APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1994, p. 2324-2329 Vol. 60, No. 70099-2240/94/$04.00+0

Identification of int and attP on the Genome of LactococcalBacteriophage Tuc2009 and Their Use for Site-Specific Plasmid

Integration in the Chromosome of Tuc2009-ResistantLactococcus lactis MG1363

MAARTEN VAN DE GUCHTE,"12t CHARLES DALY,' 2 GERALD F. FITZGERALD,2*AND ELKE K. ARENDT' 2t

National Food Biotechnology Centre' and Food Microbiology Depatment,2 University College, Cork, Ireland

Received 13 December 1993/Accepted 13 April 1994

The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 ispresented. In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids,representing the Tuc2009 integrase, was identified. The nucleotide sequence of the bacteriophage attachmentsite, attP, and the sequences ofattB, attL, and attR in the lysogenic host Lactococcus lactis subsp. cremoris UC509were determined. A sequence almost identical to the UC509 attB sequence was found to be present in theplasmid-free Tuc2009-resistant L. lactis subsp. cremoris MG1363. This site could be used for the site-specificintegration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, therebydemonstrating that the application of chromosomal insertion vectors based on bacteriophage integrationfunctions is not limited to the prophage-cured original host strain of the phage.

Lysogeny is a widespread phenomenon in lactococci (18). Inmilk fermentations, in which relatively undefined mixed-strainstarter cultures are commonly used, bacteriophages can readilybe isolated from a large number of strains after induction byUV irradiation or exposure to mitomycin C (12). Despite thecommon occurrence of lysogenic strains and the potentialthreat lysogenic phages impose on dairy fermentations, rela-tively little is known about the molecular biology of theseagents. So far, research attention has focused mainly on thehost and its role in the permission of, and resistance to, lyticphage development (18).

It is only recently that more detailed information on lysog-eny in lactic acid bacteria has become available. Phage (attP)and bacterial (attB) attachment sites have been described for4adh and its lysogenic host Lactobacillus gasseri ADH (22), aswell as for OLC3 and its host Lactococcus lactis subsp. cremorisIMN-C3 (19). In the latter system the integrase-encoding gene,int, whose product was shown to be essential for the establish-ment of the lysogenic state, has also been identified.

Tuc2009, the subject of the present study, is a small-isometric-headed temperate bacteriophage (4), originally iso-lated from its lysogenic host L. lactis subsp. cremoris UC509,following induction by UV irradiation (11). Unlike ~LC3, thisphage uses a head-full mechanism of DNA packaging (4).One of the several benefits of analyzing temperate lactococ-

cal bacteriophages at a molecular level is that their naturalintegration system, once identified and characterized, can beused to construct integration vectors for use in stabilizingindustrially important traits, many of which are plasmid en-

* Corresponding author. Mailing address: Department of FoodMicrobiology, University College, Cork, Ireland. Phone: 353 21276871. Fax: 353 21 275934.

t Present address: Laboratoire de Genetique Microbienne, InstitutNational de la Recherche Agronomique, Domaine de Vilvert, 78352Jouy-en-Josas Cedex, France.

t Present address: Food Technology Department, University Col-lege, Cork, Ireland.

coded. Classically, plasmid integration into lactococcal chro-mosomes has relied on crossover events between homologousregions on the plasmid and the chromosome, mediated by thehost recombination system. However, an alternative strategy,which makes use of bacteriophage-encoded site-specific inte-gration functions and a prophage-cured derivative of theoriginal lysogenic host, has been shown to be feasible (22). Inthe present study, it is shown that this procedure holds thepotential for more widespread application.

MATERIALS AND METHODS

Bacteriophage, bacterial strains, and plasmids. The bacte-riophage, bacterial strains, and plasmids used in this study arelisted in Table 1. Plasmid pSK+203 contains a 13.7-kbTuc2009 PvuII fragment cloned into the EcoRV site of pBlue-scriptlISK+ (Stratagene, La Jolla, Calif.). The Tuc2009 int-attP region is located near one end (see Fig. 3) of thisfragment, adjacent to the EcoRI site of pBluescriptlISK+. A2.8-kb EcoRI fragment containing the int-attP region (see Fig.3) was isolated from pSK+203 and inserted in the EcoRI siteof pCI341 (16) to generate pIN1 (see Fig. 5). pCI341 carriesthe pBR322 (7) origin of replication and does not replicate inL. lactis (10, 16). pIN1 was constructed in E. coli MC1061 andsubsequently introduced into L. lactis subsp. cremoris MG1363by electroporation as described by Holo and Nes (17).

Media. Escherichia coli was grown in TY broth (23) or onTY solidified with 1.5% agar. L. lactis was grown in glucoseM17 broth (25) or on glucose M17 solidified with 1.5% agar.When appropriate, chloramphenicol was added to these mediato a final concentration of 50 jig/ml for E. coli or 5 ,ug/ml forL. lactis.DNA manipulations. Plasmid DNA was isolated by the

method of Birnboim and Doly (6). L. lactis chromosomal DNAwas isolated by a modified version of the Anderson and McKaymethod (3). Restriction enzymes and T4 DNA ligase werepurchased from Boehringer GmbH, Mannheim, Germany, andused as specified by the supplier. ECL DNA labeling anddetection reagents were purchased from Amersham Corp.,

2324

on August 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from

SITE-SPECIFIC INTEGRATION USING int AND attP 2325

TABLE 1. Phage, bacterial strains, and plasmids used in this study

Phage, bacterial strain, or Relevant features Source orplasmid reference

PhageTuc2009 Isolated after induction from 4, 11

L. lactis UC509Bacteria

L. lactis subsp. cremorisUC509 Lysogenic host of Tuc2009 11UC526 Indicator strain for Tuc2009 11MG1363 Plasmid-free strain, resistant 13, 14

to Tuc2009MG1363::pIN1 MG1363 with integrated This work

pIN1E. coli MC1061 ara leu lacAX74galUgalK 9

hsdR strAPlasmidspSK+203 Ampr, pBluescriptIISK+, 4

carrying a 13.7-kbTuc2009 PvuII fragment

pCI341 Cmr, pBR322 origin of 16replication, nonreplicatingin L. lactis

pIN1 Cmr, pCI341 derivative, This workcarrying the Tuc2009 int-attP region

Amersham, United Kingdom, and used in DNA hybridizationsas specified by the supplier. Oligonucleotides were synthesizedby using a model 391 DNA synthesizer (Applied BiosystemsInc., Foster City, Calif.). Taq DNA polymerase was purchasedfrom Promega, Madison, Wis. PCR-mediated DNA amplifica-tion was performed by using a model 480 DNA thermal cycler(Perkin-Elmer Cetus, Norwalk, Conn.) (30 cycles of 94°C for30 s, 52°C for 1 min, and 70°C for 2 or 3.5 min). attL and attRsequences were amplified by inverse PCR (24) with primers(Table 2) based on Tuc2009 sequences (see Fig. 3). Chromo-somal DNA of the lysogenic host L. lactis UC509, cut withTaqI and religated under dilute conditions, was used as thetemplate in these reactions.DNA sequence analysis. DNA sequence analysis was per-

formed by using an Applied Biosystems 373A automated DNAsequencer. Bacteriophage Tuc2009 sequences were obtained

by sequencing plasmids containing cloned Tuc2009 fragmentsor by direct sequencing of the phage DNA. Bacterial attach-ment sites were sequenced on PCR-generated fragments. Tothis end, PCR products were purified by using Microcon 100microconcentrators (Amicon, Inc. Beverly, Mass.). Data basesearches were performed by using the programs FASTA (21)and BLAST (2).

Nucleotide sequence accession number. The nucleotide se-quence of the phage Tuc2009 int-attP region has been assignedGSDB accession no. L31348.

RESULTS

Identification of the bacteriophage Tuc2009 int gene. Theentire genome of the temperate lactococcal bacteriophageTuc2009 was cloned in E. coli (4). DNA sequence analysis ofthe cloned Tuc2009 fragments revealed the presence of a1,122-bp open reading frame, int, which, on the basis ofhomology searches, is likely to encode the phage integrase(Fig. 1). The inferred amino acid sequence shows a high degreeof similarity (69% in 362 amino acids; 21% identity) to theintegrase of Staphylococcus aureus bacteriophage L54a (28).Furthermore, in the C-terminal part of the sequence a numberof amino acids which are well conserved in the integrase familyof site-specific recombinases can be identified (Fig. 1) (1, 5).This is consistent with the observation that the integrases fromdifferent bacteriophages, although quite diverse in their N-terminal parts, can be aligned with respect to their C termini(5). The integrase-encoding sequence is preceded by near-consensus promoter and Shine-Dalgamo sequences (Fig. 1)(15, 20, 27). The putative -35 promoter region is flanked byinverted repeats (Fig. 1, IR1), which may be involved inregulation of the expression of the int gene. Downstream of thecoding sequence, a putative transcription terminator was iden-tified (Fig. 1, IR2; AG = -16.4 kcallmol [-68.6 kJ/mol]) (26).

Localization of the attachment site, attP. A comparison ofthe genetic organization of the integrase genes and the bacte-riophage attachment sites, attP, in different phages shows thatthe latter may be situated either directly upstream or directlydownstream of the int locus (28). To determine the organiza-tion of the two sites in Tuc2009, we probed bacteriophage andUC509 chromosomal DNA digests with three different PCR-generated probes (Table 2) that hybridize to either int (Fig. 2,

TABLE 2. PCR primers used in this study

No. Sequence (5S-3') Origin' Used for amplification of:

1 CGGTTCCATGCTATGCCCAT p attR (inverse PCR [Fig. 3])2 GGTTTCATACTAGAGTTAGA p attR (inverse PCR [Fig. 3])3 TAACACTATCAAGTGTCGCA p attR (inverse PCR [Fig. 3])4 GGCTATCACACAGCAAACCT p attR (inverse PCR [Fig. 3])5 CAACAATACTATCAGAAACA p attL (inverse PCR [Fig. 3])6 TCAAATGACAACATCCTCGT p attL (inverse PCR [Fig. 3])7 GGCAAGAATTAGCACGTCAG p attL (inverse PCR [Fig. 3])8 CCAAAGTTTACTTTAACCGT p attL (inverse PCR [Fig. 3])9 CCTATTCGCTTGAAAGTCCC c attB, attL (in combination with primer 5)10 CGATTGAAGATATGCTGGTA c attB, auR (in combination with primer 4)11 GGCACCTATCTCAGCGATCT * attR in MG1363::pIN1 (in combination with primer 10)12 CTGCGCACTTCTTATCTTCT p Probe 1 (Fig. 2)13 CCTAACCTAGCTTTTAACGA p Probe 1 (Fig. 2)14 CTACAAAGTCCGATGCACAA p Probe 2 (Fig. 2)15 CTAATTGCTTGTATTCATCA p Probe 2 (Fig. 2)16 CCTGTGAAGCTAATAACCTG p Probe 3 (Fig. 2)17 GGTAAAGAAGCCAATGAAGT p Probe 3 (Fig. 2)

a p, phage; c, chromosomal; *, sequence complementary to pCI341.

VOL. 60, 1994

on August 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from

IR1 IR11 aaaaaatccgctcaagtttgacgacaaggggcggatttaaactataaaatagtataaaggcttttaacaagc

-35 -1073 tttttactataccattttatcagaaatqaggtataaaaagcaaatATGGCTACATATCAAAAGCGTGGTAAA

SD M A T Y Q K R G K

145 ACTTGGCAGTATTCAATATCAAGAACAAAACAAGGACTTCCTCGTCTAACAAAGGGTGGTTTTTCTACAAAGT W Q Y S I S R T K Q G L P R L T K G G F S T K

217 TCCGATGCACAAGCTGAAGCAATGGATATTGAAAGCAAACTAAAAAAAGGATTTATTGTTGACCCCATTAAGS D A Q A E A M D I E S K L K K G F I V D P I K

289 CAAGAAATTTCCGAATATTTTAAAGACTGGATGGAACTTTATAAGAAAAATGCAATTGATGAAATGACTTATQ E I S E Y F K D W M E L Y K K N A I D E M T Y

361 AAAGGTTATGAGCAAACGTTAAAATATTTAAAAACCTATATGCCAAATGTTTTAATTTCCGAAATAACAGCAK G Y E Q T L K Y L K T Y M P N V L I S E I T A

433 TCTTCTTATCAAAGAGCGCTAAATAAATTTGCTGAAACACACGCCAAAGCATCTACAAAAGGGTTTCATACTS S Y Q R A L N K F A E T H A K A S T K G F H T

505 AGAGTTAGAGCATCTATTCAACCACTCATTGAAGAGGGACGACTGCAAAAAGATTTTACCACTCGTGCAGTAR V R A S I Q P L I E E G R L Q K D F T T R A V

577 GTTAAAGGTAATGGAAATGATAAAGCCGAGCAAGACAAGTTTGTAAATTTTGATGAATACAAGCAATTAGTTV K G N G N D K A E Q D K F V N F D E Y K Q L V

649 GATTATTTCAGAAATAGGCTTAATCCAAACTATTCATCTCCCACTATGCTGTTTATAATTTCAATTACTAGCD Y F R N R L N P N Y S S P T M L F I I S I T S

721 ATGAGAGCCAGTGAAGCTTTTGGCTTAGTCTGGGATGATATTGATTTTAATAATAACACTATCAAGTGTCGCM R A S E A F G L V W D D I D F N N N T I K C R

793 AGAACTTGGAATTACAGAAATAAAGTAGGTGGTTTCAAAAAGCCCAAAACAGATGCTGGAATAAGAGATATTR T W N Y R N K V G G F K K P K T D A G I R D I

865 GTTATAGATGATGAAAGTATGCAATTGCTAAAAGATTTTAGAGAACAGCAAAAAACATTATTTGAAAGCTTGV I D D E S M Q L L K D F R E Q Q K T L F E S L

937 GGTATAAAACCGATACATGACTTTGTTTGTTATCATCCTTATAGAAAAATAATAACTCTCTCAGCTTTGCAAG I K P I H D F V C Y H P Y R K I I T L S A L Q

1009 AATACATTAGATCATGCATTGAAAAAACTAAATATTTCTACTCCACTTACTGTACACGGTTTAAGGCATACTN T L D H A L K K L N I S T P L T V H G L R H T

1081 CATGCTTCTGTTCTCCTCTATCATGGAGTTGATATCATGACTGTTTCAAAACGCCTAGGACACGCAAGTGTGH A S V L L Y H G V D I M T V S K R L G H A S V

1153 GCTATCACACAGCAAACCTATATCCATATTATAAAAGAGCTAGAAAATAAAGATAAGGATAAAATAATTGAGA I T Q Q T Y I H I I K E L E N K D K D K I I E

I I1225 CTACTAATGGAGTTATAAttttcttacaacaaaaatacaacaaatcattaaaaatcaagaataaagccatta

L L M E L - aaaagaatgttgtttttatgttgtttagtaatttttagttcttatttcggtaatid

1297 tctaaagcacatctatcctttactatgtagttcttcatgaacgataataaacaataatagaataaatataaaagatttcgtgtagataggaaatgatacatcaaQaaQtacttgctattatttgttattatcttatttatattt

IR2 IR21369 ataaaaacggcttaatatagccgtttttcctattttaaaagtgactaaaaaacatagaaataaaaagctcat

tatttttgccgaattatatcggcaaaaaggataaaattttcactgattttttgtatctttatttttcgagtaI I Ral I

1441 acaacaaaattacaacatatcattatgtttctgatagtattgttgtaaatttacaacaaacaaaaaaagccgtgttgttttaatgttgtatagtAATACAAAGACTATCATAACAACATTT_A_TGTTGTTTGTTTTTTTCGGC

I II1513 ctccgaagaatggcttgactctaggaataggatgaaatctcacaaacatcccgactatattatagcacaaaa

GAGGCTTCTTACCGAACTGAGATCCTTATCCTACTTTAGAGTGTTTGTAGGGCTGATATAATATCGTGTTTTII

1585 aaagcgccccagttaggagagggacgctaaggaatgaatttattaaaatggattcagcattcatataatataTTTCGCGGGGTCAATCCTCTCCCTGCGATTCCTTACTTAAATAATTTTACCTAAGTCGTAAGTAtattatat

1657 tccttataaaatataaaaaaatccccgcgtcagagcttgtctgtccttaatggatacgaggatgttgtcattaaQaatattttatatttttttaggggcgcagtctcgaacagacaggaattacctatgctcctacaacagtaaSD I -10 I -35

1729 tgataaagatattacaacaaaatattttgctacttgtcaataaaaaatgttatttgaataaattatatttaaactatttcstataatgttgttttataaaacgatgaacagttattttttacaat aaacttatttaatataaatt

1801 aaaaacacccgccgaagcggggttaattttaataatttagtgtttgaccagcgtaaatcaaattagggtttgtttttRtq3qcaacttcgccccaattaaAATTATTAAATCACAAACTGGTCGCATTTAGTTTAATCCCAAAC

IR3 IR3

2326

on August 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from

SITE-SPECIFIC INTEGRATION USING int AND attP 2327

FIG. 1. Nucleotide sequence of the phage Tuc2009 int-attP region. int is nucleotides 118 to 1239. The inferred amino acid sequence is givenbelow the DNA sequence. Residues which are highly or fully conserved in the integrase family of site-specific recombinases (1, 5) are underlinedand doubly underlined, respectively. orfx comprises nucleotides 1648 to 1466 (printed in capital letters). Nucleotides 1872 to 1832 represent the3' end of lys, which encodes the Tuc2009 lysin (4). Putative -35 and -10 promoter sequences, as well as Shine-Dalgarno (SD) sequences and startcodons, are underlined. IR1, IR2, and IR3 indicate inverted repeats. I and II indicate repeated sequences. pal, 20-bp palindromic sequence; id,identity segment shared by attP, attL, attR, and attB.

probe 2) or open reading frames upstream (probe 1) ordownstream (probe 3) of int. The results presented in Fig. 2show that the attP site is located on an 0.8-kb EcoRV-HindIIIfragment downstream of and overlapping the integrase gene.Some nonspecific hybridization can be seen in the lanescontaining the Tuc2009 DNA, although the probes weredeliberately chosen to cover coding sequences in order tominimize this. However, the results clearly show that probe 1hybridizes to identical EcoRV and HindIII fragments inTuc2009 and in the chromosome of UC509 but to differentEcoRI fragments, thereby locating the attP site between theEcoRV site and the EcoRI site on the map shown in Fig. 2.These results were corroborated by the results obtained fol-lowing hybridization with probe 2. In accordance with theseresults, probe 3 hybridized to different EcoRV and EcoRIfragments in the phage and the UC509 genomes. IdenticalHindIII fragments were found to hybridize to this probe,however, thereby delimiting the area in which attP is located tothe EcoRV-HindIII fragment mentioned above.

attL, attR, and affB in L. lactis subsp. cremoris UC509.Inverse PCR with UC509 chromosomal DNA, which had beencut with TaqI and religated under dilute conditions, anddifferent primer combinations allowed a more precise localiza-

orfYprobe I

Hindlll HinlIEcoRVI I~EoR

HindIll EcoRI

int lys

probe 2 probe 3attP

tion of attP (Fig. 3), as well as the amplification and sequencingof attL and attR. On the basis of the chromosomal DNAsequences, we designed two new primers and used them toamplify and sequence the attB region of L. lactis UC509. Thefact that this region could be amplified in a PCR with UC509chromosomal DNA as the template shows that in the lysogenicstrain UC509, uninterrupted attB sites are present, presumablyas a result of spontaneous excision of the phage.

attP, attB, attL, and attR appear to share a 9-bp sequence,TTCTTCATG (Fig. 4), representing the site of recombinationbetween the phage genome and the chromosome. In the regionsurrounding this identity segment in Tuc2009, the sequenceATTACAACAAAAT, or part thereof, occurs several times(Fig. 1): in direct repeats, either in tandem or not, in invertedrepeats, and as part of a 20-bp palindromic sequence. A secondsequence, GC lTlTITGT, is present in one direct repeat.Superimposed on this highly structured sequence is an openreading frame of unknown function (Fig. 1, orf x), which runsin the opposite direction to int. Putative promoter and Shine-Dalgamo sequences, resembling the consensus sequences (15,20, 27), are present upstream of this open reading frame.Downstream, a putative bidirectional terminator (Fig. 1, IR2;AG = -16.4 kcal/mol) (26) may serve to terminate transcrip-tion of this gene as well as that of int.

attB in L. lactis subsp. cremoris UC526. An attB regionidentical to that in UC509 was found to be present in UC526,the strain used for lytic propagation of Tuc2009 (Fig. 4).

attB in L. lactis subsp. cremoris MG1363 and site-specificplasmid integration. The primers used to amplify the attBregion in L. lactis subsp. cremoris UC509 and UC526 could beused to amplify an equivalent region of the chromosome ofthe plasmid-free, Tuc2009-resistant L. lactis subsp. cremoris

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7

probe I probe 2 probe 3

FIG. 2. Localization of attP. The figure shows a restriction map of

part of the Tuc2009 genome and the results obtained following probingof Tuc2009 DNA and UC509 chromosomal DNA with different

probes. The positions of the three PCR-generated probes are indi-

cated. Each probe is situated in a different open reading frame: orfy(unpublished results), int, and lys (4). Probe sizes: 1, 330 bp; 2, 420 bp;3, 360 bp. Lanes: 1, DNA molecular size marker III (BoehringerGmbH) (21,226, 5,148, 4,973, 4,268, 3,530, 2,027, 1,904, 1,584, 1,375,947, 831, 564, and 125 bp); 2 and 3, EcoRV digests of Tuc2009 andUC509 DNAs, respectively; 4 and 5, EcoRI digests of Tuc2009 andUC509 DNAs, respectively; 6 and 7, Hindlll digests of Tuc2009 andUC509 DNAs, respectively.

EcoRi (vector) 2.8 kb

Taql_PvuII Hind,IIHindlEoir >EcoR

orfy int

EcoR I--_ -L

EcoRIHindIIlI Taql

_I-40<~ lys

1 2 3 4 5 6 7 8

FIG. 3. Localization of attP and amplification of attL and attR. Thefigure shows a restriction map of part of the Tuc2009 genome.Arrowheads 1 to 8 represent primers complementary to phage DNAsequences and point in the direction 5'--3'. The asterisk representsattP, the site of recombination with the host chromosome. UC509chromosomal DNA which had been cut with TaqI and religated wasused as the template in PCRs with the different primers. The use ofprimer 1 together with one of the primers 2, 3, or 4 resulted in theamplification of attR. The use of primer 8 together with one of theprimers 5, 6, or 7 resulted in the amplification of attL. attP is thereforelocated between primers 4 and 5 on the phage genome. The designa-tion of attR and attL was chosen in accordance with the system ofLillehaug and Birkeland (19). The dashed line represents the situationin pSK+203, where the int-attP region is flanked by two EcoRI sites.

VOL. 60, 1994

on August 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from

2328 VAN DE GUCHTE ET AL.

TUC2009, attP: AGCACATCTATCCTTTACTATGTAGTTCTTCATGAACGATAATAAACAATAATAGAATAUC509, attL:

UC509, attR:

UC509, attB:

UC526, attB:

MG1363, attB:

AACTAAAGAATTGCAAACCTTGTTATTCTTCATGAACGATAATAAACAATAATAGAATAAGCACATCTATCCTTTACTATGTAGTTCTTCATGGGAGCTCAAGAAGAATAAAAGACAAAACTAAAGAATTGCAAACCTTGTTATTCTTCATGGGAGCTCAAGAAGAATAAAAGACAAAACTAAAGAATTGCAAACCTTGTTATTCTTCATGGGAGCTCAAGAAGAATAAAAGACAAAACTAAAGAATTGCAAACTTTGTTATTCTTCATGGGAGCTCAAGAAGAATAAAAGACAA

FIG. 4. Nucleotide sequences of attachment sites. The identity segment, present in all the attachment sites shown, is underlined. In the MG1363attB site, the one nucleotide differing from the corresponding sequences in UC509 and UC526 is doubly underlined.

MG1363. DNA sequencing of this PCR product showed it tobe nearly identical to the corresponding UC509 fragment: inthe sequences surrounding the attB identity segment, only onebase substitution was found (Fig. 4). This observationprompted us to test whether site-specific recombination be-tween attP and attB could take place in this strain. To this end,plasmid pIN1 (Fig. 5), which contains the Tuc2009 int-attPregion inserted in plasmid pCI341 (16) (see Materials andMethods), was introduced into MG1363 by electroporation.Chloramphenicol-resistant transformants were obtained, albeitat a low frequency (1.4 x 103 CFU/,ug of DNA; the transfor-mation frequency for a replicating control plasmid was 1.3 x107 CFU/pug of DNA), and subsequently analyzed by PCRamplification of the attachment sites (results not shown). Asexpected, no transformants were obtained when plasmidpCI341 was used. In MG1363::pINl, both attL and attR couldbe amplified by using the primer combinations 5+9 (Table 2)and 4+10, respectively, whereas attB could not be amplifiedanymore. These results were corroborated by the amplificationof attR with primers 10 (chromosomal DNA sequence) and 11(pCI341 sequence), which unambiguously showed that plasmidpIN1 had been integrated in the chromosome. DNA sequenc-ing of the attL and attR sites finally confirmed that recombi-nation between the plasmid and the chromosome had occurredwithin the 9-bp identity segment, as expected. The chloram-phenicol resistance marker appeared to be stably maintainedduring propagation in glucose M17 broth in the absence ofchloramphenicol. After 150 generations, chloramphenicol re-sistance was still 100% as judged by the results of plating onmedia with and without chloramphenicol.

DISCUSSION

The DNA sequence representing part of the genome of thetemperate lactococcal bacteriophage Tuc2009, comprising theattP and int loci, is presented. In addition, the sequences ofattL, attR, and attB in the lysogenic host L. lactis subsp.cremoris UC509 were determined.

int potentially encodes a protein of 374 amino acids, theTuc2009 integrase. The attP region shows sequence character-istics similar to those reported to be present in the attachmentsites of other bacteriophages (19, 22), in that it is rich inrepeated sequences. The DNA sequence of the int-attP regionappears to be nearly identical to the corresponding regionrecently reported for bacteriophage 4LC3 (19). The attL, attR,and attB sequences in both systems are identical. Althoughboth Tuc2009 and 4LC3 are small-isometric-headed, temper-ate bacteriophages and both lysogenize L. lactis subsp. cremorisstrains (4, 19), these phages show considerable differences. Themost prominent of these is the DNA-packaging mechanism,which involves a cos site in 4LC3 (19), as opposed to ahead-full mechanism giving rise to circularly permuted mole-cules with terminal redundancy in Tuc2009 (4). Furthermore,the genome size of (LC3 is 33 kb, whereas the unit genome

size of Tuc2009 is estimated at 39 kb. Also, the Tuc2009 DNAsequence presented here, despite its near identity to the (LC3sequence, shows significant differences. Throughout the regionof Tuc2009 analyzed, single-base-pair differences with thefLC3 sequence exist. As a result, the deduced amino acidsequence of the integrase differs in five residues, although thesame length is maintained. Toward the end of the sequencepresented in Fig. 1, the number of differences from the 4LC3sequence increases, and from approximately nucleotide 1630(Fig. 1) the homology between the two sequences seems to belost. As a result, the start of the putative orfx (Fig. 1) and thepreceding Shine-Dalgarno and promoter sequences are notpresent in the 4LC3 sequence.

In this study, it is shown that the attB sequence, which iscommon to L. lactis subsp. cremoris UC509 and IMN-C3, isalso present in L. lactis subsp. cremoris MG1363. As pointedout by Lillehaug and Birkeland (19), attB seems to be part ofa coding sequence (a configuration not uncommon for bacte-rial attachment sites [8]) with an as yet unknown function,which could explain the conservation of the attB sequence,assuming that the target gene is important to the bacterium.The integration of plasmid pIN1 at the attB site in the MG1363chromosome shows that this strain, although resistant to phageTuc2009 and not directly related to the original lysogenic hostof Tuc2009, contains the elements (attB, host factor[s]) re-quired for integration of the phage in the chromosome. Thereason for the low efficiency of integration is not clear. On thebasis of homology searches, an excisionase (xis) gene could notbe identified in Tuc2009. An open reading frame upstream ofint, which would be the most likely candidate for xis, wasinactivated during the construction of pIN1 (results notshown). Furthermore, the stability of the integrated plasmidseems to indicate that a functional xis gene is not present onpIN1.

In contrast to expectations based on the presumed specificity

FIG. 5. Plasmid pIN1. The Tuc2009-derived int, attP (*) and lys (3'end) loci are indicated. cat, chloramphenicol acetyltransferase. Theplasmid size is 6 kb.

APPL. ENVIRON. MICROBIOL.

on August 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from

SITE-SPECIFIC INTEGRATION USING int AND attP 2329

of lysogenic lactococcal phages for particular hosts, the use ofintegration systems which use phage-encoded integration func-tions is shown not to be limited to the prophage-cured originalhost strain of the phage. As a result, these systems may findmore widespread application in the lactococci than was origi-nally envisioned.

ACKNOWLEDGMENTS

We thank Aine Healy for the synthesis of oligonucleotides and LiamBurgess for photographic work.

This work was supported by BioResearch Ireland and the EuropeanUnion BRIDGE (contract BIOT-CT-91-0263).

REFERENCES1. Abremski, K. E., and R. H. Hoess. 1992. Evidence for a second

conserved arginine residue in the integrase family of recombina-tion proteins. Protein Eng. 5:87-91.

2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman.1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.

3. Anderson, D. G., and L. L. McKay. 1983. A simple and rapidmethod for isolating large plasmid DNA from lactic streptococci.Appl. Environ. Microbiol. 46:549-552.

4. Arendt, E. K., C. Daly, G. F. Fitzgerald, and M. van de Guchte.1994. Molecular characterization of lactococcal bacteriophageTuc2009 and identification and analysis of genes encoding lysin, aputative holin, and two structural proteins. Appl. Environ. Micro-biol. 60:1875-1883.

5. Argos, P., A. Landy, K. Abremski, J. B. Egan, E. Haggard-Ljungquist, R. H. Hoess, M. L. Kahn, B. Kalionis, S. V. L.Narayana, L. S. Pierson, N. Sternberg, and J. M. Leong. 1986. Theintegrase family of site-specific recombinases: regional similaritiesand global diversity. EMBO J. 5:433-440.

6. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

7. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. V. Betlach, H. L.Heynecker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977.Construction and characterization of new cloning vehicles. II. Amultipurpose cloning system. Gene 2:95-113.

8. Campbell, A. M. 1992. Chromosomal insertion sites for phages andplasmids. J. Bacteriol. 174:7495-7499.

9. Casadaban, M., and S. N. Cohen. 1980. Analysis of gene controlsignals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol.138:179-207.

10. Casey, J., C. Daly, and G. F. Fitzgerald. 1991. Chromosomalintegration of plasmid DNA by homologous recombination inEnterococcus faecalis and Lactococcus lactis subsp. lactis hostsharboring Tn919. Appl. Environ. Microbiol. 57:2677-2682.

11. Costello, V. A. 1988. Characterization of bacteriophage-host inter-actions in Streptococcus cremoris UC503 and related lactic strep-tococci. Ph.D. thesis. National University of Ireland, UniversityCollege, Cork.

12. Davidson, B. E., I. B. Powell, and A. J. Hillier. 1990. Temperate

bacteriophages and lysogeny in lactic acid bacteria. FEMS Micro-biol. Rev. 87:79-90.

13. Gasson, M. J. 1983. Plasmid complements of Streptococcus lactisNCDO 712 and other lactic streptococci after protoplast-inducedcuring. J. Bacteriol. 154:1-9.

14. Godon, J.-J., C. Delorme, S. D. Ehrlich, and P. Renault. 1992.Divergence of genomic sequences between Lactococcus lactissubsp. lactis and Lactococcus lactis subsp. cremoris. Appl. Environ.Microbiol. 58:4045-4047.

15. Harley, C. B., and R. P. Reynolds. 1987. Analysis of E. colipromoter sequences. Nucleic Acids Res. 15:2343-2361.

16. Hayes, F., C. Daly, and G. F. Fitzgerald. 1990. Identification of theminimal replicon of Lactococcus lactis subsp. lactis UC317 plasmidpCI305. Appi. Environ. Microbiol. 56:202-209.

17. Holo, H., and I. F. Nes. 1989. High-frequency transformation byelectroporation, of Lactococcus lactis subsp. cremoris grown withglycine in osmotically stabilized media. Appl. Environ. Microbiol.55:3119-3123.

18. Klaenhammer, T. R., and G. F. Fitzgerald. 1993. Bacteriophagesand bacteriophage resistance, p. 106-168. In M. Gasson, and W.de Vos (ed.), Genetics and biotechnology of lactic acid bacteria.Blackie Academic & Professional, London.

19. Lillehaug, D., and N. K. Birkeland. 1993. Characterization ofgenetic elements required for site-specific integration of thetemperate lactococcal bacteriophage 4~LC3 and construction ofintegration-negative 4~LC3 mutants. J. Bacteriol. 175:1745-1755.

20. Moran, C. P., N. Lang, S. F. J. le Grice, G. Lee, M. Stephens, A. L.Sonenshein, J. Pero, and R. LosicL 1982. Nucleotide sequencesthat signal the initiation of transcription and translation in Bacillussubtilis. Mol. Gen. Genet. 186:339-346.

21. Pearson, W. R., and D. J. Lipman. 1988. Improved tools forbiological sequence comparison. Proc. Natl. Acad. Sci. USA85:2444-2448.

22. Raya, R. R., C. Fremaux, G. L. De Antoni, and T. R. Klaenham-mer. 1992. Site-specific integration of the temperate bacterio-phage 4adh into the Lactobacillus gasseri chromosome and mo-lecular characterization of the phage (attP) and bacterial (attB)attachment sites. J. Bacteriol. 174:5584-5592.

23. Rottlander, E., and T. A. Trautner. 1970. Genetic and transfectionstudies with Bacillus subtilis phage SP50. J. Mol. Biol. 108:47-60.

24. Silver, J., and V. Keerikatte. 1989. Novel use of polymerase chainreaction to amplify cellular DNA adjacent to an integratedprovirus. J. Virol. 63:1924-1928.

25. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium forlactic streptococci and their bacteriophages. Appl. Microbiol.29:807-813.

26. Tinoco, I., P. N. Borer, B. Dengler, M. D. Levine, 0. C. Uhlenbeck,D. M. Crothers, and J. Gralla. 1973. Improved estimation ofsecondary structure in ribonucleic acids. Nature (London) NewBiol. 246:40-41.

27. van de Guchte, M., J. Kok, and G. Venema. 1992. Gene expressionin Lactococcus lactis. FEMS Microbiol. Rev. 88:73-92.

28. Ye, Z. H., and C. Y. Lee. 1989. Nucleotide sequence and geneticcharacterization of staphylococcal bacteriophage L54a int and xisgenes. J. Bacteriol. 171:4146-4153.

VOL. 60, 1994

on August 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from