endonuclease (33) as described above //; vitro methylase activity was observed if the DNA was protected against cleavage with the purified LlaM endonuclease //; vivo LlaAl methylase activity was determined by plaque assays as described previously (23)

Transformation. Protoplast transformation of Lactococcus was conducted as previously described (23) L. laclis MG1614 was grown with 4 % glycine and transformed by elec- troporation using a Bio-Rad Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, Calif, USA) as described by Holo and Nes (1 ) E. coli was transformed by the CaCl ₂ standard procedure as decribed by Sambrook et al (37)

DNA isolation and cloning. Plasmid DNA was extracted from L. lactis by the method of Andresen et al (1) and further purified by CsCl-EtBr gradients (37). Plasmid was isolated from Exoli by alkaline lysis (37) and further purified by QIAGEN kit (QIAGEN Inc , Chatsworth, USA) or by CsCl-EtBr gradient (37) Phage DNA was purified by the met- hods described by Sambrook et al (37). Deletions were performed with the 'Erase-a-base' system (Promega Corp., Madison, USA) Blunt ends were created by filling in with Kle- now fragment of DNA polymerase (Boehringer Mannheim, Mannheim, Germany) Re¬ striction endonuclease, T4 ligase, and Calf Intestine Alkaline Phosphase (CIP), were pur¬ chased from Boehringer Mannheim (Mannheim, Germany) or New England Biolabs (Beverly, USA) All enzymes and kits were used according to the manufacturers recom¬ mendations

DNA sequencing The nucleotide sequence was determined by the dideoxynucleotide chain termination method (39) using double-stranded DNA as template Restriction frag- ments and deleted fragments in pBluescriptIISK+ were sequenced in both directions with the Sequenase version 2 0 Kit (United States Biochemical, Cleveland, Ohio) using [ ^?5 S]- dATP (Amersham, England) and standard primers (Stratagene). A few non-overlapping sequences were sequenced directly on the intact pFW094 plasmid using synthetic primers purchaced from P. Hobolt (Department of Microbiology, The Technical University, Den- mark) Compressed DNA sequences were resolved by using AmpliCycle Sequencing kit (Perkin Elmer) Computer analysis was based on GCG sequence analysis program (Version 7 0) (10)

Sequences comparison. The sequence of the LlaM gene was compared with R-M genes from the GenBank and the EMBL data bank.

Results

Identification and isolation of the R-M encoding plasmid pFW094. Total plasmid DNA from L. lactis W9 was isolated and protoplast transformed together with pVS2

(chloramphenicol marker) into the plasmid-free strain L. lactis MG1614 (23). Cm ^R trans-

formants were tested for phage resistance with phage jj50/MG1614 Phage jj50 was pro¬ pagated and plaque assayed on L. lactis MG1614 and on transformants with increased phage resistance (data not shown) By this method two R-M coding plasmids, pFW093 and pFW094, 12 7 kbp and 15 5 kbp, respectively, were identified The transformants, TW093 and TW094, carrying pFW093 and pFW094, respectively, were isolated after cu¬ ring of ρVS2 Transformants TW093 and TW094 restricted phage jj 50 with an EOP of 10 ^"1 and 10 ^"5 respectively, indicating that both plasmids code for phage resistance mecha¬ nisms

Cell extracts from L. lactis W9, TW093 and TW094 were screened for type II endonucle¬ ase activity Only L. laclis W9 and the transformant TW094 expressed type II activity, designated LlaM The endonuclease was purified and its recognition sequence determined as earlier described (34) The LlaM system recognized the sequence 5'-!GATC-3' (34) LlaM is an isoschizomer ofMbol from Moraxe/la bovis and Dpnll from Streptococcus pneuniomae λ DNA and plasmid DNA isolated from a ^* Ecoli strains were refractory to digestion with the restriction endonuclease LlaM, indicating that methylation of the adenine in the recognition sequence protected against cleavage by the LlaM endonuclease

Plasmid pFW094 was stably maintained during at least 500 generations in L lactis MG 1614 without any loss of the capability to restrict phages

Cloning of the R-M system. A restriction map of pFW094 was constructed as shown in Figure 1 Different restriction fragments were ligated into the shuttle vector pSA3 and transformed into L. lactis MG1614 selecting for Ery ^R Transformants were examined for expression of R-M activity, and the resulting EPOs of phages jj50 and c2 are shown in Table 4

Table 4. Restriction activity of Lactococcus lactis MG1614 harbouring different plas¬ mids on the phages jj50 and c2.

[ ] denotes the plasmid

Plasmid pSNAl was constructed by cloning a 6,0 kbp EcoRV fragment from pFW094 into pSA3 /,. laclis MG1614 [pSNAl] restricted phages jj50 and c2 to an EOP of 10 ^"7 and 10^, respectively (Table 4), showing that both the endonuclease and methylase activity was expressed by the plasmid in L. lactis MG1614 (Figure 1 ) Deletions of the H elll- EcoRV ₂ fragment from pSNAl , resulted in plasmid pSNA2, which did not restrict phages in L. lactis MG1614 (EOP increased from 10 ^'7 resp 10 ^"6 to 10 ^' '), but it had methylase ac¬ tivity (Figure 1 ) This showed that the Hαelll site was located within the gene encoding the endonuclease LlaM To localize the ZVαAI methylase, the subclones, pSNA3, pSNA4 and pSNA5 were constructed by cloning of restriction fragments of pSNAl into pSA3, as shown in Figure 1 The plasmid pSNA3 contained the 3 1 kbp Bgfll-SaiβAt fragment, ρSNA4 the 4 6 kbp Bgfll-BstXl fragment, and pSNA5 the 2 1 kbp Sau3A ₂ -EcoRV ₂ fragment, all inserted into pSA3 None of the three clones restricted phages and only pSNA4 expressed methylase activity This indicates that the the SaiβA-BstXl fragment is part of the methylase gene

Plasmid pNAl was constructed by cloning the 6 0 kbp EcoRW fragment in pBluescriptl- ISK+ in Ecoli XLl-Blue Deletions were made from the EcoRVi site of plasmid pNAl

From one of the generated derivatives pNA6, the overhangs of a 3 7 kbp Z?.wHII-EcσRV ₂ fragment was filled in and cloned into the EcoRV site of pSA3 yielding pSNAό. L. laclis MG1614 [pSNA6] restricted phage jj50 with an ΕOP of 10 ^"7 (Table 2), indicating that the 3.7 kbp jB.wHII-EcøRV∑ fragment is the smallest fragment obtained that contains all the information necessary for the expression of the R-M system in Laclococciis

E.colι TCI 685 (da ) was transformed with plasmids pSNAl and pSNA6 None of the transformants expressed endonuclease activity as determined by plaque assay with phage λ b2 or by assaying E. coli cell extracts for endonuclease activity (data not shown) Howe- ver, when phage λ b2 DNA was propagated through TCI 685 containing pSNAl or pSNAό, the λ DNA was refractory to digestion with the purified endonuclease LlaM, in¬ dicating that the phage DNA was fully methylated and that plasmid pSNAl and pSNAό had expressed methylase activity

Deposit. Lactococcus lactis MG1614 transformed with plasmid pSNAl comprising the LlaM R-M system has been deposited at the Belgian Coordinated Collections of Micro¬ organisms (BCCM), Laboratorium voor Microbiologie - Bacterienverzameling (LMG), Universiteit Gent, Belgium, under the accession number LMG P- 15720

Gene organization and D A sequence. Subfragments of the 6 0 kbp EcoRV fragment were cloned in the cloning vector pBluescriptIISK+ and transformed into E.coli XL1- Blue. Several of the clones were deleted in one direction as described in Material and Met¬ hods. The nucleotide sequence of a 3 7 kbp region containing the LlaM R-M genes was determined on both strands Three open reading frames, ORFl , ORF2 and ORF3, were located on the same strand of DNA (SΕQ ID No 1) The ORFl was located between nt 769 to 1620 The ORFl contained an initiation codon (ATG) and a putative but unusual ribosomal binding site (RBS) (GGTATAA) located at position 755 to 761 If the initiation codon at position 769 is used, ORFl should encode a protein of 284 amino acid Since the plasmid pSNA4, containing ORFl and part of ORF2, expressed LlaM methylase activity (Figure 1), ORFl must encode a methylase, M.LIaMA. The ORF2 is presumably positio¬ ned 29 bp downstream of ORFl and correspond to position 1649 to 2419 The initiation

codon at position 1649 is preceded by a RBS (GGAGG) that conforms well to the con¬ sensus RBS (GGAGG) positioned 7 bp upstream of a putative ATG start codon If this start codon is used, the ORF2 should encode a protein of 257 amino acids However, the¬ re is another initiation codon at position 1613, but it is not preceded by a consensus RBS If this is used as the start codon, the ORF2 should encode a protein of 269 amino acids It is not yet known which of the two initiation codons is actually used The ORF3 is located between nucleotides 2412 and 3323 with a 8 bp overlap to the ORF2 and is preceeded by a possible RBS 7 bp upstream (AAGGAG) The protein which could be translated from ORF3 starting at position 2412 would contain 304 amino acids Deletions of the region downstream to the Hαellli site at position 3203 abolished endonuclease activity in plasmid pSNA2, showing that ORF3 code for the endonuclease, R,/,/αAI A putative promoter for the LlaM R-M system could be located from position 699 to 726 The putative promoter region might contain a -10 region (TATTTA), which has good homology to the consen¬ sus, and a -35 region (TTAAGA) with low homology to consensus, 16 bp upstream Downstream the llaM gene with a 6 bp overlap is a putative rho-independent transcriptio- nal terminator structure with a -G = -26 kJ/mol No terminator-like structures were found downstream of ORFl and ORF2 This indicated that the LlaM R-M system consisted of three ORFs, possibly transcribed as a single polycistronic mRNA ORFl codes for a methylase, M LlaAl A, and ORF3 for a restriction endonuclease, R,L/aM

Comparison of amino acid sequences. No primary sequence homology was found bet¬ ween the restriction endonuclease R,Z./αAI and the methylase M L/aMA or the deduced protein encoded by ORF2, and no sequence similarities besides motifs I and II (se below) were found between the methylase M L/aMA and the deduced ORF2 encoded protein The deduced protein encoded by ORFl contained the motif I, PFXGXGAhXXG, and the motif II, DhVhXDPPYh, often found in adenine methylases (29) indicating that this ORF most likely codes for an adenine methylase The same motifs were found in the deduced protein from ORF2 indicating that this ORF most likely also encode an adenine methylase The deduced amino acid sequences from the ZVaAI R-M system were compared to the isoschizomeric systems, Dpn l from Dip/ococciis pneumomae (28) and Mbo from Mo-

raxe/la bovis (47) Based on amino acid sequence alignments of the three R-M systems, LlaM, Dpnll and Mbol, the calculated identity is shown in Table 5

Table 5. Amino acid sequence identity between the LlaAl R-M system and the Dpnll and Mbol R-M systems.

The three R-M systems are very homologous, especially the two methylases from the Dpnll and the deduced M LlaMA and ORF2 encoded protein of the LlaM systems were highly homologous (Table 5) The identity between the methylase M LlaMA and DpnM was 75%, and between the methylase DpnA and the protein from ORF2 86% The results are consistent with the proposal that both ORFl and ORF2 encodes adenine methylases designated M LlaMA and M Lla AIB, respectively, and similar to DpnM and DpnA Less amino acid identity was found between the methylases from the LlaAl and the Mbol sy¬ stems, 45% and 50% identity, respectively, (Table 5) The identity between the M Lla¬ MA and the Dam methylase from Eco/i was even lower (32%, data not shown) As ex¬ pected the identity between the endonuclease from the three R-M systems was not as significant as between the methylases However, the identity between RJ./oAl, and RMbol and R,E>/.v/II was 36% and 32%, respectively (Table 5) The LlaM endonuclease did not show any strong homology to other endonucleases from the GenBank database The LlaM and the Dpnll systems have the same gene organization Genes encoding methylases are located upstream of the gene encoding the endonuclease, whereas in the Mbol system, the gene encoding the endonuclease is located between the two methylases

Discussion.

LlaM is the first R-M system from Lactococcus lactis, with known recognition sequence, which has been cloned and completely sequenced The LlaM system has a recognition se- quence 5-4GATC-3' deviating from the recognition sequences of ScvFI and L/a , recog¬ nizing 5-CC-INGG-3' and 5'-CClWGG-3', respectively (27), in being more AT rich This may have practical implication, since Lactococcus has AT rich DNA (34-40 % CG (40)) The genes were localized to a 3 7 kbp fragment Interestingly, when the 3 7 kbp fragment cloned in pSA3 (pSNAό) was introduced into L. laclis MG1614, it restricted phage jj50 with an even higher efficiency (10 ^"7 ) than the wild-type plasmid This may be due to a slightly higher plasmid copy number of pSNAό compared to pFW094 and therefor a hig¬ her level of expression of the LlaM genes involved in the R-M phage resistance mecha¬ nism In E. coli TCI 685, however, pSNA6 only expressed methylase activity Whether this is due to instability of the mRNA or to a regulatory mechanism of the LlaM endonu- clease, that does not function in E. coli, is not known

We found that the R-M system consisted of three ORF's putatively transcribed on a poly- cistronic mRNA The gene organization two methylase genes followed by the endonucle¬ ase gene, is the same as for the genes encoding the Dpnll R-M system (28) but not like the Mbol system (47), which has the endonuclease gene surrounded by the two methylase ge¬ nes

The two ZVCTAI methylases showed a very high degree of identity (75 and 86%) to the two methylases from the E»/ //II R-M systems From the Dpnll R-M system it has been found, that one of the Dpnll methylases, DpnM, is an N6-adenine methylase that methylate he- mimethylated DNA (9) in the sequence 5'-GATC-3', whereas the other methylase, DpnA, methylate single stranded DNA (4) The sensitivity of the LlaM endonuclease to dam methylation and the high identity between the DpnM and M L/aMA suggests that M LlaMA also is a N6-adenine methylase methylating hemi-methylated DNA The high identity between the DpnA and MJVαAIB suggests that M ZVαrAIB may act like DpnA and methylate single stranded DNA The homologies found between Dam, DpnM, MboA

and M,Z./ AI indicate a strong relationship between the four 5 -GATC-3' N6-Adenine methylases Despite the fact, that the Dam methylase has an other biological function than DpnM, M MboA and M.LlaM, it appears that the four methylases originate from a com¬ mon ancestor and that the methylases have diverged into different biological functions No homology besides the two motifs I and II was found between the two LlaM methylases, M.LlaMA and M.LIaMB, indicating that the two LlaM methylases are not a result of ge¬ ne duplication, but have evolved independently of each other

The homology between the endonuclease from LlaM and those from Dpnll (32%) and Mbol (36%) is unusually high for isoschizomers, indicating a common ancestor Similar results have been seen before (52), but most often no similarities are seen between isoschi¬ zomers (52)

EXAMPLE 2

Here we report the cloning and nucleotide sequence of the genes coding for the LlaBl sy¬ stem from Laclococciis laclis subsp demons W56 The LlaBl endonuclease is an isoschizomer to Sfcl from Enlei(κ:occus faeciimi L. lactis W56 has previously been isola¬ ted from the Danish mixed Cheddar starter, TK5 (22) It was shown that /,. laclis W56 harbors at least tliree plasmids, pJW563, pJW565, and pJW566, which encode distinct R- M systems (23) It was found that transformants harbouring the plasmid pJW563 expres¬ sed type II activity, named ZVαBI (34)

Materials and Methods.

Strains, phages, plasmids and growth conditions. The strains Ixictococciis lactis subsp demons W56, obtained from E Waagner Nielsen (22) and Ixictococciis lactis subsp demons MG1614, originally classified as Ixictococciis lactis subsp lactis (12, 14), obtai¬ ned from Atte von Wright, were grown at 30 °C in Ml 7 media (Oxoid) supplemented with 0,5 % glucose (GM 17) and 5 mM CaCl ₂ when phages were used The antibiotics we¬ re purchased from Sigma and were used at the following concentrations chloramphenicol,

10 μg/ml, erythromycin and tetracycline, 5 μg/ml Eschencia coli strains XL 1 -Blue (Stratagene) and HB101 (3) were grown at 37 °C in LB supplemented with chloramphe¬ nicol, erythromycin, tetracycline or ampicillin at 10, 100, 12,5 and 100 μg/ml, respectively, when needed The isometric headed phage jj50 and the prolate headed phage c2 (25) were propagated and titrated by the method of Terzaghi and Sandine (34) λ Λ2 phage was pro¬ pagated as described by Sambrook (37) The plasmids used in this study are shown in Table 6

Table 6. Plasmids.

Plasmid Relevant characteristics Source or reference

pJW563 rVm ^* (23)

pSA3 shuttle vector (7) pBluescriptIISK+ Stratagene

pUC7,erm pUC7 1 1 kbp H///PI pIL253 e/w W M de Vos, NIZO, Ede, The Netherlands pJWCl pJW563 cam cassette in lal site this work pJWC2 pJWC 1 deletion of 1 2 kbp B ll this work fragment pJWEl pJWCl 1 1 kbp ery cassette mBgUl this work site

pSNBl pSA3 4 0 kbp H//;dIII pJW563 this work

pSK-c l pBluescriptHSK+ 3 9 kbp pVC5 cam Finn K Vogensen pAG55 pSK-cml 3 1 kbp H//;dIII this work pJW565, 64 kbp E oRl pJW563

Preparation of cell extracts. A 500 ml fresh over-night culture of L. lactis was harvested by centrifugation at 8 000 x g washed twice in 10 ml cold lysis buffer (50 mM Tris-ΗCI

pH 7 6, 10 mM MgCl ₂ , 25 mM NaCl, 7 M mercaptoethanol) and suspended in 10 ml cold lysis buffer A French press (Aminco, USA) was used to disrupt the cells at 1 000 psi (6,9 MPa) The crude extracts were centrifugated for 30 min at 40 000 x g before glycerol was added to the supernatant to a final concentration of 20% Aliquots of the extract were stored at -20 °C

Determination of endonuclease activity. Type II endonuclease activity in vitro was de¬ termined by incubating cell extract or purified enzyme (34) with pBluescriptIISK+ or pha¬ ge λ DNA in 50 mM Tris-HCl pH 7 9, 10 mM NaCl, 10 mM MgCl ₂ , 100 μg/ml BSA 0 001 M DTT After 2 hrs incubation at 37 °C the reaction mixture was analyzed by hori¬ zontal electrophoresis in agarose gel with TAE buffer (37) The /// vivo endonuclease ac¬ tivity was determined as an average of three independent determinations of the efficiency of plating (EOP) performed as described earlier (23)

Transformation. L. lactis MG1614 was grown with 4 % glycine and transformed by electroporation using a Bio-Rad Gene Pulser apparatus (Bio-Rad Laboratories, Ri¬ chmond, USA) as described by Holo and Nes (19) E. coli strains were transformed by the CaCl ₂ standard procedure (37)

DNA isolation and cloning. Plasmid DNA was extracted from L. lactis strains by the method of Andresen el al (\ ) and purified by the CsCl gradient method (37) Plasmid DNA isolation from E.coli was performed by alkaline lysis (37) DNA was further purified by QIAGEN coloums (QIAGEN Inc , Chatsworth, USA) or by CsCl-EtBr gradients (37) Phage DNA was isolated as described by Sambrook et al. (37) Restriction endonucleases, T4 ligase, and Calf Intestine Alkaline Phosphase (CIP) were purchased from Boehringer Mannheim (Mannheim, Germany) or New England Biolabs (Beverly, USA) Deletions of subcloned fragments were obtained by using the 'Erase-a-base' system (Promega Corp , Madison, USA) All enzymes and kits were used according to the manufacturer's recom¬ mendations

DNA sequencing The nucleotide sequence was determined by the dideoxynucleotide chain termination method (39) using double-stranded DNA as templates Restriction fragments and deleted fragments cloned in pBluescriptlISK+ were sequenced using the Sequenase version 2 0 kit (United States Biochemical, Cleveland, Ohio, USA) The sequ- encing was done in both directions using [ ³⁵ S]-dATP (Amersham, England) and standard primers (Stratagene) complementary to the region of the plasmid upstream of the deleted fragments A few regions were sequenced by PCR directly from the intact pJW563 plas¬ mid using synthetic primers provided by P Hobolt (Department of Microbiology, The Technical University, Denmark) In case of compression, DNA sequencing was carried out with AmpliCycle™ Sequencing kit (Perkin Elmer) All computer analysis was done with GCG sequence analysis program (Version 7 0) (10)

Sequence comparisons. The sequence of the LlaBl genes was compared to R-M genes from the GenBank and the EMBL data bank

Results

Cloning and localization of the LlaBl R-M system Cell extracts from /,. lactis W56, Tl 1 [pJW563], T21 5 [pJW565] and T46 22 [pJW566] were screened for type II endo- nuclease activity Only /,. lactis W56 and the transformant T 1 1 expressed type II activity, designated LlaBl A restriction map of the plasmid pJW563 was made (Figure 2) Direct cloning of the entire R-M system in Eco/i in different vectors was not successful (data not shown) Therefor cloning of pJW563 was carried out in L. lactis MG 1614 Due to pro¬ blems in cloning the entire system in L. laclis MG1614 it was decided to determine the lo- cation of the genes for the R-M system on the plasmid pJW563 A chloramphenicol resi¬ stance cassette was inserted into one of the Cla sites, resulting in the plasmid pJWC I (data not shown) L. lactis MG1614[pJWCl] expressed R-M activity like the wild type plasmid Bidirectional deletions were made showing that the R-M system was located around the Bglll site (data not shown) An erythromycin cassette with compatible BaniHl linker ends was inserted into the unique Bglll site of pJW563, and the resulting plasmid, pJWEl, was electroporated into L. lactis MG1614 (Figure 2) Cell lysate of the transfor-

mant did not express any LlaBl endonuclease activity and the transformant did not restrict phages However, the transformant L. laclis MG1614[pJWEl] expressed methylase ac¬ tivity found by the fact that phages, propagated on the transformant, was not restricted by transformants containing pJW563 Deletion of the 1 2 kbp Bell fragment from pJWEl re- suited in plasmid pJWE2, which showed neither endonuclease nor methylase activity (Figure 2) These results indicated that the endonuclease gene was located near the Bglll site, whereas at least a part of the methylase gene was located on the 1 2 kbp Bell frag¬ ment The entire R-M system was then cloned in the vector pSA3 on a 4 0 kbp Hwdlll fragment containing the BgHl and Belli sites, resulting in the plasmid pSNB 1 Crude cell extracts prepared from L. lactis MG1614[pSNBl] transformants expressed LlaBl endo¬ nuclease activity (data not shown) and restricted phage jj50 with an (EOP) of 10 ^"4 and phage c2 with and EOP of 10 ^"; Phage jj50 propagated on L. lactis MG I614[pSNBl] was not restricted by the L. lactis MG1614[pJW563] strain, indicating that phage jιj50 DNA had been methylated, suggesting that the plasmid pSNBl carried the genes encoding both the endonuclease and methylase from the LlaBl R-M system and that they function as a phage resistance mechanism It was not possible to transform E. coli ΗB101 with the plasmid pSNBl, which harboured the H///dIII-fragment cloned in pSA3, indicating that the entire LlaBl R-M system is lethal to E. coli The LlaBl encoding genes were also clo¬ ned as a 64 kbp EcoRl fragment from pJW563 in a BluescriptIISK+ derivative carrying a cassette encoding chloramphenicol resistance and a replicon from pJW565, resulting in plasmid pAG55 L. laclis MG1614[pAG55] restricted phage jj50 at the same order of magnitude as the wild-type plasmid pJW563

Deposit. Lactococcus lactis subsp lactis MG16I4 transformed with plasmid pAG55 comprising the LlaBl R-M system has been deposited at the Belgian Coordinated Collec¬ tions of Microorganisms (BCCM), Laboratorium voor Microbiologie - Bacterienverzame- ling (LMG), Universiteit Gent, Belgium, under the accession number LMG P- 15719

Gene organization and DNA sequence. It was not possble to clone the 40 kbp H//;dIII fragment in pBluescriptlISK+ in Ecofi XLl-Blue, indicating again that the endonuclease expression may be lethal to E. co Several of the clones were deleted in one direction as

decribed in Materials and Methods. The nucleotide sequence of the 4.0 kbp Hindlϊl frag¬ ment containing the Ll Bl R-M genes was determined on both strands Two major ORFs, ORFl and ORF2, were found in the sequence (SEQ ID No 9): ORFl comprised 1740 bp, with a coding potential for a 580 aa protein, and ORF2 comprised 897 bp, capable of coding for a 299 aa protein The two ORFs were separated by 302 bp and transcribed di¬ vergently. Both ORFs were preceded by putative Shine-Dalgarno sequences 7 bp in front of the ATG start codon. Putative -10 (sequence TATAAT and TATAAG) and -35 (sequence TTGACT and TCGTAA) consensus regions were found upstream of both ORFs The sequenced region had only one Bglll site, and it was located 138 bp downstre- am of the putative start codon in ORF2. This Bglll site was inactivated in plasmids pJWEl and pJWCl, which did not express endonuclease activity, showing that ORF2 codes for the LlaBl endonuclease. The 1.2 kbp i?c7I fragment, which was deleted in plasmid pJWE2 (Figure 2), was found at position 1305 to 2520 covering 856 bp downstream in ORFl, showing that ORFl codes for the methylase, M,LlaBl. A secondary structure indicating a putative terminator loop was found downstream of the methylase gene with a 2 bp over¬ lap The results show that the LlaBl R-M system consists of a methylase, M-LlaBI, putati¬ ve of a 580 aa protein (65 kDa) and an endonuclease, R.LlaBI, putative of a 299 aa prote¬ in (33 kDa), transcribed divergently.

The r-llaBI gene is preceded by one short ORF, which extends over 90 bp and may code for a small protein of 30 amino acids. This ORF is aligned in the same orientation as the -llaBI gene and separated therefrom by 1 10 bp.

Comparison of amino acid sequences. No primary sequence similarities were found between the restriction endonuclease RLlaBl and the corresponding methylase M-ZVαBI, or with other type II restriction endonucleases. The deduced amino acid sequence of the ML/άBl methylase was compared with the amino acid sequence of other methylases in the data banks. The motif II, DhVhXDPPYh, which is common to all known adenine and cy¬ tosine methylases (29) was found in the sequence from nucleotide 1717 to 1688. The se- quence lacked the motifs common to cytosine methylases (30). A motif similar to motif III (21), which is associated with adenine methylases like Eco571, Pstl, PaeRll and BstiBl,

recognizing the sequence CTxxAG, was also found in LlaBl Since these comparisons of the amino acid sequence of the MLlaBl methylase to other type II methylases revealed a significant similarity to N6-adenine methylases and a lack of the numerous conserved mo¬ tifs common to cytosine methylases, the MLlaBl methylase most likely is a N6-adenine methylase

Discussion.

The R-M coding plasmid, pfW563, isolated from L. lactis W56, was previously reported to restrict the isometric headed phage jj 50 with an efficiency of plating (EOP) of 10 ^"1 and the prolate headed phage with an EOP of 10 ^'2 , clearly showing that it encodes a phage re¬ sistance mechanism (23) The system exhibiting type II endonuclease activity, designated R LlaBl, was purified and its recognition sequence determined to be 5'-CiTRYAG-3' (34) Two plasmids, pSNBl and pAG55, harbouring the genes encoding the LlaBl R-M system, have been constructed In L. lactis MG1614 the plasmids pSNB 1 and pAG55 had the ability to restrict lactococcal phages with an EOP at the same level as found for the wild-type plasmid, pJW563 This showed that the genes can be cloned and used to increa¬ se the phage resistance in Ixictococciis strains

By cloning and sequencing the LlaBl R-M system, it was found that the R-M system con¬ sists of two ORFs putatively transcribed divergently The methylase is encoded by an ORF of 1740 bp capable to code for a protein of 580 amino acid, while the putative endonucle¬ ase is encoded by an ORF of 897 bp capable to code for a protein of 299 amino acids The size of the methylase (65 kDa) is considerably larger than most of the sequenced methyla- ses, indicating that the M-ZVαBI methylase may be a monomer The deduced size of the restriction endonuclease (33 kDa) is comparable to the sizes of other endonucleases (51) Probably the endonuclease functions as a dimer like many other type II endonucleases The missing primary sequence similarities between the MLlaBl and the corresponding RLlaBl supports the general assumption that type II restriction endonucleases and methylases are evolutionary unrelated and interact with target DNA sequences by different mechanisms (49)

Preceding the rllaBI gene was a small ORF (90 bp) potentially encoding a protein of 30 amino acids. Probably this protein is too small to act as a trans-acting positive regulator of the r llaBI gene, similar found in the Pλmll system (43) and other systems with divergently transcribed genes.

The motif II is presumably involved in the general steps of DNA methylation, probably in the transfer of the methyl group (29). The structural similarity of the methylases recogni¬ zing the sequence CTXXAG suggests that motif III may be involved in the sequence re¬ cognition of the methylases. From cytosine methylases, however, experimental evidence suggests that the large amount of conserved motifs (29) may be involved in the proper folding of the protein, while the variable regions may be responsible for sequence specifici¬ ty (35). It cannot be excluded that motif III is involved in the folding of the methylases

During the cloning of the LlaBl system it was found that the plasmid pJW563 was resi¬ stant to digestion by the Pstl restriction endonuclease (data not shown) although subclones of pJW563 containing fragments of the MLlaBl methylase were not resistant to Pstl re¬ striction. The Pstl endonuclease recognises 5'-CTGCA-lG-3' and cuts as indicated by the arrow. LlaBl can recognise the same sequence, 5'-C4 GCAG-3' (and 5'-ClTATAG-3'), but will cut the recognition sequence at a different place (LlaBl generates 5'- overhangs while Pstl gives 3 -overhangs). This indicates, that the adenine in the Pstl recognition se¬ quence has been methylated by the MLlaBl methylase. This result, together with the ho¬ mology found between the M-ZVαBI methylase and other adenine methylases, and the lack of homology to common motifs in cytosine methylases, indicate that the M/,/αBI methyla- se is a N6-adenine methylase.

The average G+C content of the LlaBl genes is 27,8% (31,5% for the r llaBI and 25,7% for m llaBl), which is much lower than the 34 to 43 % G+C content normally found in lactococci by measuring the melting temperature (40). This may indicate that the ZVαBI R- M system originates from genus other than Lactococcus.

EXAMPLE 3

L. lactis W39 has previously been isolated from the Danish mixed Cheddar starter, TK5 (33) We found, as shown in Table 2, that L. lactis W39 expressed type II endo- nuclease activity, which we designated LlaOl Here we report the cloning and nu¬ cleotide sequence of the genes coding for another type II R-M system from L. laclis W39, designated LlaOll, with an endonuclease having a different restriction pattern from that of LlaDl

Materials and Methods

Strains, phages, plasmids and growth conditions. The strains and bacteriophages used in this study are listed in Table 7 The L. lactis strains were grown at 30 °C in Ml 7 media (Oxoid) supplemented with 0,5 % glucose (GM17) and 5mM CaCl ₂ when phages were used Escherichia coli (E. coli) strains were grown at 37 °C in LB The antibiotics (Sigma) were used at the following concentrations in L. lactis chloram¬ phenicol, 6 μg/ml, in E. colt chloramphenicol, 20 μg/ml, tetracycline, 12,5 μg/ml, and ampicillin, 100 μg/ml Lactococcal phage propagation and plaque assays were carryed out as described by Terzaghi and Sandine (44) The plasmids used in this study are shown in Table 8

Table 7. Bacterial strains and bactenophages used

Table 8. Plasmids used in this study.

Preparation of cell extracts. A 1 1 fresh over-night culture ofE. lactis was harvested and washed once in 10 ml cold lysis buffer (50 mM Tris-HCl pH 7 6, 10 mM MgCl ₂ , 25 mM NaCl, 7 mM mercaptoethanol) and suspended in 12-15 ml cold lysis buffer. A French press (Aminco, USA) was used to disrupt the cells at 1500 psi The crude ex- tracts were centrifiigated for 2 hrs at 180 000 X g before glycerol was added to the supernatant to a final concentration of 20% Aliquots of the extract were stored at -20 ^β C

Determination of endonuclease activity. Type II endonuclease activity m vitro was determined by incubating cell extract or partially purified enzyme with plasmid DNA in NEBuffer 2 (10 mM Bis-Tris-propane-HCI pH 7 0, 10 mM MgCl ₂ , 1 mM DTT) from Biolabs (New England, USA) After 1 hr incubation at 37 °C, the reaction mix¬ ture was analyzed by electrophoresis in agarose gel with TAE buffer (37) The m vivo endonuclease activity was determined as an average of three independent determina- tion of the efficiency of plating (EOP) performed as described earlier (23)

Purification of restriction endonucleases Cell extract, made as described, was pu¬ rified by an one-step FPLC chromatographic procedure using a Mono Q column in buffer A (50 mM Tris-HCl pH 7.6, 10 mM MgCl ₂ , 5 mM mercaptoethanol) The en- zyme was eluted with 1 M KCI in buffer A and collected in small fractions, which were assayed for endonuclease activity.

Determination of methylase activity Plasmid DNA was incubated with purified LlάDll endonuclease as described above Methylase activity was observed if the plasmid DNA encoding the methylase was protected against cleavage with the puri¬ fied L/aDll endonuclease /// vivo LlaDll methylase activity was determined by plaque assays

Transformation. L. laclis LM2301 was grown with 3 % glycine and transformed by electroporation using a Bio-Rad Gene Pulser apparatus (Bio-Rad Laboratories, Rich-

mond, USA) as described by Holo and Nes (19). E. coli strains were transformed by the CaCl ₂ standard procedure (37).

DNA isolation and cloning. Plasmid DNA was extracted from L. lactis strains by the method of Andresen et al ( ) and purified by the CsCl gradient method (37). Plasmid DNA isolation from E.coli was performed by alkaline lysis and CsCl gradient method (37) or by QIAGEN columns (QIAGEN Inc., Chatsworth, USA). Restriction endo¬ nucleases, T4 ligase, and Calf Intestine Alkaline Phosphase (CIP) were purchased from Boehringer Mannheim (Mannheim, Germany), Amersham (Buckinghamshire, UK) or New England Biolabs (Beverly, USA). All enzymes and kits were used ac¬ cording to the manufacturers recommendations

DNA sequencing. Double-stranded DNA templates for sequencing were obtained by subcloning various DNA fragments in pBluescript II SK+. Deletions of the entire fragments were obtained by using the 'Erase-a-base' system (Promega Corp., Madi¬ son, USA). The nucleotide sequence was determined by standard dideoxy sequencing using Auto Read™ Sequencing kit (Hoefer Pharmacia Biotech Inc., San francisco, USA) The fragments were sequenced on both strands using universal and reverse primers (Stratagene) Computer analyses were performed with the GCG sequence analysis program (Version 8.0) (10).

Sequence comparisons. The sequence of the L/aDl genes was compared with R-M genes from the NCBI database.

Results

Identification of a plasmid encoding the LlaOll R-M system. Total plasmid DNA from L. lactis W39 was cotransformed with pVS2 into L. lactis MG1614 selecting for chloramphenicol resistance (23). The phage sensitivity of the transformants were determined by crossstriking with phage jj50. It was found that cell extract from transformant L. lactis 39.26, which harboured plasmid pHW393 besides pVS2, ex-

pressed type II endonuclease activity designated LlaOl L. lactis 39 26 restricted the small isometric headed phages jj50 and p2 with an efficiency of plating (EOP) of 10 ^"4 and the prolate headed phage c2 with an EOP of 10 ^"2 Phages propagated on the transformant circumvented the restriction, showing that plasmid pHW393 encodes a R-M system The transformant 39 26 was cured for plasmid pVS2 by treatment with novobioein, and pHW393 was retransformed into L lactis LM2301 This transfor¬ mant T39 3 restricted the phages jj50, p2 and c2 with the same EOP as tranformant 39 26

Cloning and localization of the Lla ll R-M system. A restriction map of the plasmid pHW393 was made (Figure 3) Two plasmids, designated pCADl and pCAD2, containing a 2 4 kbp Pstl-EcoRl fragment and a 5 4 kbp Λ7>αI-EcσRI frag¬ ment, respectively, in the shuttle vector pCI3340, were constructed and transformed into L. lactis LM2301 Both transformants restricted phages jj50 and p2 with an ΕOP of 10 ^'3 Phages propagated on transformant L. lactis LM2301 [pCADl] circumvented the restriction, showing that plasmid pCADl encodes a R-M system, which was des¬ ignated LlaDll It was only possible to transform E. coli XLl-Blue MRF' with plas¬ mid pCADl , when plasmid pCAD2 was used for transformation only deleted plasmids were obtained

Deposit. Laclococciis lactis subsp demons LM2301 transformed with plasmid pCADl comprising the LlaDll R-M system has been deposited at the Belgian Coordinated Collec¬ tions of Microorganisms (BCCM), Laboratorium voor Microbiologie - Bacterienverzame- ling (LMG), Universiteit Gent, Belgium, under the accession number LMG P- 16901

Determination of the recognition site. The restriction endonuclease, LlaDll, of the transformant L. lactis LM2301 [pCADl] was partially purified from cell extract The restriction patterns of λ DNA digested with the endonucleases from L. laclis LM2301 [pCADl] and L. lactis 39 26 were different from each other (data not shown) When pBluescript IISK+ and pSA3 were digested with the LlaDll and BsoFl restriction en¬ donucleases identical restriction pattern were obtained (Figure 4), showing that the

restriction endonucleases LlaDll and BsoFl expressed the same type II activity and are isoschizomers. BsoFl recognized and cleaved the sequence 5'-GCiNGC-3' (31).

Gene organization and DNA sequence. The Pstl-EcoRl fragment was subcloned as Pstl-Xhol and Λ7?σI-EcσRI fragments in pBluescript II SK+ resulting in plasmids pSADl and pSAD2, respectively. Deletions were made as described in Materials and Methods. The nucleotide sequence of the 2.4 kb fragment revealed 2 major open reading frames (ORFs) (SEQ ID No. 12). ORFl was putatively 540 bp with a coding potential for a protein of 180 amino acids (SEQ ID No. 13), and ORF2 was 951 bp capable of coding for a protein of 316 amino acids (SEQ ID No. 14). The two ORFs are separated by 108 bp and arranged tandemly with ORFl preceding ORF2 ORF2 was preceded by a putative Shine-Dalgarno sequence with good identity to consensus, 8 bp in front of the ATG start codon Plasmid pSAD2, harbouring only ORF2, was resistent to digestion by the ZVαDII endonuclease, showing that ORF2 had its own promotor and encodes a methyltransferase, which can be expressed in E. coli (data not shown) The results show that the ZVαDII R-M system consists of two consecu¬ tively transcribed genes, where ORF2 carry the gene for a methyltransferase, MZVαDII

Comparison of amino acid sequences. The deduced amino acid sequences of ORFl and ORF2 were compared with the amino acid sequences of other methylases in the databases. The first ten amino acids encoded by the putative ORFl may be doubtful as the sequencing first gave base no. 744 in SEQ ID No 12 as TT. However, from base no. 773 this reading frame gives a high homology with the endonuclease of the Bsp6l R-M system. The deduced amino acid sequence of ORF2 showed 60% identity and 76% similarity to the methylase from the Bspβl R-M system (31 ) and it contained several amino acid sequence motifs characteristic for C-5-cytosine methyltransferases (50). This also indicates that ORF2 codes for a C-5-cytosine methyltransferase.

Discussion.

The 8.9 kb naturally occurring plasmid pH 393 was isolated from L. laclis W39 Transformants L. lactis LM2301 [pHW393] and L. lactis LM2301 [pCADl] were both able to restrict phages and this restriction was circumvented by propagation of surviving phages on the respective transformants, showing that plasmid pHW393 and the 2.4 kbp Psll-EcόRl fragment in pCADl both code for a restriction modification system, and that both plasmids can be used to increase the level of phage defence in Laclococciis laclis. The 2.4 kbp stl-EcoRI fragment cloned in pCI3340 had in Lac- tococciis the ability to restrict lactococcal phages with one order of magnitude lower EOP than found for the wild-type plasmid, pHW393, indicating that the expression of the cloned ZVαDII R-M system may be depending of the plasmid copy number, or it may require some additional factors or that there are two R-M systems present on plasmid pHW393. Since plasmid pHW393 was found to express ZVαDI endonuclesase activity, and plasmid pCADl containing the 2.4 kb Psll-EcoRl fragment thereof ex¬ presses LlaDll activity, plasmid pHW393 must encode two type II R-M systems. This is the first time that two type II R-M systems have been found on the same plasmid.

The 2.4 kbp Pstl-EcoRl fragment harbours two tandemly arranged genes, LlaDϊlR and ZVαDIIM, which encode a restriction endonuclease and a C-5-cytosine methyl¬ transferase, respectively. The RLlaDll gene precedes the M LlaDll gene and they are separated by 108 bp. Since plasmid pSAD2 harbouring only the M LlaDll gene ex¬ pressed methylase activity in E. coli, these genes are most likely transcribed as two monocistronic mRNAs.

The endonuclease of the LlaDll R-M system is an isoschizomer of BsoFl recognizing the sequence 5'-GC4NGC-3', showing that the Z./αDII R-M system is a type II sys¬ tem. This is the first time a R-M system, which recognizes the sequence 5'-GC-!NGC- 3', has been identified and cloned in Lactococcus lactis.

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15 Hayes, F, Daly, C , and Fitzgerald, G F Identification of the minimal replicon of Laclococciis laclis subsp lactis UC317 plasmid pCI305 Appl Environ Microbi- ol 56 (1990) 202-209

16 Higgins, D L , Sanozky-Dawes, R B and Klaenhammer, T R Restriction and modification activities from Streptococcus laclis ME2 are encoded by a self- transmissible plasmid, pTN20, that forms cointegrates during mobilization of lac- tose-fermenting ability J Bacteriol 170 (1988) 3435-3442

Hill, C , Pierce, K and Klaenhammer, T R. The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modifi¬ cation (R7M ^* ) and abortive infection (Hsp ⁺ ) Appl Environ Microbiol 55 (1989) 2416-2419

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Josephsen, J and Vogensen, F.K Identification of three different plasmid enco¬ ded restriction/modification systems in Streptococcus laclis subsp cremo s W56 FEMS Microbiol Lett 59 (1989) 161-166

24 Josephsen, J and Klaenhammer, T R Stacking of three different restriction and modification systems in Lactococdis lactis by cotransformation Plasmid 23 (1990) 71-75

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Jytte Josephsen

(B) STREET: Magnoliavej 34

(C) CITY: Frederiksberg

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2000

(A) NAME: Niels Randel Nyengaard

(B) STREET: Jaegergaardsgade 3 st. th.

(C) CITY: Copenhagen N

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2200

(A) NAME: Finn Kvist Vogensen

(B) STREET: Nordre Fasanvej 28

(C) CITY: Frederiksberg

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2000

(A) NAME: Annette Madsen

(B) STREET: Thyrasgade 4, Apt. 605

(C) CITY: Copenhagen N

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2200

(ii) TITLE OF INVENTION: Plasmid-derived type II restriction-modification systems from Lactococcus lactis

(iii) NUMBER OF SEQUENCES: 14

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: DK 0179/95

(B) FILING DATE: 17-FEB-1995

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3695 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Lactococcus lactis subsp. cremoris

(B) STRAIN: W9

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 769..1620

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_start= 769

/product= "LlaAl -GATC- N-6-adenine methylase A"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 1

/standard_name= "Gene coding for M.LlaAIA"

/label= m-llaAIA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1613..2419

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_start= 1613

/product= "LlaAl -GATC- adenine methylase B"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 2

/standard_name= "Gene coding for M.LlaAIB"

/label= m-llaAIB

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:2412..3323

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION:/codon_start= 2412

/product= "LlaAl restriction endonuclease"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 3

/standard_name= "Gene coding for LlaAl restriction endonuclease"

/label= r-llaAI

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATATAAGATA TATAAATCAG TTCGCCTTTT TCTACTCCGT TCTAAAATCT TAAAATCAAG 60

GTCAAAAGAA AAAGTCAAAA CCATTGAATT GAGGTTCTAA AATTAAACTC CCTGCGTTGC 120

TCTTGGCTGC CGCTTGTACA CTCTGATTTT ATATTAGATA CATTCTGCCA TTAAAAAGAA 180

CTCCTAACGG TCGTGGCTAC TTTGTTTAGT CTAAACGCTT TAAATAGTCC TACAAGCTCA 240

TATTTTGCCT TTTAAGCGAT TTTAAACGTG AGTTAGTAAT AATTATCATG GATAAAAGAA 300

AAAGCCCTTA AATAGGCTTG TATGTAATTG ACTAAAACGT ACAATTTAGC TTTTAAATAT 360

GACCCTTATT TATGACCTGC TCTAACCTCA CTATTCATCA GCATTCAAAA AAGAGGTCAA 420

AACTGTTAAG TTATGAGCTG AATAGATTTT ATTAAATTTT ATTTGGTTTA AAAGACCAAT 480

TATCTATTTT TTAACAAACA CTAAAATAGA TTTTTTGGAA AACTTTGCAA CAGAACCAGC 540

AATCTGATGT TGCGAGATGG ACGTTCTTTC GGTTTTGAAC CTCAAGGGGA ACACTCGTTT 600

GATAAAGCGT CTCAATGGTT GTCAGTAAAC AAACAAAAAC TTTTGGAAGT GTGCTATTAT 660

AAGTCATATA AGTCGTGCGC TTTCTAATGC TTAGTGCTTT AAGATTAGGA TAGCACGACT 720

TATTTATTTT CCAATAAAAT TAACTAGCAA TTCGGGTATA ATATATTT ATG AAT TTA 777

Met Asn Leu 1

TTA CAA AAA AAC AAG ATC AAC TTA CGT CCG TTT ACT AAA TGG ACA GGT 825 Leu Gin Lys Asn Lys lie Asn Leu Arg Pro Phe Thr Lys Trp Thr Gly 5 10 15

GGG AAA AGG CAA CTA CTG CCA CAC ATT CAA TAC CTA ATG CCA GAA AAA 873 Gly Lys Arg Gin Leu Leu Pro His lie Gin Tyr Leu Met Pro Glu Lys 20 25 30 35

TAC AAT CAT TTT TTC GAA CCT TTT ATT GGT GGT GGC GCT TTG TTT TTT 921 Tyr Asn His Phe Phe Glu Pro Phe lie Gly Gly Gly Ala Leu Phe Phe 40 45 50

GAA CTC GCT CCT CAA AAA GCA GTT ATT AAC GAC TTC AAT TCT GAG CTT 969 Glu Leu Ala Pro Gin Lys Ala Val lie Asn Asp Phe Asn Ser Glu Leu 55 60 65

ATA AAC TGT TAC CGG CAG ATG AAA GAT AAT CCT GAG CAA TTG ATA GAA 1017 lie Asn Cys Tyr Arg Gin Met Lys Asp Asn Pro Glu Gin Leu lie Glu 70 75 80

TTG TTG ACT AAT CAT CAG CGG GAA AAT TCT AAA GAA TAT TAT TTA GAC 1065 Leu Leu Thr Asn His Gin Arg Glu Asn Ser Lys Glu Tyr Tyr Leu Asp 85 90 95

TTA CGT TCT TCT GAT AGA GAT GGA AGA ATT GAT AAG ATG AGC GAA GTT 1113 Leu Arg Ser Ser Asp Arg Asp Gly Arg lie Asp Lys Met Ser Glu Val 100 105 110 115

GAA CGT GCT GCT AGA ATT ATG TAT ATG CTA CGT GTT GAT TTT AAT GGT 1161 Glu Arg Ala Ala Arg lie Met Tyr Met Leu Arg Val Asp Phe Asn Gly 120 125 130

TTA TAT CGT GTT AAT TCG AAA AAC CAG TTT AAT GTG CCT TAT GGA AGA 1209 Leu Tyr Arg Val Asn Ser Lys Asn Gin Phe Asn Val Pro Tyr Gly Arg 135 140 145

TAT AAA AAT CCT AAG ATA GTT GAT AAA GAA TTG ATT GAA AGT ATT TCC 1257 Tyr Lys Asn Pro Lys lie Val Asp Lys Glu Leu lie Glu Ser lie Ser 150 155 160

GAG TAC TTG AAT AAC AAT TCT ATT AAG ATC ATG AGT GGA GAT TTT GAA 1305 Glu Tyr Leu Asn Asn Asn Ser lie Lys lie Met Ser Gly Asp Phe Glu 165 170 175

AAA GCC GTT AAA GAA GCA CAG GAT GGA GAT TTT GTT TAT TTC GAC CCT 1353 Lys Ala Val Lys Glu Ala Gin Asp Gly Asp Phe Val Tyr Phe Asp Pro 180 185 190 195

CCA TAC ATT CCA CTT TCT GAA ACT AGC GCC TTT ACT TCT TAT ACA CAC 1401 Pro Tyr lie Pro Leu Ser Glu Thr Ser Ala Phe Thr Ser Tyr Thr His 200 205 210

GAA GGC TTT AGC TAC GAA GAT CAA GTT AGG CTA AGA GAT TGT TTC AAA 1449 Glu Gly Phe Ser Tyr Glu Asp Gin Val Arg Leu Arg Asp Cys Phe Lys 215 220 225

CAG TTA GAT TCA AAA GGG GTA TTC GTC ATG CTT TCA AAT TCT TCA AGC 1497 Gin Leu Asp Ser Lys Gly Val Phe Val Met Leu Ser Asn Ser Ser Ser 230 235 240

CCT TTA GCG GAG GAA TTA TAT AAA GAT TTT TAC ATC CAT AAA ATT GAA 15 5 Pro Leu Ala Glu Glu Leu Tyr Lys Asp Phe Tyr lie His Lys lie Glu 245 250 255

GCT ACT CGA ACA AAT GGG GCT AAA TCA TCT AGT CGT GGA AAA ATC ACT 1593 Ala Thr Arg Thr Asn Gly Ala Lys Ser Ser Ser Arg Gly Lys lie Thr 260 265 270 275

GAA ATC ATC GTA ACC AAT TAT GGC AAT TAACGAATAT AAGTATGGAG 1640 Glu He He Val Thr Asn Tyr Gly Asn 280

GTGTTTTAAT GATAAAACCA TACTATGAAA AAGAAAACGC AATTCTCGTT CACGCAGATT 1700

CATTTAAATT ATTAGAAAAA ATTAAACCTG AAAGCATGGA CATGATATTT GCTGACCCTC 1760

CTTACTTTTT AAGTAATGGA GGAATGTCAA ATTCAGGTGG TCAAATTGTT TCTGTTGATA 1820

AAGGGGATTG GGATAAAATT TCTTCATTTG AAGAAAAACA TGACTTTAAT AGACGTTGGA 1880

TTAGGTTAGC AAGATTGGTT TTAAAACCCA ACGGAACTAT TTGGGTTTCC GGAAGCCTTC 1940

ATAACATATA TTCTGTCGGG ATGGCGCTGG AACAGGAAGG TTTCAAAATC TTAAATAATA 2000

TAACTTGGCA AAAGACAAAT CCTGCACCTA ATCTATCATG TCGGTACTTC ACCCACTCTA 2060

CAGAGACAAT TTTATGGGCA AGAAAGAACG ATAAAAAATC TCGCCATTAT TATAACTATG 2120

AATTGATGAA AGAGTTTAAT GACGGGAAAC AAATGAAAGA TGTTTGGACA GGTAGTCTGA 2180

CAAAAAAATC AGAAAAATGG GCTGGGAAAC ATCCAACTCA GAAGCCAGAG TATATTTTAG 2240

AACGGATAAT CTTAGCTAGT ACAAAGGAAA ATGATTATAT TTTAGACCCT TTCGTCGGAA 2300

GTGGAACTAC TGGTGTAGTA GCCAAGAGAT TGGGGCGTAA ATTTATTGGG ATTGATTCTG 2360

AGAAAGAATA TCTTAAAATT GCTAAAAAAA GGCTAAATAA AGGAGCAACA TATGGACTTT 2420

AATAATTACA TCGGTTTAGA ATCTGACGAT AGATTAAATG CTTTTATGGC AACACTTTCC 2480

GTAACTAATA GAACTCCCGA ATACTACGTG AACTGGGAAA AAGTTGAACG TGAAACACGA 2540

AAATTTGAAT TAGAACTAAA TACTTTAAAC TATCTCATTG GGAAAGAAGA TATTTATAGT 2600

GAAGCACTTG AACTATTTAC CAATCAACCT GAATTGCTTA AAGCTATTCC TAGTTTGATT 2660

GCTAGTAGAG ATACATCTTT AGATATACTA AACATTGACG AAAATGATGA TATGAGTTTT 2720

GAACAACTTA ACTTTCTTGT TATCGACGAA AATTGTATCG CTGATTATGT AGACTTTATT 2780

AACCAGGCAG GTTTACTAGA TTTTCTACAG AATAAAGCAA AACGTTCTCT GGTAGACTAT 2840

GTGTATGGTG TTGAAGCAGG GCTTGATAGC AATGCTCGAA AAAACCGAAG CGGTACAACC 2900

ATGGAGGGGA TTTTAGAACG TACTGTTTCA AAAATAGCTC AAGAGAAAGG GCTTGAATGG 2960

AAGCCACAGG CAACCGCTTC TTTTATCAAG TCTCAATGGG ACATAGAAGT CCCTGTAGAC 3020

AAATCAAAAA GACGCTTTGA TGCAGCAGTT TACTCTCGTG CGCTCAATAA GGTTTGGCTC 3080

ATAGAAACAA ATTACTACGG CGGTGGAGGA AGTAAACTCA AAGCAGTTGC TGGAGAATTT 3140

ACAGAATTGA GTCAGTTTGT AAAAACATCA AAAGATAATG TTGAATTTGT ATGGGTAACA 3200

GACGGCCAAG GGTGGAAATT TTCCCGCTTA CCACTTGCAG AAGCTTTCGG ACACATCGAT 3260

AACGTTTTCA ATCTAACCAT GTTGAAAGAA GGTTTCTTGT CTGATTTATT CGAAAAAGAA 3320

ATTTAAAAAG ACAGAGAATC TCTGTCTTTT TAAATTTCAA TTCCTTCCTT CTGCTAGCTA 3380

TAACTTTCCA AAAAACCTGA AAAACGGTTC TGTTGCAATT GTATGTGGGG TCGGAACTTA 3440

CTACTATATC ATGAGAAATG AAGATTAAAG TTGAAACAAA AAAACAGATT ATTTTAAAAT 3500

GTAAATCTGT TTTTGTTTGG GCTGATTTTA TCACACCAAT TCTATGTTCA GAAAATGGTC 3560

ATTTTCTGGA CACTCTTCTT TTGTTATTAA AACTCTCAAA ATCATTTACA TTTATTGTTC 3620

ATTAACCCAT AATTTATTCT ATGTTCATTT ATAGATATCG AATTCCTGCA GGGCCCTCCA 3680

CTAGTTCTAG AGGCG 3695

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 284 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Asn Leu Leu Gin Lys Asn Lys He Asn Leu Arg Pro Phe Thr Lys 1 5 10 15

Trp Thr Gly Gly Lys Arg Gin Leu Leu Pro His He Gin Tyr Leu Met 20 25 30

Pro Glu Lys Tyr Asn His Phe Phe Glu Pro Phe He Gly Gly Gly Ala 35 40 45

Leu Phe Phe Glu Leu Ala Pro Gin Lys Ala Val He Asn Asp Phe Asn 50 55 60

Ser Glu Leu He Asn Cys Tyr Arg Gin Met Lys Asp Asn Pro Glu Gin 65 70 75 80

Leu He Glu Leu Leu Thr Asn His Gin Arg Glu Asn Ser Lys Glu Tyr 85 90 95

Tyr Leu Asp Leu Arg Ser Ser Asp Arg Asp Gly Arg He Asp Lys Met 100 105 110

Ser Glu Val Glu Arg Ala Ala Arg He Met Tyr Met Leu Arg Val Asp 115 120 125

Phe Asn Gly Leu Tyr Arg Val Asn Ser Lys Asn Gin Phe Asn Val Pro 130 135 140

Tyr Gly Arg Tyr Lys Asn Pro Lys He Val Asp Lys Glu Leu He Glu 145 150 155 160

Ser He Ser Glu Tyr Leu Asn Asn Asn Ser He Lys He Met Ser Gly 165 170 175

Asp Phe Glu Lys Ala Val Lys Glu Ala Gin Asp Gly Asp Phe Val Tyr 180 185 190

Phe Asp Pro Pro Tyr He Pro Leu Ser Glu Thr Ser Ala Phe Thr Ser 195 200 205

Tyr Thr His Glu Gly Phe Ser Tyr Glu Asp Gin Val Arg Leu Arg Asp 210 215 220

Cys Phe Lys Gin Leu Asp Ser Lys Gly Val Phe Val Met Leu Ser Asn 225 230 235 240

Ser Ser Ser Pro Leu Ala Glu Glu Leu Tyr Lys Asp Phe Tyr He His 245 250 255

Lys He Glu Ala Thr Arg Thr Asn Gly Ala Lys Ser Ser Ser Arg Gly 260 265 270

Lys He Thr Glu He He Val Thr Asn Tyr Gly Asn 275 280

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3695 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Lactococcus lactis subsp. cremoris

(B) STRAIN: W9

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1613..2419

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_start= 1613

/product= "LlaAl -GATC- adenine methylase B"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 2

/standard_name= "Gene coding for M.LlaAIB"

/label= m-llaAIB

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ATATAAGATA TATAAATCAG TTCGCCTTTT TCTACTCCGT TCTAAAATCT TAAAATCAAG 60

GTCAAAAGAA AAAGTCAAAA CCATTGAATT GAGGTTCTAA AATTAAACTC CCTGCGTTGC 120

TCTTGGCTGC CGCTTGTACA CTCTGATTTT ATATTAGATA CATTCTGCCA TTAAAAAGAA 180

CTCCTAACGG TCGTGGCTAC TTTGTTTAGT CTAAACGCTT TAAATAGTCC TACAAGCTCA 240

TATTTTGCCT TTTAAGCGAT TTTAAACGTG AGTTAGTAAT AATTATCATG GATAAAAGAA 300

AAAGCCCTTA AATAGGCTTG TATGTAATTG ACTAAAACGT ACAATTTAGC TTTTAAATAT 360

GACCCTTATT TATGACCTGC TCTAACCTCA CTATTCATCA GCATTCAAAA AAGAGGTCAA 420

AACTGTTAAG TTATGAGCTG AATAGATTTT ATTAAATTTT ATTTGGTTTA AAAGACCAAT 480

TATCTATTTT TTAACAAACA CTAAAATAGA TTTTTTGGAA AACTTTGCAA CAGAACCAGC 540

AATCTGATGT TGCGAGATGG ACGTTCTTTC GGTTTTGAAC CTCAAGGGGA ACACTCGTTT 600

GATAAAGCGT CTCAATGGTT GTCAGTAAAC AAACAAAAAC TTTTGGAAGT GTGCTATTAT 660

AAGTCATATA AGTCGTGCGC TTTCTAATGC TTAGTGCTTT AAGATTAGGA TAGCACGACT 720

TATTTATTTT CCAATAAAAT TAACTAGCAA TTCGGGTATA ATATATTTAT GAATTTATTA 780

CAAAAAAACA AGATCAACTT ACGTCCGTTT ACTAAATGGA CAGGTGGGAA AAGGCAACTA 840

CTGCCACACA TTCAATACCT AATGCCAGAA AAATACAATC ATTTTTTCGA ACCTTTTATT 900

GGTGGTGGCG CTTTGTTTTT TGAACTCGCT CCTCAAAAAG CAGTTATTAA CGACTTCAAT 960

TCTGAGCTTA TAAACTGTTA CCGGCAGATG AAAGATAATC CTGAGCAATT GATAGAATTG 1020

TTGACTAATC ATCAGCGGGA AAATTCTAAA GAATATTATT TAGACTTACG TTCTTCTGAT 1080

AGAGATGGAA GAATTGATAA GATGAGCGAA GTTGAACGTG CTGCTAGAAT TATGTATATG 1140

CTACGTGTTG ATTTTAATGG TTTATATCGT GTTAATTCGA AAAACCAGTT TAATGTGCCT 1200

TATGGAAGAT ATAAAAATCC TAAGATAGTT GATAAAGAAT TGATTGAAAG TATTTCCGAG 1260

TACTTGAATA ACAATTCTAT TAAGATCATG AGTGGAGATT TTGAAAAAGC CGTTAAAGAA 1320

GCACAGGATG GAGATTTTGT TTATTTCGAC CCTCCATACA TTCCACTTTC TGAAACTAGC 1380

GCCTTTACTT CTTATACACA CGAAGGCTTT AGCTACGAAG ATCAAGTTAG GCTAAGAGAT 1440

TGTTTCAAAC AGTTAGATTC AAAAGGGGTA TTCGTCATGC TTTCAAATTC TTCAAGCCCT 1500

TTAGCGGAGG AATTATATAA AGATTTTTAC ATCCATAAAA TTGAAGCTAC TCGAACAAAT 1560

GGGGCTAAAT CATCTAGTCG TGGAAAAATC ACTGAAATCA TCGTAACCAA TT ATG 1615

Met 285

GCA ATT AAC GAA TAT AAG TAT GGA GGT GTT TTA ATG ATA AAA CCA TAC 1663 Ala He Asn Glu Tyr Lys Tyr Gly Gly Val Leu Met He Lys Pro Tyr 290 295 300

TAT GAA AAA GAA AAC GCA ATT CTC GTT CAC GCA GAT TCA TTT AAA TTA 1711 Tyr Glu Lys Glu Asn Ala He Leu Val His Ala Asp Ser Phe Lys Leu 305 310 315

TTA GAA AAA ATT AAA CCT GAA AGC ATG GAC ATG ATA TTT GCT GAC CCT 1759 Leu Glu Lys He Lys Pro Glu Ser Met Asp Met He Phe Ala Asp Pro 320 325 330

CCT TAC TTT TTA AGT AAT GGA GGA ATG TCA AAT TCA GGT GGT CAA ATT 1807 Pro Tyr Phe Leu Ser Asn Gly Gly Met Ser Asn Ser Gly Gly Gin He 335 340 345

GTT TCT GTT GAT AAA GGG GAT TGG GAT AAA ATT TCT TCA TTT GAA GAA 1855 Val Ser Val Asp Lys Gly Asp Trp Asp Lys He Ser Ser Phe Glu Glu 350 355 360 365

AAA CAT GAC TTT AAT AGA CGT TGG ATT AGG TTA GCA AGA TTG GTT TTA 1903 Lys His Asp Phe Asn Arg Arg Trp He Arg Leu Ala Arg Leu Val Leu 370 375 380

AAA CCC AAC GGA ACT ATT TGG GTT TCC GGA AGC CTT CAT AAC ATA TAT 1951 Lys Pro Asn Gly Thr He Trp Val Ser Gly Ser Leu His Asn He Tyr 385 390 395

TCT GTC GGG ATG GCG CTG GAA CAG GAA GGT TTC AAA ATC TTA AAT AAT 1999 Ser Val Gly Met Ala Leu Glu Gin Glu Gly Phe Lys He Leu Asn Asn 400 405 410

ATA ACT TGG CAA AAG ACA AAT CCT GCA CCT AAT CTA TCA TGT CGG TAC 2047 He Thr Trp Gin Lys Thr Asn Pro Ala Pro Asn Leu Ser Cys Arg Tyr 415 420 425

TTC ACC CAC TCT ACA GAG ACA ATT TTA TGG GCA AGA AAG AAC GAT AAA 2095 Phe Thr His Ser Thr Glu Thr He Leu Trp Ala Arg Lys Asn Asp Lys 430 435 440 445

AAA TCT CGC CAT TAT TAT AAC TAT GAA TTG ATG AAA GAG TTT AAT GAC 2143 Lys Ser Arg His Tyr Tyr Asn Tyr Glu Leu Met Lys Glu Phe Asn Asp 450 455 460

GGG AAA CAA ATG AAA GAT GTT TGG ACA GGT AGT CTG ACA AAA AAA TCA 2191 Gly Lys Gin Met Lys Asp Val Trp Thr Gly Ser Leu Thr Lys Lys Ser 465 470 475

GAA AAA TGG GCT GGG AAA CAT CCA ACT CAG AAG CCA GAG TAT ATT TTA 2239 Glu Lys Trp Ala Gly Lys His Pro Thr Gin Lys Pro Glu Tyr He Leu 480 485 490

GAA CGG ATA ATC TTA GCT AGT ACA AAG GAA AAT GAT TAT ATT TTA GAC 2287 Glu Arg He He Leu Ala Ser Thr Lys Glu Asn Asp Tyr He Leu Asp 495 500 505

CCT TTC GTC GGA AGT GGA ACT ACT GGT GTA GTA GCC AAG AGA TTG GGG 2335 Pro Phe Val Gly Ser Gly Thr Thr Gly Val Val Ala Lys Arg Leu Gly 510 515 520 525

CGT AAA TTT ATT GGG ATT GAT TCT GAG AAA GAA TAT CTT AAA ATT GCT 2383 Arg Lys Phe He Gly He Asp Ser Glu Lys Glu Tyr Leu Lys He Ala 530 535 540

AAA AAA AGG CTA AAT AAA GGA GCA ACA TAT GGA CTT TAATAATTAC 2429

Lys Lys Arg Leu Asn Lys Gly Ala Thr Tyr Gly Leu

545 550

ATCGGTTTAG AATCTGACGA TAGATTAAAT GCTTTTATGG CAACACTTTC CGTAACTAAT 2489

AGAACTCCCG AATACTACGT GAACTGGGAA AAAGTTGAAC GTGAAACACG AAAATTTGAA 2549

TTAGAACTAA ATACTTTAAA CTATCTCATT GGGAAAGAAG ATATTTATAG TGAAGCACTT 2609

GAACTATTTA CCAATCAACC TGAATTGCTT AAAGCTATTC CTAGTTTGAT TGCTAGTAGA 2669

GATACATCTT TAGATATACT AAACATTGAC GAAAATGATG ATATGAGTTT TGAACAACTT 2729

AACTTTCTTG TTATCGACGA AAATTGTATC GCTGATTATG TAGACTTTAT TAACCAGGCA 2789

GGTTTACTAG ATTTTCTACA GAATAAAGCA AAACGTTCTC TGGTAGACTA TGTGTATGGT 2849

GTTGAAGCAG GGCTTGATAG CAATGCTCGA AAAAACCGAA GCGGTACAAC CATGGAGGGG 2909

ATTTTAGAAC GTACTGTTTC AAAAATAGCT CAAGAGAAAG GGCTTGAATG GAAGCCACAG 2969

GCAACCGCTT CTTTTATCAA GTCTCAATGG GACATAGAAG TCCCTGTAGA CAAATCAAAA 3029

AGACGCTTTG ATGCAGCAGT TTACTCTCGT GCGCTCAATA AGGTTTGGCT CATAGAAACA 3089

AATTACTACG GCGGTGGAGG AAGTAAACTC AAAGCAGTTG CTGGAGAATT TACAGAATTG 3149

AGTCAGTTTG TAAAAACATC AAAAGATAAT GTTGAATTTG TATGGGTAAC AGACGGCCAA 3209

GGGTGGAAAT TTTCCCGCTT ACCACTTGCA GAAGCTTTCG GACACATCGA TAACGTTTTC 3269

AATCTAACCA TGTTGAAAGA AGGTTTCTTG TCTGATTTAT TCGAAAAAGA AATTTAAAAA 3329

GACAGAGAAT CTCTGTCTTT TTAAATTTCA ATTCCTTCCT TCTGCTAGCT ATAACTTTCC 3389

AAAAAACCTG AAAAACGGTT CTGTTGCAAT TGTATGTGGG GTCGGAACTT ACTACTATAT 3449

CATGAGAAAT GAAGATTAAA GTTGAAACAA AAAAACAGAT TATTTTAAAA TGTAAATCTG 3509

TTTTTGTTTG GGCTGATTTT ATCACACCAA TTCTATGTTC AGAAAATGGT CATTTTCTGG 3569

ACACTCTTCT TTTGTTATTA AAACTCTCAA AATCATTTAC ATTTATTGTT CATTAACCCA 3629

TAATTTATTC TATGTTCATT TATAGATATC GAATTCCTGC AGGGCCCTCC ACTAGTTCTA 3689

GAGGCG 3695

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 269 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala He Asn Glu Tyr Lys Tyr Gly Gly Val Leu Met He Lys Pro 1 5 10 15

Tyr Tyr Glu Lys Glu Asn Ala He Leu Val His Ala Asp Ser Phe Lys 20 25 30

Leu Leu Glu Lys He Lys Pro Glu Ser Met Asp Met He Phe Ala Asp 35 40 45

Pro Pro Tyr Phe Leu Ser Asn Gly Gly Met Ser Asn Ser Gly Gly Gin 50 55 60

He Val Ser Val Asp Lys Gly Asp Trp Asp Lys He Ser Ser Phe Glu 65 70 75 80

Glu Lys His Asp Phe Asn Arg Arg Trp He Arg Leu Ala Arg Leu Val 85 90 95

Leu Lys Pro Asn Gly Thr He Trp Val Ser Gly Ser Leu His Asn He 100 105 110

Tyr Ser Val Gly Met Ala Leu Glu Gin Glu Gly Phe Lys He Leu Asn 115 120 125

Asn He Thr Trp Gin Lys Thr Asn Pro Ala Pro Asn Leu Ser Cys Arg 130 135 140

Tyr Phe Thr His Ser Thr Glu Thr He Leu Trp Ala Arg Lys Asn Asp 145 150 155 160

Lys Lys Ser Arg His Tyr Tyr Asn Tyr Glu Leu Met Lys Glu Phe Asn 165 170 175

Asp Gly Lys Gin Met Lys Asp Val Trp Thr Gly Ser Leu Thr Lys Lys 180 185 190

Ser Glu Lys Trp Ala Gly Lys His Pro Thr Gin Lys Pro Glu Tyr He 195 200 205

Leu Glu Arg He He Leu Ala Ser Thr Lys Glu Asn Asp Tyr He Leu 210 215 220

Asp Pro Phe Val Gly Ser Gly Thr Thr Gly Val Val Ala Lys Arg Leu 225 230 235 240

Gly Arg Lys Phe He Gly He Asp Ser Glu Lys Glu Tyr Leu Lys He 245 250 255

Ala Lys Lys Arg Leu Asn Lys Gly Ala Thr Tyr Gly Leu 260 265

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3695 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Lactococcus lactis subsp. cremoris

(B) STRAIN: W9

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1649..2419

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION:/codon_start= 1649

/product= "LlaAl -GATC-adenine methylase B"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 2

/standard_name= "Gene coding for M.LlaAIB"

/label= m-llaAIB

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATATAAGATA TATAAATCAG TTCGCCTTTT TCTACTCCGT TCTAAAATCT TAAAATCAAG 60

GTCAAAAGAA AAAGTCAAAA CCATTGAATT GAGGTTCTAA AATTAAACTC CCTGCGTTGC 120

TCTTGGCTGC CGCTTGTACA CTCTGATTTT ATATTAGATA CATTCTGCCA TTAAAAAGAA 180

CTCCTAACGG TCGTGGCTAC TTTGTTTAGT CTAAACGCTT TAAATAGTCC TACAAGCTCA 240

TATTTTGCCT TTTAAGCGAT TTTAAACGTG AGTTAGTAAT AATTATCATG GATAAAAGAA 300

AAAGCCCTTA AATAGGCTTG TATGTAATTG ACTAAAACGT ACAATTTAGC TTTTAAATAT 360

GACCCTTATT TATGACCTGC TCTAACCTCA CTATTCATCA GCATTCAAAA AAGAGGTCAA 420

AACTGTTAAG TTATGAGCTG AATAGATTTT ATTAAATTTT ATTTGGTTTA AAAGACCAAT 480

TATCTATTTT TTAACAAACA CTAAAATAGA TTTTTTGGAA AACTTTGCAA CAGAACCAGC 540

AATCTGATGT TGCGAGATGG ACGTTCTTTC GGTTTTGAAC CTCAAGGGGA ACACTCGTTT 600

GATAAAGCGT CTCAATGGTT GTCAGTAAAC AAACAAAAAC TTTTGGAAGT GTGCTATTAT 660

AAGTCATATA AGTCGTGCGC TTTCTAATGC TTAGTGCTTT AAGATTAGGA TAGCACGACT 720

TATTTATTTT CCAATAAAAT TAACTAGCAA TTCGGGTATA ATATATTTAT GAATTTATTA 780

CAAAAAAACA AGATCAACTT ACGTCCGTTT ACTAAATGGA CAGGTGGGAA AAGGCAACTA 840

CTGCCACACA TTCAATACCT AATGCCAGAA AAATACAATC ATTTTTTCGA ACCTTTTATT 900

GGTGGTGGCG CTTTGTTTTT TGAACTCGCT CCTCAAAAAG CAGTTATTAA CGACTTCAAT 960

TCTGAGCTTA TAAACTGTTA CCGGCAGATG AAAGATAATC CTGAGCAATT GATAGAATTG 1020

TTGACTAATC ATCAGCGGGA AAATTCTAAA GAATATTATT TAGACTTACG TTCTTCTGAT 1080

AGAGATGGAA GAATTGATAA GATGAGCGAA GTTGAACGTG CTGCTAGAAT TATGTATATG 1140

CTACGTGTTG ATTTTAATGG TTTATATCGT GTTAATTCGA AAAACCAGTT TAATGTGCCT 1200

TATGGAAGAT ATAAAAATCC TAAGATAGTT GATAAAGAAT TGATTGAAAG TATTTCCGAG 1260

TACTTGAATA ACAATTCTAT TAAGATCATG AGTGGAGATT TTGAAAAAGC CGTTAAAGAA 1320

GCACAGGATG GAGATTTTGT TTATTTCGAC CCTCCATACA TTCCACTTTC TGAAACTAGC 1380

GCCTTTACTT CTTATACACA CGAAGGCTTT AGCTACGAAG ATCAAGTTAG GCTAAGAGAT 1440

TGTTTCAAAC AGTTAGATTC AAAAGGGGTA TTCGTCATGC TTTCAAATTC TTCAAGCCCT 1500

TTAGCGGAGG AATTATATAA AGATTTTTAC ATCCATAAAA TTGAAGCTAC TCGAACAAAT 1560

GGGGCTAAAT CATCTAGTCG TGGAAAAATC ACTGAAATCA TCGTAACCAA TTATGGCAAT 1620

TAACGAATAT AAGTATGGAG GTGTTTTA ATG ATA AAA CCA TAC TAT GAA AAA 1672

Met He Lys Pro Tyr Tyr Glu Lys 270 275

GAA AAC GCA ATT CTC GTT CAC GCA GAT TCA TTT AAA TTA TTA GAA AAA 1720 Glu Asn Ala He Leu Val His Ala Asp Ser Phe Lys Leu Leu Glu Lys 280 285 290

ATT AAA CCT GAA AGC ATG GAC ATG ATA TTT GCT GAC CCT CCT TAC TTT 1768 He Lys Pro Glu Ser Met Asp Met He Phe Ala Asp Pro Pro Tyr Phe 295 300 305

TTA AGT AAT GGA GGA ATG TCA AAT TCA GGT GGT CAA ATT GTT TCT GTT 1816 Leu Ser Asn Gly Gly Met Ser Asn Ser Gly Gly Gin He Val Ser Val 310 315 320 325

GAT AAA GGG GAT TGG GAT AAA ATT TCT TCA TTT GAA GAA AAA CAT GAC 1864 Asp Lys Gly Asp Trp Asp Lys He Ser Ser Phe Glu Glu Lys His Asp 330 335 340

TTT AAT AGA CGT TGG ATT AGG TTA GCA AGA TTG GTT TTA AAA CCC AAC 1912 Phe Asn Arg Arg Trp He Arg Leu Ala Arg Leu Val Leu Lys Pro Asn 345 350 355

GGA ACT ATT TGG GTT TCC GGA AGC CTT CAT AAC ATA TAT TCT GTC GGG 1960 Gly Thr He Trp Val Ser Gly Ser Leu His Asn He Tyr Ser Val Gly 360 365 370

ATG GCG CTG GAA CAG GAA GGT TTC AAA ATC TTA AAT AAT ATA ACT TGG 2008 Met Ala Leu Glu Gin Glu Gly Phe Lys He Leu Asn Asn He Thr Trp 375 380 385

CAA AAG ACA AAT CCT GCA CCT AAT CTA TCA TGT CGG TAC TTC ACC CAC 2056 Gin Lys Thr Asn Pro Ala Pro Asn Leu Ser Cys Arg Tyr Phe Thr His 390 395 400 405

TCT ACA GAG ACA ATT TTA TGG GCA AGA AAG AAC GAT AAA AAA TCT CGC 2104 Ser Thr Glu Thr He Leu Trp Ala Arg Lys Asn Asp Lys Lys Ser Arg 410 415 420

CAT TAT TAT AAC TAT GAA TTG ATG AAA GAG TTT AAT GAC GGG AAA CAA 2152 His Tyr Tyr Asn Tyr Glu Leu Met Lys Glu Phe Asn Asp Gly Lys Gin 425 430 435

ATG AAA GAT GTT TGG ACA GGT AGT CTG ACA AAA AAA TCA GAA AAA TGG 2200 Met Lys Asp Val Trp Thr Gly Ser Leu Thr Lys Lys Ser Glu Lys Trp 440 445 450

GCT GGG AAA CAT CCA ACT CAG AAG CCA GAG TAT ATT TTA GAA CGG ATA 2248 Ala Gly Lys His Pro Thr Gin Lys Pro Glu Tyr He Leu Glu Arg He 455 460 465

ATC TTA GCT AGT ACA AAG GAA AAT GAT TAT ATT TTA GAC CCT TTC GTC 2296 He Leu Ala Ser Thr Lys Glu Asn Asp Tyr He Leu Asp Pro Phe Val 470 475 480 485

GGA AGT GGA ACT ACT GGT GTA GTA GCC AAG AGA TTG GGG CGT AAA TTT 2344 Gly Ser Gly Thr Thr Gly Val Val Ala Lys Arg Leu Gly Arg Lys Phe 490 495 500

ATT GGG ATT GAT TCT GAG AAA GAA TAT CTT AAA ATT GCT AAA AAA AGG 2392 He Gly He Asp Ser Glu Lys Glu Tyr Leu Lys He Ala Lys Lys Arg 505 510 515

CTA AAT AAA GGA GCA ACA TAT GGA CTT TAATAATTAC ATCGGTTTAG 2439 Leu Asn Lys Gly Ala Thr Tyr Gly Leu 520 525

AATCTGACGA TAGATTAAAT GCTTTTATGG CAACACTTTC CGTAACTAAT AGAACTCCCG 2499

AATACTACGT GAACTGGGAA AAAGTTGAAC GTGAAACACG AAAATTTGAA TTAGAACTAA 2559

ATACTTTAAA CTATCTCATT GGGAAAGAAG ATATTTATAG TGAAGCACTT GAACTATTTA 2619

CCAATCAACC TGAATTGCTT AAAGCTATTC CTAGTTTGAT TGCTAGTAGA GATACATCTT 2679

TAGATATACT AAACATTGAC GAAAATGATG ATATGAGTTT TGAACAACTT AACTTTCTTG 2739

TTATCGACGA AAATTGTATC GCTGATTATG TAGACTTTAT TAACCAGGCA GGTTTACTAG 2799

ATTTTCTACA GAATAAAGCA AAACGTTCTC TGGTAGACTA TGTGTATGGT GTTGAAGCAG 2859

GGCTTGATAG CAATGCTCGA AAAAACCGAA GCGGTACAAC CATGGAGGGG ATTTTAGAAC 2919

GTACTGTTTC AAAAATAGCT CAAGAGAAAG GGCTTGAATG GAAGCCACAG GCAACCGCTT 2979

CTTTTATCAA GTCTCAATGG GACATAGAAG TCCCTGTAGA CAAATCAAAA AGACGCTTTG 3039

ATGCAGCAGT TTACTCTCGT GCGCTCAATA AGGTTTGGCT CATAGAAACA AATTACTACG 3099

GCGGTGGAGG AAGTAAACTC AAAGCAGTTG CTGGAGAATT TACAGAATTG AGTCAGTTTG 3159

TAAAAACATC AAAAGATAAT GTTGAATTTG TATGGGTAAC AGACGGCCAA GGGTGGAAAT 3219

TTTCCCGCTT ACCACTTGCA GAAGCTTTCG GACACATCGA TAACGTTTTC AATCTAACCA 3279

TGTTGAAAGA AGGTTTCTTG TCTGATTTAT TCGAAAAAGA AATTTAAAAA GACAGAGAAT 3339

CTCTGTCTTT TTAAATTTCA ATTCCTTCCT TCTGCTAGCT ATAACTTTCC AAAAAACCTG 3399

AAAAACGGTT CTGTTGCAAT TGTATGTGGG GTCGGAACTT ACTACTATAT CATGAGAAAT 3459

GAAGATTAAA GTTGAAACAA AAAAACAGAT TATTTTAAAA TGTAAATCTG TTTTTGTTTG 3519

GGCTGATTTT ATCACACCAA TTCTATGTTC AGAAAATGGT CATTTTCTGG ACACTCTTCT 3579

TTTGTTATTA AAACTCTCAA AATCATTTAC ATTTATTGTT CATTAACCCA TAATTTATTC 3639

TATGTTCATT TATAGATATC GAATTCCTGC AGGGCCCTCC ACTAGTTCTA GAGGCG 3695

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 257 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met He Lys Pro Tyr Tyr Glu Lys Glu Asn Ala He Leu Val His Ala 1 5 10 15

Asp Ser Phe Lys Leu Leu Glu Lys He Lys Pro Glu Ser Met Asp Met 20 25 30

He Phe Ala Asp Pro Pro Tyr Phe Leu Ser Asn Gly Gly Met Ser Asn 35 40 45

Ser Gly Gly Gin He Val Ser Val Asp Lys Gly Asp Trp Asp Lys He 50 55 60

Ser Ser Phe Glu Glu Lys His Asp Phe Asn Arg Arg Trp He Arg Leu 65 70 75 80

Ala Arg Leu Val Leu Lys Pro Asn Gly Thr He Trp Val Ser Gly Ser 85 90 95

Leu His Asn He Tyr Ser Val Gly Met Ala Leu Glu Gin Glu Gly Phe 100 105 110

Lys He Leu Asn Asn He Thr Trp Gin Lys Thr Asn Pro Ala Pro Asn 115 120 125

Leu Ser Cys Arg Tyr Phe Thr His Ser Thr Glu Thr He Leu Trp Ala 130 135 140

Arg Lys Asn Asp Lys Lys Ser Arg His Tyr Tyr Asn Tyr Glu Leu Met 145 150 155 160

Lys Glu Phe Asn Asp Gly Lys Gin Met Lys Asp Val Trp Thr Gly Ser 165 170 175

Leu Thr Lys Lys Ser Glu Lys Trp Ala Gly Lys His Pro Thr Gin Lys 180 185 190

Pro Glu Tyr He Leu Glu Arg He He Leu Ala Ser Thr Lys Glu Asn 195 200 205

Asp Tyr He Leu Asp Pro Phe Val Gly Ser Gly Thr Thr Gly Val Val 210 215 220

Ala Lys Arg Leu Gly Arg Lys Phe He Gly He Asp Ser Glu Lys Glu 225 230 235 240

Tyr Leu Lys He Ala Lys Lys Arg Leu Asn Lys Gly Ala Thr Tyr Gly 245 250 255

Leu

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3695 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Lactococcus lactis subsp. cremoris

(B) STRAIN: W9

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:2412..3323

(C) IDENTI ICATION METHOD: experimental

(D) OTHER INFORMATION:/codon_start= 2412

/product---- "LlaAl restriction endonuclease"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 3

/standard_name= "Gene coding for LlaAl restriction endonuclease"

/label= r-llaAI

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

ATATAAGATA TATAAATCAG TTCGCCTTTT TCTACTCCGT TCTAAAATCT TAAAATCAAG 60

GTCAAAAGAA AAAGTCAAAA CCATTGAATT GAGGTTCTAA AATTAAACTC CCTGCGTTGC 120

TCTTGGCTGC CGCTTGTACA CTCTGATTTT ATATTAGATA CATTCTGCCA TTAAAAAGAA 180

CTCCTAACGG TCGTGGCTAC TTTGTTTAGT CTAAACGCTT TAAATAGTCC TACAAGCTCA 240

TATTTTGCCT TTTAAGCGAT TTTAAACGTG AGTTAGTAAT AATTATCATG GATAAAAGAA 300

AAAGCCCTTA AATAGGCTTG TATGTAATTG ACTAAAACGT ACAATTTAGC TTTTAAATAT 360

GACCCTTATT TATGACCTGC TCTAACCTCA CTATTCATCA GCATTCAAAA AAGAGGTCAA 420

AACTGTTAAG TTATGAGCTG AATAGATTTT ATTAAATTTT ATTTGGTTTA AAAGACCAAT 480

TATCTATTTT TTAACAAACA CTAAAATAGA TTTTTTGGAA AACTTTGCAA CAGAACCAGC 540

AATCTGATGT TGCGAGATGG ACGTTCTTTC GGTTTTGAAC CTCAAGGGGA ACACTCGTTT 600

GATAAAGCGT CTCAATGGTT GTCAGTAAAC AAACAAAAAC TTTTGGAAGT GTGCTATTAT 660

AAGTCATATA AGTCGTGCGC TTTCTAATGC TTAGTGCTTT AAGATTAGGA TAGCACGACT 720

TATTTATTTT CCAATAAAAT TAACTAGCAA TTCGGGTATA ATATATTTAT GAATTTATTA 780

CAAAAAAACA AGATCAACTT ACGTCCGTTT ACTAAATGGA CAGGTGGGAA AAGGCAACTA 840

CTGCCACACA TTCAATACCT AATGCCAGAA AAATACAATC ATTTTTTCGA ACCTTTTATT 900

GGTGGTGGCG CTTTGTTTTT TGAACTCGCT CCTCAAAAAG CAGTTATTAA CGACTTCAAT 960

TCTGAGCTTA TAAACTGTTA CCGGCAGATG AAAGATAATC CTGAGCAATT GATAGAATTG 1020

TTGACTAATC ATCAGCGGGA AAATTCTAAA GAATATTATT TAGACTTACG TTCTTCTGAT 1080

AGAGATGGAA GAATTGATAA GATGAGCGAA GTTGAACGTG CTGCTAGAAT TATGTATATG 1140

CTACGTGTTG ATTTTAATGG TTTATATCGT GTTAATTCGA AAAACCAGTT TAATGTGCCT 1200

TATGGAAGAT ATAAAAATCC TAAGATAGTT GATAAAGAAT TGATTGAAAG TATTTCCGAG 1260

TACTTGAATA ACAATTCTAT TAAGATCATG AGTGGAGATT TTGAAAAAGC CGTTAAAGAA 1320

GCACAGGATG GAGATTTTGT TTATTTCGAC CCTCCATACA TTCCACTTTC TGAAACTAGC 1380

GCCTTTACTT CTTATACACA CGAAGGCTTT AGCTACGAAG ATCAAGTTAG GCTAAGAGAT 1440

TGTTTCAAAC AGTTAGATTC AAAAGGGGTA TTCGTCATGC TTTCAAATTC TTCAAGCCCT 1500

TTAGCGGAGG AATTATATAA AGATTTTTAC ATCCATAAAA TTGAAGCTAC TCGAACAAAT 1560

GGGGCTAAAT CATCTAGTCG TGGAAAAATC ACTGAAATCA TCGTAACCAA TTATGGCAAT 1620

TAACGAATAT AAGTATGGAG GTGTTTTAAT GATAAAACCA TACTATGAAA AAGAAAACGC 1680

AATTCTCGTT CACGCAGATT CATTTAAATT ATTAGAAAAA ATTAAACCTG AAAGCATGGA 1740

CATGATATTT GCTGACCCTC CTTACTTTTT AAGTAATGGA GGAATGTCAA ATTCAGGTGG 1800

TCAAATTGTT TCTGTTGATA AAGGGGATTG GGATAAAATT TCTTCATTTG AAGAAAAACA 1860

TGACTTTAAT AGACGTTGGA TTAGGTTAGC AAGATTGGTT TTAAAACCCA ACGGAACTAT 1920

TTGGGTTTCC GGAAGCCTTC ATAACATATA TTCTGTCGGG ATGGCGCTGG AACAGGAAGG 1980

TTTCAAAATC TTAAATAATA TAACTTGGCA AAAGACAAAT CCTGCACCTA ATCTATCATG 2040

TCGGTACTTC ACCCACTCTA CAGAGACAAT TTTATGGGCA AGAAAGAACG ATAAAAAATC 2100

TCGCCATTAT TATAACTATG AATTGATGAA AGAGTTTAAT GACGGGAAAC AAATGAAAGA 2160

TGTTTGGACA GGTAGTCTGA CAAAAAAATC AGAAAAATGG GCTGGGAAAC ATCCAACTCA 2220

GAAGCCAGAG TATATTTTAG AACGGATAAT CTTAGCTAGT ACAAAGGAAA ATGATTATAT 2280

TTTAGACCCT TTCGTCGGAA GTGGAACTAC TGGTGTAGTA GCCAAGAGAT TGGGGCGTAA 2340

ATTTATTGGG ATTGATTCTG AGAAAGAATA TCTTAAAATT GCTAAAAAAA GGCTAAATAA 2400

AGGAGCAACA T ATG GAC TTT AAT AAT TAC ATC GGT TTA GAA TCT GAC GAT 2450 Met Asp Phe Asn Asn Tyr He Gly Leu Glu Ser Asp Asp 260 265 270

AGA TTA AAT GCT TTT ATG GCA ACA CTT TCC GTA ACT AAT AGA ACT CCC 2498 Arg Leu Asn Ala Phe Met Ala Thr Leu Ser Val Thr Asn Arg Thr Pro 275 280 285

GAA TAC TAC GTG AAC TGG GAA AAA GTT GAA CGT GAA ACA CGA AAA TTT 2546 Glu Tyr Tyr Val Asn Trp Glu Lys Val Glu Arg Glu Thr Arg Lfe Phe 290 295 300

GAA TTA GAA CTA AAT ACT TTA AAC TAT CTC ATT GGG AAA GAA GAT ATT 2594 Glu Leu Glu Leu Asn Thr Leu Asn Tyr Leu He Gly Lys Glu Asp He

305 310 315

TAT AGT GAA GCA CTT GAA CTA TTT ACC AAT CAA CCT GAA TTG CTT AAA 2642 Tyr Ser Glu Ala Leu Glu Leu Phe Thr Asn Gin Pro Glu Leu Leu Lys 320 325 330

GCT ATT CCT AGT TTG ATT GCT AGT AGA GAT ACA TCT TTA GAT ATA CTA 2690 Ala He Pro Ser Leu He Ala Ser Arg Asp Thr Ser Leu Asp He Leu 335 340 345 350

AAC ATT GAC GAA AAT GAT GAT ATG AGT TTT GAA CAA CTT AAC TTT CTT 2738 Asn He Asp Glu Asn Asp Asp Met Ser Phe Glu Gin Leu Asn Phe Leu 355 360 365

GTT ATC GAC GAA AAT TGT ATC GCT GAT TAT GTA GAC TTT ATT AAC CAG 2786 Val He Asp Glu Asn Cys He Ala Asp Tyr Val Asp Phe He Asn Gin 370 375 380

GCA GGT TTA CTA GAT TTT CTA CAG AAT AAA GCA AAA CGT TCT CTG GTA 2834 Ala Gly Leu Leu Asp Phe Leu Gin Asn Lys Ala Lys Arg Ser Leu Val 385 390 395

GAC TAT GTG TAT GGT GTT GAA GCA GGG CTT GAT AGC AAT GCT CGA AAA 2882 Asp Tyr Val Tyr Gly Val Glu Ala Gly Leu Asp Ser Asn Ala Arg Lys 400 405 410

AAC CGA AGC GGT ACA ACC ATG GAG GGG ATT TTA GAA CGT ACT GTT TCA 2930 Asn Arg Ser Gly Thr Thr Met Glu Gly He Leu Glu Arg Thr Val Ser 415 420 425 430

AAA ATA GCT CAA GAG AAA GGG CTT GAA TGG AAG CCA CAG GCA ACC GCT 2978 Lys He Ala Gin Glu Lys Gly Leu Glu Trp Lys Pro Gin Ala Thr Ala 435 440 445

TCT TTT ATC AAG TCT CAA TGG GAC ATA GAA GTC CCT GTA GAC AAA TCA 3026 Ser Phe He Lys Ser Gin Trp Asp He Glu Val Pro Val Asp Lys Ser 450 455 460

AAA AGA CGC TTT GAT GCA GCA GTT TAC TCT CGT GCG CTC AAT AAG GTT 3074 Lys Arg Arg Phe Asp Ala Ala Val Tyr Ser Arg Ala Leu Asn Lys Val 465 470 475

TGG CTC ATA GAA ACA AAT TAC TAC GGC GGT GGA GGA AGT AAA CTC AAA 3122 Trp Leu He Glu Thr Asn Tyr Tyr Gly Gly Gly Gly Ser Lys Leu Lys 480 485 490

GCA GTT GCT GGA GAA TTT ACA GAA TTG AGT CAG TTT GTA AAA ACA TCA 3170 Ala Val Ala Gly Glu Phe Thr Glu Leu Ser Gin Phe Val Lys Thr Ser 495 500 505 510

AAA GAT AAT GTT GAA TTT GTA TGG GTA ACA GAC GGC CAA GGG TGG AAA 3218 Lys Asp Asn Val Glu Phe Val Trp Val Thr Asp Gly Gin Gly Trp Lys 515 520 525

TTT TCC CGC TTA CCA CTT GCA GAA GCT TTC GGA CAC ATC GAT AAC GTT 3266 Phe Ser Arg Leu Pro Leu Ala Glu Ala Phe Gly His He Asp Asn Val 530 535 540

TTC AAT CTA ACC ATG TTG AAA GAA GGT TTC TTG TCT GAT TTA TTC GAA 3314 Phe Asn Leu Thr Met Leu Lys Glu Gly Phe Leu Ser Asp Leu Phe Glu 545 550 555

AAA GAA ATT TAAAAAGACA GAGAATCTCT GTCTTTTTAA ATTTCAATTC 3363

Lys Glu He 560

CTTCCTTCTG CTAGCTATAA CTTTCCAAAA AACCTGAAAA ACGGTTCTGT TGCAATTGTA 3423

TGTGGGGTCG GAACTTACTA CTATATCATG AGAAATGAAG ATTAAAGTTG AAACAAAAAA 3483

ACAGATTATT TTAAAATGTA AATCTGTTTT TGTTTGGGCT GATTTTATCA CACCAATTCT 3543

ATGTTCAGAA AATGGTCATT TTCTGGACAC TCTTCTTTTG TTATTAAAAC TCTCAAAATC 3603

ATTTACATTT ATTGTTCATT AACCCATAAT TTATTCTATG TTCATTTATA GATATCGAAT 3663

TCCTGCAGGG CCCTCCACTA GTTCTAGAGG CG 3695

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 304 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Asp Phe Asn Asn Tyr He Gly Leu Glu Ser Asp Asp Arg Leu Asn 1 5 10 15

Ala Phe Met Ala Thr Leu Ser Val Thr Asn Arg Thr Pro Glu Tyr Tyr 20 25 30

Val Asn Trp Glu Lys Val Glu Arg Glu Thr Arg Lys Phe Glu Leu Glu 35 40 45

Leu Asn Thr Leu Asn Tyr Leu He Gly Lys Glu Asp He Tyr Ser Glu 50 55 60

Ala Leu Glu Leu Phe Thr Asn Gin Pro Glu Leu Leu Lys Ala He Pro 65 70 75 80

Ser Leu He Ala Ser Arg Asp Thr Ser Leu Asp He Leu Asn He Asp 85 90 95

Glu Asn Asp Asp Met Ser Phe Glu Gin Leu Asn Phe Leu Val He Asp 100 105 110

Glu Asn Cys He Ala Asp Tyr Val Asp Phe He Asn Gin Ala Gly Leu 115 120 125

Leu Asp Phe Leu Gin Asn Lys Ala Lys Arg Ser Leu Val Asp Tyr Val 130 135 140

Tyr Gly Val Glu Ala Gly Leu Asp Ser Asn Ala Arg Lys Asn Arg Ser 145 150 155 160

Gly Thr Thr Met Glu Gly He Leu Glu Arg Thr Val Ser Lys He Ala 165 170 175

Gin Glu Lys Gly Leu Glu Trp Lys Pro Gin Ala Thr Ala Ser Phe He 180 185 190

Lys Ser Gin Trp Asp He Glu Val Pro Val Asp Lys Ser Lys Arg Arg 195 200 205

Phe Asp Ala Ala Val Tyr Ser Arg Ala Leu Asn Lys Val Trp Leu He 210 215 220

Glu Thr Asn Tyr Tyr Gly Gly Gly Gly Ser Lys Leu Lys Ala Val Ala 225 230 235 240

Gly Glu Phe Thr Glu Leu Ser Gin Phe Val Lys Thr Ser Lys Asp Asn 245 250 255

Val Glu Phe Val Trp Val Thr Asp Gly Gin Gly Trp Lys Phe Ser Arg 260 265 270

Leu Pro Leu Ala Glu Ala Phe Gly His He Asp Asn Val Phe Asn Leu 275 280 285

Thr Met Leu Lys Glu Gly Phe Leu Ser Asp Leu Phe Glu Lys Glu He 290 295 300

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3706 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(Vi) ORIGINAL SOURCE:

(A) ORGANISM: Lactococcus lactis subsp. cremoris

(B) STRAIN: W56

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATIONcomplement (422..2161)

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_start= 422

/product= "LlaBl methylase"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 1

/standard_name= "Gene coding for LlaBl methylase"

/label= -llaBI

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:2464..3360

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATIO :/codon_start= 2464

/product= "LlaBl endonuclease" /evidence= EXPERIMENTAL /gene= "ORF" /number= 2

/standard_name= "Gene coding for LlaBl endonuclease"

/label= r-llaBI

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GAATTCGCAA GGTCTTTTAT AGATATAATT CCTAGCTTAT TTAAACGTTT CTCAGTTCGT 60

TTTCCAATGC CCCAGAAGGC AGTAATCTTG GACTAATGTC AAATAAATAG ACACGAAAAA 120

GTAAATAATA TACCCGAATT GCTAGTTAAT TTCATTGGAA AATAAATAAG TCGTGCTATC 180

CTAATCTTAA ACCACTAAGC ATTAGAAAGC GCACGACTTA TAACACTGAA AAGCTTTCGG 240

TTTCTAGTAT TATGCTCGGT CTTGGAGGTT TGGCAAGCTC TATGTTTGCT ACGCTTCGTG 300

GCGCAAACGA CCTTGTTGGG GGAGTGTTTC ACTTCCCCCG AAACCCCCTT AAAAAACTGT 360

CAAAACGTAG CCGTTTTGTA TTAAAAAAGA TCAGCAGGAG AAGCCCAGCT GATCTTTTTT 420

AATATAGTTC TTCGAACACA ATTCGTTTAT TCATATACTC TTCATAATTT ATATTTAATT 480

GGTATTTTTC AATTAAATAT AAATCAAGCT CTCTTTTAGA ATCGCAATTT TTAATAAAAG 540

TTAATTCGTC TTTTTCAAAA TGTGGAATTG AAAAATTTTT AAGATATTTT TTTTGAAAGC 600

AAAAGTATCC TCCGCCTATC ATATAACTAG TGTTTTCAAT ATAATATTTC ATAATAACTG 660

AGTTCAATAT CTTAGCTAAA ATGTCTAAAT CGATACTTTC TACTGAATTT TTTACTCCAT 720

AAATTGCATA TCCATTATTA AAAAGAGCAT AATCTGTAAA ATATACAAAG TTTGGATTCA 780

AAGAATTTGT AGGAAAAATT ATTTTAGGTA CATGGCTATT CAATGCTTGA GATCGCCCAT 840

ATTCATACCA AATGTTAACG GTTGGTTTCC CAGCATTGCG TTTACTGAGC TCGTCTTTAA 900

TAGCAATAAA ATAATTTAAA GTATTAGGGA ATTTTTCCTT CATTGAGACA ATGCTGATTG 960

GTACAGCATT GCCGTTCATA TTTTCATAAG GATATATTAT TCGATTAAAT TCGTAAAAAT 1020

TATTGTTAGT ATTAACTTTT TTTTCTCCAG ATCCTTTAAT AATAGGAATA GTTATTTCTT 1080

TCTCTATGAG AAAAGGTGTG TCATTATACT TTTTCACGAA ATATTCTTTA TCATTATTAA 1140

CTTCTTTTTT AGTATAGTCT ATTAAATAAA GTTTATCTTT TTGAGTAGCA ATACCAGTAG 1200

ATATATTCAG TGTAAAAGGT TGATTTTCTA TTTTATTTAT ATTTAATAAT TCAATTTCAT 1260

CTAACAAATT AATAGATTCA GGATTAACAT CATCATACCT AATTTGATCA AACTTGTTTT 1320

TTAATTCTTT TTTCATTGAT ACACTATTAC TATTCGACTG TATATTTTTA TATAATATAT 1380

GACTTTTTTC ACTTTTGTCT AAAAATAATA TAGCAGAATA AGTTTGAGCA TTTGAAAAAA 1440

GTTGATTATC TTTAAAATCT ATTACTTTGT ATATTGATCT AGAATCTACT AAAAGAGCCC 1500

GCAAACCAAA AGCAGATTTC ATTTTTAAAA GGTGATTTGG AACAATATAA CCAATCTTTC 1560

CATTTTCAGA AAGAATATTT AAACTTAATT CTATAAATGC GTAAAATAAA TTATAGCTCC 1620

CAGATTTGCA AGACATATAA TGTTGTTGTA AATACTTTTT TTGATTGGAG GAGAGTTCTT 1680

GTATTTTTAC ATATGGAGGA TTACCGATAA TAAAATCGAT TAAAGAAAGG CTTGGTATTA 1740

TATGTTTAGC ATACTTTAAT GCCGATAGTA GAAATTCGCC ACACCCACAA GAAAAATCAC 1800

CAATGGAGCT TTTTTTATTA ACTGACTTTA AAGTTTCTTC AACTATAAAG TCTGAAACTA 1860

GTGAGGGAGT ATATACGATT CCATTTTCTT TCTTTGAGTT CTCACTCAAA CTAGCATATA 1920

AAAATTCTTC AATATTTTTA AGAGAAAAAT GAAGCTCATT TTCTTCAATA TAATTTCTTA 1980

TGTCTAAATT TGAGTAGCCT AATAGCTCGT TTATCAAACT ATTTTTAAGG GATTCTATGG 2040

GTATTTTTTT TTCGGTGAAG TAATTTCTTA TTATCTCGCT TAATATTTCT TTGCTTGAAT 2100

ATTTATCGAG TATTTTTTTT ATAAACTCTA TATTTGTTTG TTTATCTATA ACCTCAAGCA 2160

TAATAGCACC TCATTTTTAT TTAATTATAA CTCCTAGGGT TATAAAAGTC AAGTGGAAAG 2220

GAGTAACATT ATGATTATTT TTGTTCTTAA CGAACGGCTA AAAGAACTAA ATATATCACA 2280

AAATAAGTTT GCGAAGCAAT CACATATTAG GCCGATACAA TAAATGATAT CTGCAATAAC 2340

AGTACTAAAA GAATAGAAGT TTCAACTATC AACAAAATAC TAATTCAATT AAATAAGATA 2400

GGTATTCGTA AATACTCTAT TGAAGACATA ATAAAATATA AGCATGAATA AGGAGATTTT 2460

CAT ATG AAT ATA GAT CAA GTT GCA AAT AAA ATG AAA AGG GAT TTA GAA 2508 Met Asn He Asp Gin Val Ala Asn Lys Met Lys Arg Asp Leu Glu 305 310 315

CTA GCT ATT ACT GAT CAA ATA GTT GAC GGT TCT AAA GTA AAT AAA AAA 2556 Leu Ala He Thr Asp Gin He Val Asp Gly Ser Lys Val Asn Lys Lys 320 325 330 335

GGG AAA TTA TTT TTA AAT GGA GCA GAA GCA AAA CAA TCT TTA ATT AGA 2604 Gly Lys Leu Phe Leu Asn Gly Ala Glu Ala Lys Gin Ser Leu He Arg 340 345 350

TCT AGT AAA CTT ATT AAT TAT GTT CAC GAG TTT GTA AAA CAT GAA CTA 2652 Ser Ser Lys Leu He Asn Tyr Val His Glu Phe Val Lys His Glu Leu 355 360 365

ATA AGA AAT AGT GTT GAA GAA TCT CTG ATA TTC CCC CCA TTA GGT CAG 2700 He Arg Asn Ser Val Glu Glu Ser Leu He Phe Pro Pro Leu Gly Gin 370 375 380

ACA AAC CCT GAA ATA AAA CTT ACT GGT ATG TTT AAA CAA AAG GAT CAA 2748 Thr Asn Pro Glu He Lys Leu Thr Gly Met Phe Lys Gin Lys Asp Gin 385 390 395

GAT GTT TGT GTA AAG CCT CAG GGA GTT TTA CCC GAA AGA ACT TTA ATT 2796 Asp Val Cys Val Lys Pro Gin Gly Val Leu Pro Glu Arg Thr Leu He 400 405 410 415

GGA TGG GGA CCT ATG ATA AAT TCG GGA TTA TAC TGT GAT TAT GGT CGC 2844 Gly Trp Gly Pro Met He Asn Ser Gly Leu Tyr Cys Asp Tyr Gly Arg 420 425 430

GCT TAT GCA GAA AGA GTA TTA TCT ATC AAT GTA AGA AGT CAA TTA AGT 2892 Ala Tyr Ala Glu Arg Val Leu Ser He Asn Val Arg Ser Gin Leu Ser 435 440 445

AGT CTA GAT AAA AAT TCT GAT ACG TTA TTT GAG CGG ATG TTT GCA GAA 2940 Ser Leu Asp Lys Asn Ser Asp Thr Leu Phe Glu Arg Met Phe Ala Glu 450 455 460

GCA TTA AAT TTA CAC GAG TTG TAT CCA AAA ATA GTT ATG GGA GAA GTA 2988 Ala Leu Asn Leu His Glu Leu Tyr Pro Lys He Val Met Gly Glu Val 465 470 475

TAT GTT ATT CCA GTT TAT GAA TAC GAC GAC CAA GCA ATG ATA AAT AAT 3036 Tyr Val He Pro Val Tyr Glu Tyr Asp Asp Gin Ala Met He Asn Asn 480 485 490 495

CAA GTT AAG TTC AAG TCA AGA AGA ACA AAT TTA GAA AAA TAC ATT AAT 3084 Gin Val Lys Phe Lys Ser Arg Arg Thr Asn Leu Glu Lys Tyr He Asn 500 505 510

TTT TTC TAT TAT TTA AGT GGC AGA GAT GAA CAG GAT CTT GAA GAA GAC 3132 Phe Phe Tyr Tyr Leu Ser Gly Arg Asp Glu Gin Asp Leu Glu Glu Asp 515 520 525

AAA CAA AAG TAC GAA AGG TGC GCA TTG GTT ATA ATA GAT TTT AGA GGA 3180 Lys Gin Lys Tyr Glu Arg Cys Ala Leu Val He He Asp Phe Arg Gly 530 535 540

GAT CAA GCC AAA GTC TAT AAA AAT ACT GCA GAG TTA AAA GCT AGG GGC 3228 Asp Gin Ala Lys Val Tyr Lys Asn Thr Ala Glu Leu Lys Ala Arg Gly 545 550 555

TTA GTC AGA AAT GAT TTT GAG GTT GAG TTA GCA GAA CTT TCA ACG GAT 3276 Leu Val Arg Asn Asp Phe Glu Val Glu Leu Ala Glu Leu Ser Thr Asp 560 565 570 575

AAA TTT ATT GAA GAC TTA TTA CTT ATT TAT AAT AAT AGA TTT CCT GGT 3324 Lys Phe He Glu Asp Leu Leu Leu He Tyr Asn Asn Arg Phe Pro Gly 580 585 590

TCT GTT GCG AAG TTT GAA AAT CAA ACG CGC CCT CTC TGAACTCCAA 3370

Ser Val Ala Lys Phe Glu Asn Gin Thr Arg Pro Leu 595 600

ATATCTTAGG CTGGTATTCC CATTAATACC TTGATTTCAG TAGACACCGA AAAGCCGAAG 3430

AGAGTTCCAT TTCTTCGGTT CTTTTTATAT ATTCCTCGAA TGGTCTGCAT CCCCTTAATC 3490

GTGGAAGAGG CTGTACGGAG ACTTTGATAA AATTTATTCC GTCGTTTAAT AGGTCGATGG 3550

TCTTGTTCTA TTAAATTGTT AAGATACTTC ACAGTTCGGT GCTCTGTCTT AGTATATAAA 3610

CCCACACTCT GTAACTTTCT AAAGCGGAGC CAAGAGAAGG TGCTTTATCG TGCAATTGAT 3670

GCGGACGATC AAAATATTAT TGGGAATACC TGCTTA 3706

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 580 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Leu Glu Val He Asp Lys Gin Thr Asn He Glu Phe He Lys Lys 1 5 10 15

He Leu Asp Lys Tyr Ser Ser Lys Glu He Leu Ser Glu He He Arg 20 25 30

Asn Tyr Phe Thr Glu Lys Lys He Pro He Glu Ser Leu Lys Asn Ser 35 40 45

Leu He Asn Glu Leu Leu Gly Tyr Ser Asn Leu Asp He Arg Asn Tyr 50 55 60

He Glu Glu Asn Glu Leu His Phe Ser Leu Lys Asn He Glu Glu Phe 65 70 75 80

Leu Tyr Ala Ser Leu Ser Glu Asn Ser Lys Lys Glu Asn Gly He Val 85 90 95

Tyr Thr Pro Ser Leu Val Ser Asp Phe He Val Glu Glu Thr Leu Lys 100 105 110

Ser Val Asn Lys Lys Ser Ser He Gly Asp Phe Ser Cys Gly Cys Gly 115 120 125

Glu Phe Leu Leu Ser Ala Leu Lys Tyr Ala Lys His He He Pro Ser 130 135 140

Leu Ser Leu He Asp Phe He He Gly Asn Pro Pro Tyr Val Lys He 145 150 155 160

Gin Glu Leu Ser Ser Asn Gin Lys Lys Tyr Leu Gin Gin His Tyr Met 165 170 175

Ser Cys Lys Ser Gly Ser Tyr Asn Leu Phe Tyr Ala Phe He Glu Leu 180 185 190

Ser Leu Asn He Leu Ser Glu Asn Gly Lys He Gly Tyr He Val Pro 195 200 205

Asn His Leu Leu Lys Met Lys Ser Ala Phe Gly Leu Arg Ala Leu Leu 210 215 220

Val Asp Ser Arg Ser He Tyr Lys Val He Asp Phe Lys Asp Asn Gin 225 230 235 240

Leu Phe Ser Asn Ala Gin Thr Tyr Ser Ala He Leu Phe Leu Asp Lys 245 250 255

Ser Glu Lys Ser His He Leu Tyr Lys Asn He Gin Ser Asn Ser Asn 260 265 270

Ser Val Ser Met Lys Lys Glu Leu Lys Asn Lys Phe Asp Gin He Arg 275 280 285

Tyr Asp Asp Val Asn Pro Glu Ser He Asn Leu Leu Asp Glu He Glu 290 295 300

Leu Leu Asn He Asn Lys He Glu Asn Gin Pro Phe Thr Leu Asn He 305 310 315 320

Ser Thr Gly He Ala Thr Gin Lys Asp Lys Leu Tyr Leu He Asp Tyr 325 330 335

Thr Lys Lys Glu Val Asn Asn Asp Lys Glu Tyr Phe Val Lys Lys Tyr 340 345 350

Asn Asp Thr Pro Phe Leu He Glu Lys Glu He Thr He Pro He He 355 360 365

Lys Gly Ser Gly Glu Lys Lys Val Asn Thr Asn Asn Asn Phe Tyr Glu 370 375 380

Phe Asn Arg He He Tyr Pro Tyr Glu Asn Met Asn Gly Asn Ala Val 385 390 395 400

Pro He Ser He Val Ser Met Lys Glu Lys Phe Pro Asn Thr Leu Asn 405 410 415

Tyr Phe He Ala He Lys Asp Glu Leu Ser Lys Arg Asn Ala Gly Lys 420 425 430

Pro Thr Val Asn He Trp Tyr Glu Tyr Gly Arg Ser Gin Ala Leu Asn 435 440 445

Ser His Val Pro Lys He He Phe Pro Thr Asn Ser Leu Asn Pro Asn 450 455 460

Phe Val Tyr Phe Thr Asp Tyr Ala Leu Phe Asn Asn Gly Tyr Ala He 465 470 475 480

Tyr Gly Val Lys Asn Ser Val Glu Ser He Asp Leu Asp He Leu Ala 485 490 495

Lys He Leu Asn Ser Val He Met Lys Tyr Tyr He Glu Asn Thr Ser 500 505 510

Tyr Met He Gly Gly Gly Tyr Phe Cys Phe Gin Lys Lys Tyr Leu Lys 515 520 525

Asn Phe Ser He Pro His Phe Glu Lys Asp Glu Leu Thr Phe He Lys 530 535 540

Asn Cys Asp Ser Lys Arg Glu Leu Asp Leu Tyr Leu He Glu Lys Tyr 545 550 555 560

Gin Leu Asn He Asn Tyr Glu Glu Tyr Met Asn Lys Arg He Val Phe 565 570 575

Glu Glu Leu Tyr 580

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 299 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Asn He Asp Gin Val Ala Asn Lys Met Lys Arg Asp Leu Glu Leu

1 5 10 15

Ala He Thr Asp Gin He Val Asp Gly Ser Lys Val Asn Lys Lys Gly 20 25 30

Lys Leu Phe Leu Asn Gly Ala Glu Ala Lys Gin Ser Leu He Arg Ser 35 40 45

Ser Lys Leu He Asn Tyr Val His Glu Phe Val Lys His Glu Leu He 50 55 60

Arg Asn Ser Val Glu Glu Ser Leu He Phe Pro Pro Leu Gly Gin Thr 65 70 75 80

Asn Pro Glu He Lys Leu Thr Gly Met Phe Lys Gin Lys Asp Gin Asp 85 90 95

Val Cys Val Lys Pro Gin Gly Val Leu Pro Glu Arg Thr Leu He Gly 100 105 110

Trp Gly Pro Met He Asn Ser Gly Leu Tyr Cys Asp Tyr Gly Arg Ala 115 120 125

Tyr Ala Glu Arg Val Leu Ser He Asn Val Arg Ser Gin Leu Ser Ser 130 135 140

Leu Asp Lys Asn Ser Asp Thr Leu Phe Glu Arg Met Phe Ala Glu Ala 145 150 155 160

Leu Asn Leu His Glu Leu Tyr Pro Lys He Val Met Gly Glu Val Tyr 165 170 175

Val He Pro Val Tyr Glu Tyr Asp Asp Gin Ala Met He Asn Asn Gin 180 185 190

Val Lys Phe Lys Ser Arg Arg Thr Asn Leu Glu Lys Tyr He Asn Phe 195 200 205

Phe Tyr Tyr Leu Ser Gly Arg Asp Glu Gin Asp Leu Glu Glu Asp Lys 210 215 220

Gin Lys Tyr Glu Arg Cys Ala Leu Val He He Asp Phe Arg Gly Asp 225 230 235 240

Gin Ala Lys Val Tyr Lys Asn Thr Ala Glu Leu Lys Ala Arg Gly Leu 245 250 255

Val Arg Asn Asp Phe Glu Val Glu Leu Ala Glu Leu Ser Thr Asp Lys 260 265 270

Phe He Glu Asp Leu Leu Leu He Tyr Asn Asn Arg Phe Pro Gly Ser 275 280 285

Val Ala Lys Phe Glu Asn Gin Thr Arg Pro Leu 290 295

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2354 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Lactococcus lactis subsp. cremoris

(B) STRAIN: W39

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 743..1282

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_start= 743

/product= "LlaDll restriction endonuclease"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 1

/standard_name= "Gene coding for R. LlaDll"

/label= r-llaDII

/note= "The first ten amino acids in this sequence may be doubtful as the sequencing first gave base 744 as TT. However, from base 773 this reading frame gives a high homology with the Bsp6I endonuclease. "

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1391..2341

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_start= 1391

/product= "LlaDll methylase"

/evidence= EXPERIMENTAL

/gene= "ORF"

/number= 2

/standard_name= "Gene coding for M. LlaDll"

/label= m-llaDII

/note= "The sequence shows 60 % identity and 76 * similarity with the Bsp6I methylase."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CTGCAGAAAA AAAAGTAATT GGTCGTAACG AAACACGATT ATTTTCAGAC GAGCAACTAA 60

ATCACTTATC TATTGAAGTT GAACCTTATC TATTAAAGCA AGGAAATGTA GATATAGAAG 120

AATTAGAAGA ATATCGTCAA AACTTTGAAA ACTATATAGA AGAAGTAAGA AATCAGACAA 180

ATGAAAGTTA TCAGCAACAA CTTGAAGCAG AACAATACGA ACCGCCAAAA GTTACAAAAA 240

GGGAAGCCAT GGAAGTCATG CTGACCGCTC TATTTGAAAA ATTTTTTGAA CCGCTCGACA 300

TTGAGCAATG GAACAAAGAT AAAGCGACAA CTCATTTTTC AGAATTATCA GATATGACTG 360

ATACTGATTA TATACTTGCT TGTATGAGAT TAAACAATCC TACAACTTCA TACACAAAAG 420

AAAATGATAT AATATAGATA AAATTTAGAT ATAAAAGGAA AAACGATTAG AAAGCTTTTC 480

TTTTTTATGT CTAATTATTT GATAATAGTC CACTTTAGCG AGCTTCGCAT TGTTTTAATT 540

GTCTGATAAT TAAGAATTAC AAGGCAACAA CATCTTTTAT AGATATACCA ATTACGCTTT 600

GCAAGCGGAC TGCTTCTGTC GTCAAGCAGA CCAACGAGCA TAAACAAAAA GACTTGCGAC 660

ACTACCTTAT TTCTTTTCTT TAGAAATCTC TATGGATATG ATAAAATTCT ACTTAGGGGA 720

TAAAACAACT CCAAGGTGAT TT ATG GCT TAT AAA AAG TTT GGC TAT ATT GAA 772

Met Ala Tyr Lys Lys Phe Gly Tyr He Glu 300 305

ATT GAT GAT GCC AGA ATA GAT GCA ACT TGT GAT GCT TAC TTT AAA TGG 820 He Asp Asp Ala Arg He Asp Ala Thr Cys Asp Ala Tyr Phe Lys Trp 310 315 320 325

AAA GAC CTA AAC TCC TAT ATT AAA AAC ACT AGT TCT CGA GGA ATG AAT 868 Lys Asp Leu Asn Ser Tyr He Lys Asn Thr Ser Ser Arg Gly Met Asn 330 335 340

ATG CCT GAT GCA ATT AGT GAA CCT ATG GGG TGT TAT TGC TTA GGA TAT 916 Met Pro Asp Ala He Ser Glu Pro Met Gly Cys Tyr Cys Leu Gly Tyr 345 350 355

CTA TGG AAT AGG GGC AGT GAA GTC GGT GAT GCA ACA GTC CCA ATC ACA 964 Leu Trp Asn Arg Gly Ser Glu Val Gly Asp Ala Thr Val Pro He Thr 360 365 370

AAT AAA AAA ATT GAG TTT AAG GCA ACA TCA AAG TTT GAA GGG GAT TTA 1012 Asn Lys Lys He Glu Phe Lys Ala Thr Ser Lys Phe Glu Gly Asp Leu 375 380 385

TCT TCT TTC GGA CCT AAA ACA GTC TTT GAT AAT TTA GTA TTT CTG AGA 1060 Ser Ser Phe Gly Pro Lys Thr Val Phe Asp Asn Leu Val Phe Leu Arg 390 395 400 405

TTT TAT CTT GAT GAA AAT AAA TTA TAT ATT TAT GAT TTA AAC ATT AAT 1108 Phe Tyr Leu Asp Glu Asn Lys Leu Tyr He Tyr Asp Leu Asn He Asn 410 415 420

TCA GAA GAG TTT GAG AAA TAT CCA GCA AAT AAG ACT CAA ACT ATA CAA 1156 Ser Glu Glu Phe Glu Lys Tyr Pro Ala Asn Lys Thr Gin Thr He Gin 425 430 435

GAA CAA AAA GCT GTT GGA AGG CGT CCT CAC GTG AGT TTA CAA TCT TTG 1204 Glu Gin Lys Ala Val Gly Arg Arg Pro His Val Ser Leu Gin Ser Leu 440 445 450

TTT GTA GAC GCA AAA AAC TTA CAA CCA GAT ATT ATT TTT GAT ATT AGA 1252 Phe Val Asp Ala Lys Asn Leu Gin Pro Asp He He Phe Asp He Arg 455 460 465

CGA TGT CGA ATT ATC GAA GAT AAT AGA CAC TAAACTGAAA GGGGAGTTGT 1302 Arg Cys Arg He He Glu Asp Asn Arg His 470 475

TTTATCTCCC TTTTCATCAT ATTAAAATTG CGGCGTGCCG CTTTTTGTTG TATACTATAT 1362

ATCATACTTG ATTTATAGGA GAATATTT ATG TTG AAA ATT GCT TCT TTT TTC 1414

Met Leu Lys He Ala Ser Phe Phe

GCC GGA GTT GGC GGA ATT GAT TTA GGT TTT GAA AAT GCA GGT TTC AAA 1462 Ala Gly Val Gly Gly He Asp Leu Gly Phe Glu Asn Ala Gly Phe Lys 10 15 20 .

ACA ATA TAT GCT AAT GAA TTT GAT AAT TAT GCT GCT GAT ACT TTT GAA 1510 Thr He Tyr Ala Asn Glu Phe Asp Asn Tyr Ala Ala Asp Thr Phe Glu 25 30 35 40

ATG AAC TTT GAC GTT AAG GTA GAC CGA CGT GAT ATA AAT GAT GTA CAA 1558 Met Asn Phe Asp Val Lys Val Asp Arg Arg Asp He Asn Asp Val Gin 45 50 55

GCT GAT GAA ATA CCA GAT TTT GAT ATT ATG TTA GCA GGT TTT CCT TGC 1606 Ala Asp Glu He Pro Asp Phe Asp He Met Leu Ala Gly Phe Pro Cys 60 65 70

CAA GCC TTT TCT ATT GCT GGT TAT CGT CAA GGC TTT AAC GAT GAA CAA 1654 Gin Ala Phe Ser He Ala Gly Tyr Arg Gin Gly Phe Asn Asp Glu Gin 75 80 85

GGT CGA GGT AAT CTT TTT TTT GAA CTT GTT CGT ATT TTA GAA ACA AAA 1702 Gly Arg Gly Asn Leu Phe Phe Glu Leu Val Arg He Leu Glu Thr Lys 90 95 100

AAA CCT CGT GTT GCA TTC TTT GAA AAT GTT AAA AAT CTT GTT TCT CAC 1750 Lys Pro Arg Val Ala Phe Phe Glu Asn Val Lys Asn Leu Val Ser His 105 110 115 120

GAT AGC GGG AAC ACA TTT AGA GTT ATT TGT TCT GAG TTA GAA AGA CTA 1798 Asp Ser Gly Asn Thr Phe Arg Val He Cys Ser Glu Leu Glu Arg Leu 125 130 135

GGG TAC AAG TAT CTT TTT CAA GTG TTT AAT GCT TCT GAA TAT GGA AAT 1846 Gly Tyr Lys Tyr Leu Phe Gin Val Phe Asn Ala Ser Glu Tyr Gly Asn 140 145 150

ATA CCT CAA AAT AGA GAA CGT ATC TAT ATT GTT GCT TTC AAA AAT AAA 1894 He Pro Gin Asn Arg Glu Arg He Tyr He Val Ala Phe Lys Asn Lys 155 160 165

AAA GAT TAT GCA AAT TTT GAA CTA CCA AAA TCT ATA CCT TTA AAA ACA 1942 Lys Asp Tyr Ala Asn Phe Glu Leu Pro Lys Ser He Pro Leu Lys Thr 170 175 180

ACG ATT CAC GAT GTT ATT GAT TTT TCT AAA AAA CAA GAC GAT AAG TTC 1990 Thr He His Asp Val He Asp Phe Ser Lys Lys Gin Asp Asp Lys Phe 185 190 195 200

TAC TAT ACC TCT GAA AAG AAT AAA TTT TTT GAT GAG TTA AAA GAA AAT 2038 Tyr Tyr Thr Ser Glu Lys Asn Lys Phe Phe Asp Glu Leu Lys Glu Asn 205 210 215

ATG ACT AAT CAC GAC ACT ACA TAT CAG TGG CGT AGA GTT TAT GTA AGA 2086 Met Thr Asn His Asp Thr Thr Tyr Gin Trp Arg Arg Val Tyr Val Arg 220 225 230

GAA AAC AAA AGT AAT TTA GTA CCA ACA CTA ACG GCT AAT ATG GGA ACA 2134 Glu Asn Lys Ser Asn Leu Val Pro Thr Leu Thr Ala Asn Met Gly Thr 235 240 245

GGT GGG CAT AAT GTG CCT ATA ATC CTT ACA TAT AGC GGA GAT ATT CGT 2182

Gly Gly His Asn Val Pro He He Leu Thr Tyr Ser Gly Asp He Arg 250 255 260

AAA TTA ACA CCA AGA GAA TGC TTT AAC GTT CAA GGT TTC CCA AAA GAA 2230

Lys Leu Thr Pro Arg Glu Cys Phe Asn Val Gin Gly Phe Pro Lys Glu 265 270 275 280

TAT AAA CTT CCA AAC CAA AGT AAT GGG AGA TTA TAT AAA CAA GCA GGA 2278

Tyr Lys Leu Pro Asn Gin Ser Asn Gly Arg Leu Tyr Lys Gin Ala Gly 285 290 295

AAC AGT GTT GTA GTA CCA GTT ATA GAA AGA ATT GCA AAA AAT CTT GCA 2326

Asn Ser Val Val Val Pro Val He Glu Arg He Ala Lys Asn Leu Ala

300 305 310

GAT ACT ATA GTC GAA TAACTATGAA TTC 2354

Asp Thr He Val Glu 315

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 180 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Ala Tyr Lys Lys Phe Gly Tyr He Glu He Asp Asp Ala Arg He 1 5 10 15

Asp Ala Thr Cys Asp Ala Tyr Phe Lys Trp Lys Asp Leu Asn Ser Tyr 20 25 30

He Lys Asn Thr Ser Ser Arg Gly Met Asn Met Pro Asp Ala He Ser 35 40 45

Glu Pro Met Gly Cys Tyr Cys Leu Gly Tyr Leu Trp Asn Arg Gly Ser 50 55 60

Glu Val Gly Asp Ala Thr Val Pro He Thr Asn Lys Lys He Glu Phe 65 70 75 80

Lys Ala Thr Ser Lys Phe Glu Gly Asp Leu Ser Ser Phe Gly Pro Lys 85 90 95

Thr Val Phe Asp Asn Leu Val Phe Leu Arg Phe Tyr Leu Asp Glu Asn 100 105 110

Lys Leu Tyr He Tyr Asp Leu Asn He Asn Ser Glu Glu Phe Glu Lys 115 120 125

Tyr Pro Ala Asn Lys Thr Gin Thr He Gin Glu Gin Lys Ala Val Gly 130 135 140

Arg Arg Pro His Val Ser Leu Gin Ser Leu Phe Val Asp Ala Lys Asn 145 150 155 160

Leu Gin Pro Asp He He Phe Asp He Arg Arg Cys Arg He He Glu

165 170 175

Asp Asn Arg His 180

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 317 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Leu Lys He Ala Ser Phe Phe Ala Gly Val Gly Gly He Asp Leu 1 5 10 15

Gly Phe Glu Asn Ala Gly Phe Lys Thr He Tyr Ala Asn Glu Phe Asp 20 25 30

Asn Tyr Ala Ala Asp Thr Phe Glu Met Asn Phe Asp Val Lys Val Asp 35 40 45

Arg Arg Asp He Asn Asp Val Gin Ala Asp Glu He Pro Asp Phe Asp 50 55 60

He Met Leu Ala Gly Phe Pro Cys Gin Ala Phe Ser He Ala Gly Tyr 65 70 75 80

Arg Gin Gly Phe Asn Asp Glu Gin Gly Arg Gly Asn Leu Phe Phe Glu 85 90 95

Leu Val Arg He Leu Glu Thr Lys Lys Pro Arg Val Ala Phe Phe Glu 100 105 110

Asn Val Lys Asn Leu Val Ser His Asp Ser Gly Asn Thr Phe Arg Val 115 120 125

He Cys Ser Glu Leu Glu Arg Leu Gly Tyr Lys Tyr Leu Phe Gin Val 130 135 140

Phe Asn Ala Ser Glu Tyr Gly Asn He Pro Gin Asn Arg Glu Arg He 145 150 155 160

Tyr He Val Ala Phe Lys Asn Lys Lys Asp Tyr Ala Asn Phe Glu Leu 165 170 175

Pro Lys Ser He Pro Leu Lys Thr Thr He His Asp Val He Asp Phe 180 185 190

Ser Lys Lys Gin Asp Asp Lys Phe Tyr Tyr Thr Ser Glu Lys Asn Lys 195 200 205

Phe Phe Asp Glu Leu Lys Glu Asn Met Thr Asn His Asp Thr Thr Tyr 210 215 220

Gin Trp Arg Arg Val Tyr Val Arg Glu Asn Lys Ser Asn Leu Val Pro 225 230 235 240

Thr Leu Thr Ala Asn Met Gly Thr Gly Gly His Asn Val Pro He He

245 250 255

Leu Thr Tyr Ser Gly Asp He Arg Lys Leu Thr Pro Arg Glu Cys Phe 260 265 270

Asn Val Gin Gly Phe Pro Lys Glu Tyr Lys Leu Pro Asn Gin Ser Asn 275 280 285

Gly Arg Leu Tyr Lys Gin Ala Gly Asn Ser Val Val Val Pro Val He 290 295 300

Glu Arg He Ala Lys Asn Leu Ala Asp Thr He Val Glu 305 310 315