JOURNAL OF BACTERIOLOGY, Sept. 2006, p. 6629–6639 Vol. 188, No. 180021-9193/06/$08.00�0 doi:10.1128/JB.00672-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Sequence Analysis of the Lactococcal Plasmid pNP40: a MobileReplicon for Coping with Environmental Hazards†

Jonathan O’Driscoll,1 Frances Glynn,1 Gerald F. Fitzgerald,1,2 and Douwe van Sinderen1,2*Department of Microbiology1 and Alimentary Pharmabiotic Centre,2 University College Cork, Cork, Ireland

Received 11 May 2006/Accepted 28 June 2006

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, pos-sesses a potent complement of bacteriophage resistance systems, which has stimulated its application as afitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of thisplasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocinresistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adap-tation and DNA damage repair were identified, further confirming pNP40’s contribution to environmentalstress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resultingin a “stockpiling” of bacteriophage resistance systems.

Lactococcus lactis, a gram-positive lactic acid bacterium, hasbeen extensively exploited for the production of a variety offermented dairy products. L. lactis strains exhibit biotechno-logically important activities, which contribute to the characterof the final food product, e.g., lactose utilization and proteaseproduction, and in addition encode properties that specificallyprovide a selective advantage to the bacterium itself, e.g.,heavy metal resistance, bacteriocin production and/or immu-nity, and bacteriophage resistance (47). Many of these indus-trially significant traits have been found to be encoded byplasmids, which are omnipresent among this species, with mostisolates containing multiple plasmids ranging in size from 2 to80 kb (11).

In recent decades, extensive research has established themolecular mechanisms governing many of these activities, inparticular with respect to bacteriophage resistance (47). Lacto-coccal strains used for many food fermentations are known tobe persistently challenged by phages and probably as a conse-quence have evolved numerous bacteriophage resistance strat-egies (48, 67).

Presently, there are 30 completely sequenced lactococcalplasmids, the largest being pSK11P, a 75.8-kb plasmid isolatedfrom L. lactis subsp. cremoris SK11 (47, 62). This plasmidencodes a variety of functions, including copper resistance,proteolytic activity, cold shock proteins, and cation transportactivities, and displays clear “markings” of multiple recombi-nation events that may have contributed to its evolution (62).

Previous studies of a similarly sized plasmid, pNP40, orig-inally identified in L. lactis subsp. diacetylactis DRC3 (45),revealed that this molecule, besides its encoded nisin andcadmium resistance determinants, is responsible for an im-pressive bacteriophage resistance profile (16, 19, 20, 50, 65).Two such systems, AbiE and AbiF, were found to provide

significant resistance that correlates to an abortive infectionphenotype (19).

In addition, on the basis of phenotypic evidence, the pres-ence of a third mechanism active at the stage of phage DNAinjection was proposed (20). Most recently, a fourth resistancesystem, the LlaJI restriction-modification system, was identi-fied (50).

In the present study, we report the complete sequence ofpNP40. Analysis of the sequence revealed the genetic deter-minants involved in replication and conjugation, in addition togenes responsible for previously uncharacterized functions.Furthermore, evidence is offered which attests to pNP40’s fullbacteriophage resistance potential.

MATERIALS AND METHODS

Bacteria, bacteriophage, plasmids, media and growth conditions. Details ofthe bacterial strains, bacteriophages, and plasmids used in the present study aresummarized in Table 1. All L. lactis strains were grown in M17 broth (Oxoid ltd.,Hampshire, United Kingdom) containing 0.5% glucose at 30°C. Escherichia coliwas grown at 37°C in Luria-Bertani (LB) medium (58). Where appropriate,antibiotics were added as follows: for L. lactis, tetracycline at 5 �g ml�1, chlor-amphenicol at 10 �g ml�1, and erythromycin at 1 �g ml�1; for E. coli, ampicillinat 100 �g ml�1, kanamycin at 25 �g of ml�1, chloramphenicol at 10 �g ml�1, anderythromycin at 100 �g ml�1. LB medium was supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside; 40 �g ml�1) and IPTG (iso-propyl-�-D-thiogalactopyranoside; 1 mM) where appropriate. Recombinant L.lactis cells containing pAK80 and derivatives were selected on GM17 agar (con-taining 0.5 M sucrose) with erythromycin, supplemented with X-Gal (40 �gml�1). Lactococcal bacteriophage were propagated as described previously (50).Plaque assays were conducted as described elsewhere (41) and the efficiency ofplaquing (EOP) was calculated as the ratio of the number of plaques formed ona tested host to those formed on a sensitive host.

Molecular techniques and shotgun cloning. Restriction enzymes and T4 DNAligase were purchased from New England Biolabs (Hertfordshire, United King-dom) and used according to the manufacturer’s instructions. E. coli plasmidDNA was isolated by using the SV Wizard plasmid miniprep kit (Promega,Madison, WI). Routine lactococcal plasmid DNA isolations were performed asdescribed previously (52). Isolation of pNP40 plasmid DNA for shotgun cloningwas performed as described previously (2), and the resulting DNA preparationwas purified by cesium chloride-ethidium bromide density gradient ultracentrif-ugation using standard techniques (58). Electroporation of plasmid DNA into E.coli was performed by using standard techniques (58) and in L. lactis as describedpreviously (69). Purified pNP40 DNA was digested with HindIII, EcoRI, andXbaI separately and shotgun cloned into the E. coli plasmid pBluescript KS(�).Large restriction fragments (�5 kb) were further digested with DraI, Sau3A, or

* Corresponding author. Mailing address: Department of Microbi-ology, University College Cork, Western Road, Cork, Ireland. Phone:353 214901365. Fax: 353 214903101. E-mail: d.vansinderen@ucc.ie.

† Supplemental material for this article may be found at http://jb.asm.org/.

6629

HaeII prior to cloning in pBluescript. For routine PCR applications Taq DNApolymerase (QIAGEN, West Sussex, United Kingdom) was used. For plasmidconstruction, where high fidelity was required, PCRs were conducted by usinga combination of Taq DNA polymerase and Proofstart DNA polymerase(QIAGEN) according to the manufacturer’s instructions. All PCR products werepurified by using the Jetquick PCR purification kit (Genomed, Lohne, Ger-many). The correct orientation and integrity of all constructs was verified bysequencing, performed at MWG Biotech (Ebersberg, Germany).

Sequence assembly and annotation. Sequence assembly, verification, and anal-ysis of pNP40 and plasmid constructs were achieved by using the SeqManprogram from the DNASTAR package (DNASTAR, Madison, WI). Initial an-notation was performed automatically using the open reading frame (ORF)finder tool from the National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov/) and the DNASTAR software package. Potential ORFs weresubsequently manually analyzed by database searches using the BLAST suite ofprograms (including blastp, clusters of orthologous groups [COG], conserveddomain database [CDD], and the conserved domain architecture tool [CDART])(1), which incorporate domains imported from the simple modular architectureresearch tool (SMART) (40) and the protein family database (Pfam) (15).Additional searches were performed by using the CBS prediction servers (http://www.cbs.dtu.dk/services/). The complete nucleotide sequence of pNP40 hasbeen deposited in the GenBank database under accession no. DQ534432.

Plasmid constructs. All of the oligonucleotide sequences (and coordinates)used for plasmid construction are available online (see Table S2 in the supple-mental material). For the construction of plasmid pAKC, a PCR product en-compassing the assumed cspC promoter region (using primers AKCF andAKCR) was inserted into the HindIII-BglII sites of pAK80. For pAKD, a PCRproduct encompassing the assumed cspD promoter region (using primers AKDFand AKDR) was inserted into the HindIII-BglII sites of pAK80. For pNZ-CD,

a cspCD-encompassing PCR product which also included both cspC and cspDpromoter regions (using primers cspCF and cspDR) was inserted into the NcoI-PstI sites of pNZ8048. Plasmid pAK17 was constructed by insertion of a PCRproduct encompassing the orf17 upstream region (using primers orf17F andorf17R) into the HindIII-BglII sites of pAK80. For pAK18, a PCR productencompassing the upstream orf18 region (using primers orf18F and orf18R) wasinserted into the HindIII-BglII sites of pAK80. For pAK25, a PCR productencompassing the upstream region of orf25 (using primers orf25F and orf25R)was inserted into the HindIII-BglII sites of pAK80. Plasmid pORF1 was con-structed by insertion of a PCR product encompassing orf1 and its presumedtranscriptional signals (using primers orf1F and orf1R) into the BglII site ofpPTPi. Plasmid pORF13 was constructed by insertion of an orf13 encompassingPCR product (using primers orf13F and orf13R) into the NcoI-Asp718 sites ofpNZ44. Plasmid pNZ-6.2 was constructed by insertion of a 6.2-kb NcoI fragmentof pNP40 (coordinates 21824 to 28061) into the NcoI site of pNZ8048. Forplasmid pNZ-7, a 7-kb NcoI fragment of pNP40 (coordinates 14786 to 21823)was inserted into the NcoI site of pNZ8048. Plasmid pNZ-10 was constructed byinsertion of a 10-kb NcoI fragment of pNP40 (coordinates 32175 to 42226) intothe NcoI site of pNZ8048.

Intracellular phage DNA replication. Lactococcal strains were grown to theearly logarithmic growth phase (optical density at 600 nm [OD600] of �0.3), afterwhich CaCl2 was added to a final concentration of 10 mM, and the cells wereinfected with the �sk1 or �sk1.m (as indicated) at a multiplicity of infection of0.1. Intracellular phage DNA replication was monitored as described previously(28). DNA preparations were separated by electrophoresis on a 1% agarose geland transferred to a Hybond-N� nylon membrane (Amersham, United King-dom) by capillary transfer using 10 mM NaOH. Membranes were probed with a�sk1-specific PCR product (generated by using the primers SK1F [5�-GGCTATATGCTCATATTTTGG-�3] and SK1R [5�-CTCTCACCGCCATATTGTC-�3])

TABLE 1. Bacteria, bacteriophages, and plasmids used in this study

Strain, bacteriophage,or plasmid Relevant characteristic(s)a Source or

reference

StrainsE. coli EC101 Cloning host, RepA�; Kanr 37L. lactis

MG1614 Plasmid-free derivative of L. lactis subsp. lactis 712 22MG1614/pNP40 MG1614 transconjugant containing pNP40 MFRC b

Bacteriophagessk1 Small isometric-headed phage for MG1614 7sk1.m sk1 propagated on LlaJI� host This study

PlasmidspNP40 Naturally occurring 64.9-kb lactococcal plasmid 45pNP40Soe1 pNP40 derivative with region encompassed by coordinates 31298 to 40722 deleted This studypPTPi Low-copy-number E. coli-L. lactis shuttle vector for cloning; Tcr 50pJO-J pPTPi derivative containing complete 6.2-kb LlaJI operon from pNP40 50PORF1 pPTPi derivative containing orf1 This studypNZ8048 High-copy-number E. coli-L. lactis overexpression vector, PnisA; Cmr 13pNZ-6.2 pNZ8048 derivative containing 6.2-kb NcoI fragment of pNP40 This studypNZ-7 pNZ8048 derivative containing 7-kb NcoI fragment of pNP40 This studypNZ-10 pNZ8048 derivative containing 10-kb NcoI fragment of pNP40 This studypNZ-CD pNZ8048 derivative containing cspC and cspD This studypNZ44 pNZ8048 derivative; PnisA replaced with constitutive P44 44PORF13 pNZ44 derivative containing orf13 This studypAK80 Promoter selection vector containing promoterless lacL and lacM genes; Emr 30PAKC pAK80 derivative containing 284-bp fragment of pNP40 encompassing cspC promoter This studyPAKD pAK80 derivative containing 237-bp fragment of pNP40 encompassing cspD promoter This studypAK17 pAK80 derivative containing 268-bp fragment of pNP40 encompassing orf17 (OrfU) promoter This studypAK18 pAK80 derivative containing 376 bp fragment of pNP40 encompassing orf18 (RecALP) promoter This studypAK25 pAK80 derivative containing 334-bp fragment of pNP40 encompassing orf25 (UvrA) promoter This studypPG05 pAM401 derivative containing an ScaI-NcoI fragment of pNP40, AbiE�; Cmr 19pPG23 pAM401 derivative containing an XbaI-NcoI fragment of pNP40, AbiF�; Cmr 19pVE6007 Temperature-sensitive RepA� helper plasmid; Cmr 42pOri280 RepA� integration vector; Emr 38pSoe1 pOri280 derivative containing SOEing PCR fragments from pNP40 (see Materials and Methods) This study

a Emr, erythromycin resistance; Cmr, chloramphenicol resistance; Tcr, tetracycline resistance; Kanr, kanamycin resistance.b MFRC, Moorepark Food Research Centre, Moorepark, Fermoy, Cork, Ireland.

6630 O’DRISCOLL ET AL. J. BACTERIOL.

using the ECL direct nucleic acid labeling and detection system (Amersham)according to the manufacturer’s instructions. Control phage DNA was isolatedfrom lysates as described previously (49).

Freeze-thaw challenge and DNA damage assays. Freeze-thaw challenge ex-periments were performed essentially as described elsewhere (71, 72). Briefly,cells were grown in GM17 medium at 30°C to an OD600 of 0.5 and subjected toa cold shock by rapid temperature downshift from 30 to 10°C for 0, 2, or 4 h, afterwhich 1 ml of these cultures was frozen at �20°C. After a 24-h freezing period,the cells were allowed to thaw at 30°C for 4 min with subsequent survival ofstrains assayed by determining viable counts. This freeze-thaw cycle was per-formed four times. Sensitivity to chemically induced DNA damage was assayedby inclusion of mitomycin C (MMC; 2.5 �g ml�1) in growth media. Cells weregrown to early log phase (OD600 �0.2), at which point the mutagen was added,followed by monitoring of growth by means of OD600 measurement. Viablecounts were performed at selected time points to corroborate OD600 measure-ments.

�-Galactosidase assays. �-Galactosidase assays were performed essentially asdescribed previously (30). For analysis of cold shock-induced transcription, cellscontaining various lacZ-transcriptional fusions were grown to an OD600 of 0.5and subjected to a cold shock at 10°C for 0, 2, and 4 h, after which 1-ml sampleswere harvested and analyzed for �-galactosidase activity.

For analysis of DNA damage-induced gene expression, cells containing variouslacZ transcriptional fusions were grown to OD600 of 0.2 to 0.3, followed by theaddition of MMC (2.5 �g ml�1), after which 1-ml samples were harvested at 0,15, 30, 60, and 90 min and analyzed for �-galactosidase activity.

Construction of a pNP40 deletion derivative. The deletion derivative of pNP40was constructed by targeted deletion mutagenesis as described previously (10, 37,

38). Briefly, two PCR products, A and B, flanking the region of pNP40 to bedeleted, were generated. Product A was amplified by using primers M1Fa andM1Rb, whereas product B was amplified by using primers M1Fc and M1Rd, andthe two resulting DNA fragments were joined by SOEing PCR as describedpreviously (29). This SOEing product was then inserted into the NcoI-BamHIsites of the integration vector pOri280 (RepA�) in the E. coli cloning host EC101(RepA�), to generate plasmid pSoe1. Plasmid pSoe1 was subsequently estab-lished in the MG1614/pNP40 background and selected for first and secondcrossover events. The integrity of the pNP40 deletion derivative, pNP40Soe1,was verified by PCR, Southern hybridization, and sequencing.

RESULTS AND DISCUSSION

Plasmid sequence and genetic organization. The conjugativelactococcal plasmid pNP40, originally identified in L. lactissubsp. diacetylactis DRC3 (45), was established in the plasmid-free lactococcal strain MG1614, from which plasmid DNA wassubsequently purified and sequenced (as outlined in the Ma-terials and Methods). Assembly of the resulting sequencesresulted in a single contiguous contig representative of a cir-cular 64,980-bp DNA molecule with a G�C content of32.33%. This is somewhat lower than that of L. lactis chromo-somal DNA (35.4% for IL1403 [4], 35.8% for MG1363 [34],and 35% for SK11 [Joint Genome Institute]) but is within the

FIG. 1. Genetic map of the lactococcal plasmid pNP40. Block arrows and lines represent identified ORFs. A number of functional gene clustersare indicated.

VOL. 188, 2006 LACTOCOCCAL PLASMID-ENCODED SAFEGUARD 6631

range exhibited by most sequenced lactococcal plasmids todate (30 to 40%) (47). A total of 62 ORFs were identified inthe pNP40 sequence, which were analyzed in detail below (seeMaterials and Methods, Fig. 1, and Table S1 in the supple-mental material).

pNP40 replication functions. Previous work had localizedthe minimal replicon for pNP40 to a cloned 7.6-kb EcoRIfragment, which also expressed a nisin resistance activity (seebelow) (16). Further subcloning suggested that the replicationfunctions were localized on a 5.0-kb EcoRI-XbaI fragment,corresponding to coordinates 59625 to 64637 of the pNP40sequence. This region was found to contain orf59 (designatedrepA), orf60 (designated repB), and orf61 (see Table S1 in thesupplemental material and Fig. 2A).

Analysis of the amino acid sequence of RepA revealed sim-ilarity to a number of pLS32-type theta replication proteins,including that of pCI2000 (AAF27326) from Lactococcus(73% identity). Consistent with this was the presence of areplication initiation N-terminal domain (pfam06970), which

was noted to contain a helix-turn-helix motif (amino acid [aa]68 to 89). Analysis of the coding region of RepA revealed thepresence of a 40-bp sequence directly repeated two and threequarter times (Fig. 2B; additional features are as indicated).Such repeats, also termed iterons, are common elements of theorigin of replication (ori) for many theta replicating plasmids(12, 25, 33, 35). Interestingly, the pLS32 ori is believed to besimilarly located within the coding sequence for its replicationinitiation protein, RepN (63). This intragenic iteron localitywas also noted for the pNP40 RepA homologue present onpCI2000 (31).

Located upstream of repA and transcribed divergently,repB is predicted to encode a gene product, which exhibitssignificant similarity to a number of replication-associatedproteins from gram-positive plasmids (see Table S1 in thesupplemental material), while containing SoJ (COG1192),Mrp (COG0489), and COG0455 domains, thus making it amember of the ParA ATPase family (pfam00991) involvedin plasmid and chromosome partitioning. An equivalent

FIG. 2. (A) Genetic organization of the replication region of pNP40. B. Sequence of repA and upstream and downstream regions. Translationalstart sites of repA and repB are in boldface, presumed ribosome-binding sites are underlined, and predicted consensus �10 and �35 sequences ofthe repB promoter are shaded. Inverted repeat structures are represented by opposing arrows, and the 40-bp directly repeated putative iteronsequences are indicated by the dashed arrows.

6632 O’DRISCOLL ET AL. J. BACTERIOL.

ParA-type protein has previously been associated with anactive plasmid partitioning system, and RepB likely medi-ates an equivalent stability function (31).

Immediately downstream of repB, a small coding region,orf61 is present. Homologous RepB-linked coding regionshave been noted in many theta replicons which, due to theirproximity and apparent transcriptional and/or translationalcoupling to RepB, are thought to be involved in the replica-tion-partitioning process (3, 5, 25).

The genetic organization, similarities, and iteron structure ofthe pNP40 replication region strongly suggests that this largeplasmid replicates via a theta-type mechanism. Furthermore,and typical of theta replicating plasmids, pNP40 appears tocontain determinants that contribute to its high segregationalstability (45).

Conjugal transfer determinants. In other microorganismsthe structure and function of the conjugative apparatus hasbeen examined in detail (18, 26, 36, 39). The conjugal capacityof pNP40 has previously been phenotypically demonstrated(27, 65). On the basis of similarity searches of the pNP40sequence, we predict the presence of a conjugal transfer genecluster on an approximately 17-kb section (coordinates 44565to 61395), which contains 19 ORFs (orf40 to orf58) arranged inan operon structure (Fig. 3A and Table S1 in the supplementalmaterial). The protein specified by orf58 (designated MobD)exhibits homology to the nickase-relaxase family of proteins(pfam03432), which introduce a nick at the origin of transfer(oriT) to initiate single-stranded plasmid DNA transmission

from the donor to a recipient cell. A number of inverted repeatstructures, reminiscent of an oriT, were identified downstreamof the MobD coding region which was noted to be significantlyAT-rich (�70%) (Fig. 3B). However, no obvious candidateconsensus nick site (26) is present within this putative oriT.

The pNP40 conjugation region appears to consist of mod-ules each of which displaying sequence similarity to discretesections from assumed conjugation regions from enterococcalplasmids (Fig. 3A). The notable exceptions include orf44,orf45, and orf53, which appear to represent components uniqueto pNP40. The product encoded by orf54 (designated TraF)is a membrane-spanning protein likely to be involved inmating channel formation, whereas ORF55 was noted tocontain the “antirestriction” domain COG4227, in additionto a conserved H-E-X-X-K catalytic active site, and likelyprovides a temporal protection to the transferred plasmidDNA against restriction endonucleases, allowing establish-ment in the recipient cell (70).

The orf43 and orf48 gene products correspond to the con-served TraG (cd01126) (pfam02534) and TraE (COG3451)conjugation proteins, respectively, whose conserved domainsare suggestive of a type IV secretion function (9, 23, 24, 61).

Both ORF49 and ORF50 exhibit homology to distinct re-gions within the protein product of ep0036 on pAM373(AAG40447). ORF49 contains a conserved FlgJ muramidasedomain (COG1705), whereas ORF50 contains a CHAP ami-dase domain (pfam05257), in contrast to EP0036, which con-tains both. These conserved domains are present in cell wall-

FIG. 3. (A) Genetic organization to the pNP40 conjugation region (Top). Checkered arrows represent ORFs encoding predicted membrane-spanning proteins. Conserved gene clusters (and percent identities) similar to segments of the conjugal transfer region of the enterococcal plasmidspCF10 and pAM373 are indicated. (B) Sequence of the region downstream of the mobD gene, predicted to contain the oriT. The mobDtranslational stop codon is in boldface, and the putative transcriptional terminator for the conjugation operon is indicated by opposing arrows(IR1). A perfect 22-bp inverted repeat (IR2) is indicated; this repeat was also noted to constitute two perfect 22-bp tandem repeats.

VOL. 188, 2006 LACTOCOCCAL PLASMID-ENCODED SAFEGUARD 6633

metabolizing proteins; therefore, it can be speculated thatORF49 and ORF50 participate in facilitating the passage ofDNA and/or proteins across the cell envelope by virtue of theirpeptidoglycan-degrading activity.

Cadmium resistance. Previously, the cadmium resistanceencoded by pNP40 has been demonstrated to be a selectablemarker for pNP40 dissemination to an industrial starter cul-ture (65). Furthermore, the presence of a CadA homologuewas confirmed by PCR using primers specific for the previouslypublished CadA homologue of pAH82 (51).

The cadmium resistance region of pNP40 was found to re-side in a section encompassed by coordinates 40808–43281,where two similarly oriented overlapping coding regions weredistinguished (ORF36 and ORF37). The first ORF, ORF36,corresponds to the previously identified cadA homologue (65),whose product contained conserved domains consistent withproteins involved with Cu, Cd, Co, and Zn transport and de-toxification (cdd00371), inorganic ion transport (COG2608),and cation transport (COG2217, pfam00122, pfam00702,COG0474, COG2216, and COG4087). The presence of theseconserved regions indicates that the CadA activity spectrum is

not solely restricted to cadmium efflux, as has previously beenreported for other homologues (60).

The protein encoded by orf37 was found to be 100% iden-tical to an abundance of CadC proteins, all of which wereencoded by genes adjacent to CadA homologues. CadC wasnoted to possess a number of helix-turn-helix containing con-served domains (cdd00090, smart00418, and CAG0640) typicalof homodimeric repressors, which dissociate from their targetDNA in the presence of metal ions.

Nisin resistance region. As with cadmium resistance, thenisin resistance capabilities of pNP40 have been documented(16, 45). The sequenced nisin resistance gene, designated nisR,was located on a 1.8-kb EcoRI-KpnI fragment (coordinates803 to 2584) (17). Analysis of the amino acid sequence of NisRconfirmed the presence of an N-terminal membrane-spanningdomain (aa 7 to 29) (but no signal peptide sequence) as sug-gested by Froseth and McKay (17) and further revealed the pres-ence of conserved protease-peptidase domains (smart00245 andpfam03572) spanning approximately 200 aa at the C termi-nus, which was predicted to reside outside the cell mem-brane. From this it can be inferred that the mechanism of

FIG. 4. (A) �-Galactosidase assays of the cspC promoter transcriptional fusion (present on pAKC) after 2 and 4 h of cold shock at 10°C. Thegraph depicts the fold increase in promoter activity after cold shock treatment relative to the promoter activity under non-cold-shock conditions(i.e., 30°C). Bars: 1, MG1614/pAKC (2 h at 10°C); 2, MG1614/pAKC (4 h at 10°C); 3, MG1614/pNP40/pAKC (2 h at 10°C); 4, MG1614/pNP40/pAKC (4 h at 10°C). Absolute values are listed beneath the graph. (B) �-Galactosidase assays of the cspD promoter transcriptional fusion (presenton pAKD) after 2 and 4 h of cold shock at 10°C. The graph depicts the fold increase in promoter activity after cold shock treatment relative tothe promoter activity under non-cold-shock conditions (i.e., 30°C). Bars: 1, MG1614/pAKD (2 h at 10°C); 2, MG1614/pAKD (4 h at 10°C); 3,MG1614/pNP40/pAKD (2 h at 10°C); 4, MG1614/pNP40/pAKD (4 h at 10°C). Absolute values are listed beneath the graph. (C) Survival ofMG1614 and MG1614/pNP40 frozen at �20°C after successive freeze-thaw cycles after exposure to a cold shock at 10°C for 0, 2, and 4 h asindicated.

6634 O’DRISCOLL ET AL. J. BACTERIOL.

NisR-mediated nisin resistance occurs via proteolytic deg-radation of nisin.

Located immediately downstream and convergently ori-ented to the nisR gene, orf1 is predicted to encode an integralmembrane protein containing a conserved, C-terminally lo-cated Abi, CAAX amino-terminal protease domain (pfam02517),which is typical of this diverse family of metal-dependent mem-brane proteases that are involved in protein and peptide modifi-cation and secretion (53). It has been suggested that these pro-teins may also play a role in bacteriocin maturation and transportand in resistance (14, 53).

Cold shock determinants. The protein products of two sim-ilarly oriented open reading frames, ORF38 and ORF39, areidentical or nearly identical to the cold shock proteins CspDand CspC, respectively, of lactococcal chromosomal or plasmidorigin. In addition, both conserved “cold shock” DNA- andRNA-binding domains (pfam00313 and smart00357, respec-tively) were observed within the pNP40-encoded CspC andCspD amino acid sequences.

Previous studies on the chromosomal CspC and CspD ho-mologues have illustrated the physiological response mediatedby these cold shock proteins; expression of CspD was enhancedby cold shock and resulted in increased survival at low tem-peratures, whereas only a modest increase in CspC expressionaccompanied cold shock, which was found to directly alter thelevels of other cold shock proteins (71–73).

Transcriptional fusions of both the cspC and the cspD pro-moter regions of pNP40 showed that both promoters wereinduced by cold shock in a manner that was independent of thepresence of pNP40 (Fig. 4A and B). The presence of these coldshock-related determinants on pNP40 suggests that this plas-mid mediates an enhancement of the cold shock response. Ananalysis of the freeze-thaw survival capacity of MG1614 con-taining pNP40 compared to the plasmid-free strain illustrateda small but appreciable increase in survival (Fig. 4C), which

was also observed with a construct containing the cspC andcspD genes cloned in tandem (pNZ-CD; data not shown).

The full phenotypic potential associated with these pNP40-encoded determinants, while clearly inducible, is likely only tobe significantly detectable in a host (perhaps the originalpNP40-containing host [45]) that does not encode chromo-somal or plasmid cspC and cspD copies (or possibly underalternative stress or cold shock conditions).

DNA damage repair. pNP40 was found to possess threeORFs—ORF17, ORF18, and ORF25—which were predictedto encode proteins involved in DNA repair. The RecA(ORF18) and UmuC-like (ORF17) homologues, designatedRecALP and OrfU, respectively, have been described previ-ously (19, 21) (see Table S1 in the supplemental material andFig. 1).

Analysis of the amino acid sequence of RecALP revealed thepresence of multiple conserved domains characteristic of bac-terial RecA homologues involved in homologous recombina-tion, DNA repair, and SOS response induction (cd00983,cd01393, cd01120, and pfam00154). Adjacent and similarly ori-ented with respect to recALP, the orf17 gene encodes theUmuC-like protein, OrfU, which contains two conserved do-mains: the IMS (impB/mucB/samB) family (pfam00817; UVprotection) and DinP (COG0389; DNA repair DNA poly-merases). The UmuC family of proteins are an essential com-ponent of the DNA damage mutagenesis mechanism of E. coliand constitute the catalytic subunit of DNA polymerase V,which possesses a translesion DNA synthesis activity at theexpense of normal replicative fidelity (54, 55, 64, 68).

The product of orf25 was predicted to harbor an UvrA ex-inuclease domain (COG0178) containing conserved, inter-rupted, N- and C-terminal ABC-ATPase motifs and similarlylocated zinc fingers. UvrA-type proteins are believed to com-prise the ATPase subunit of the UvrABC nucleotide excisionrepair system (66). BLAST searches of the ORF25 (designated

FIG. 4—Continued.

VOL. 188, 2006 LACTOCOCCAL PLASMID-ENCODED SAFEGUARD 6635

UvrA) amino acid sequence revealed 64% identity to UvrA ofLactobacillus plantarum WCFS1 (CAD63845) and 62% iden-tity to UvrA of L. caseii ATCC 334 (ZP_00386320).

The presence of pNP40-encoded components of DNA re-pair systems prompted us to investigate the growth and survivalof a pNP40-containing host in response to chemical-inducedDNA damage. To this end, MG1614 containing pNP40 waschallenged with MMC, and its growth profile was monitored.As can be seen from Fig. 5A, a significant difference in thegrowth profile was evident for the pNP40-containing host com-pared to that of the control strain in response to MMC. ThepNP40-containing strain not only reached a higher final opticaldensity but also did not lyse to the same extent as the controlstrain. The optical density values described were corroboratedby viable plate counts (data not shown).

To establish whether the expression of orfU, recALP, anduvrA was induced in response to MMC, transcriptional fusionswere constructed (see Materials and Methods). Of these, onlythe upstream regions of orfU and uvrA were found to containactive (but relatively weak) promoters (in the absence ofMMC) in a lactococcal host. The upstream recALP region ap-parently did not contain any active transcriptional signals (inthe presence or absence of MMC). Given that recALP-specific

mRNA was detectable by dot blot analysis and the apparentlyidentical recALP and orfU transcriptional yields (21), it is likelythat these genes constitute a dicistronic operon transcribedfrom the orfU promoter. This may account for the inability ofa cloned recALP to complement a chromosomal recA mutant(21), since recALP expression may not be entirely synchronizedwith that of the chromosomal version (see below).

As can be seen from Fig. 5B, expression from the orfUpromoter was induced up to threefold when monitored for 90min after exposure to MMC (which was not influenced by thepresence of pNP40 [data not shown]), whereas no such in-crease in transcriptional activity was observed for the uvrApromoter. The latter promoter appears to be constitutive dur-ing exponential growth and increases in activity up to two- tothreefold during early stationary phase in the absence of aDNA-damaging agent (data not shown). This lack of inductionof uvrA in response to DNA damage, although in contrast tothat observed for E. coli (32) and B. subtilis (8), is consistentwith that noted for a uvrA homologue of Pseudomonas aerugi-nosa (56).

Analysis of the sequence of the inducible orfU promoterregion revealed the presence of a conserved “HdiR” box (59)(Fig. 5C). HdiR is a LexA-like DNA damage regulator of L.

FIG. 5. (A) Growth of MG1614 and MG1614/pNP40 in response to MMC. The arrow on the graph indicates when MMC was added to theculture. (B) �-Galactosidase assay of the orfU promoter transcriptional fusion (pAK17) in the MG1614 host background at 15, 30, 60, and 90 minafter exposure to MMC. The graph depicts the fold increase in promoter activity (and the P value) in the presence of MMC relative to the promoteractivity at each time point in the absence of MMC. (C) Sequence of orfU, umuC (IL1403), and hdiR upstream regions. The �10 and �35 sequencesare shaded, and the orfU start codon is in boface. Sequences corresponding to the core HdiR binding motif (ATCAGW5CTGAT) are underlined.

6636 O’DRISCOLL ET AL. J. BACTERIOL.

lactis MG1363 and was shown to regulate the expression of theorfU homologue (umuC) from L. lactis IL1403 in addition to itsown gene in response to MMC (59). Interestingly, MG1363does not possess an umuC homologue (59). It is thereforelikely that the observed induction of orfU from pNP40 in thepresence of MMC is mediated by HdiR derepression. TheuvrA promoter lacks “HdiR box”-resembling sequences, whichis consistent with its expression pattern.

Insertion sequence elements: did pNP40 evolve as a result ofcointegration? Insertion sequence (IS) elements are known tocontribute significantly to species evolution (47, 57). Six ISelements were identified on the pNP40 sequence, only two ofwhich (encoded by orf7 and orf35) appeared to be complete.Both ORF7 (227 aa) and ORF35 (194 aa) were found to behighly similar to ISS1 of the lactococcal plasmid pTD1(CAA44601) and contained conserved integrase (pfam00665)and transposase (COG3316) domains. This almost exact ISS1duplication on pNP40 suggests that the intervening region be-tween orf7 and orf35 may have been acquired by an insertionevent.

ISS1 is a member of the IS6 family, whose insertion se-quences thus far have been noted to give rise exclusively tocointegrant replicon fusions (43). Downstream of the abiFgene within the ISS1-flanked section of pNP40, a 23-bp se-quence repeated three and a half times (reminiscent of theiteron-containing origin of replication from some theta plas-mids) has previously been reported (19). In addition, the orf8-to-orf10 region (in particular orf10) appear(s) to encode plas-mid stability and maintenance determinants. The presence ofthese coding regions, and the iteron-like sequences mentionedabove suggest that this section may have at some stage beencapable of autonomous replication. These observations pro-vide evidence which corroborates suggestions that pNP40 mayhave evolved as a result of a cointegration event (19).

The four remaining IS elements of pNP40 (orf23, orf24,orf26, and orf62) all encode apparent truncated and/or inacti-vated derivatives of members of the IS3 family (whose geneticorganization usually consists of two genes translated togetheras a single polypeptide via translational slippage (6, 43).

The fourth bacteriophage-resistance phenotype: injectionblocking versus synergy. Two abortive infection phage resis-tance systems (AbiE and AbiF) (19) and a putative DNApenetration blocking system (20) have been reported to resideon pNP40. Evidence for the latter system was initially based onthe enhanced pNP40-mediated resistance to �c2 compared tothe level of resistance afforded by AbiF alone, with the ob-served phenotype noted to act prior to AbiF-mediated cellkilling. The discovery of an active pNP40-encoded restriction-modification system, LlaJI (50) (which fulfills most of the pen-etration blocking phenotypes (20), made it essential to redefinethe residual pNP40-mediated phage resistance activity. A smallisometric-headed phage, �sk1, was selected for this analysissince this phage could grow with apparently equal efficiency ona host containing either AbiE or AbiF alone. Although �sk1was restricted by a host containing the LlaJI system, propaga-tion of surviving phage on the same LlaJI-containing host

FIG. 6. Intracellular sk1 DNA replication in MG1614 (A) and MG1614/pNP40 (B). (A) Lane 1 contains purified sk1 control DNA, whereaslanes 2 to 5 represent total DNA samples isolated at 0, 20, 40, and 60 min postinfection, respectively, as indicated above each lane. Lysis hadinitiated with this host after 40 min. (B) Lane 1 contains purified sk1 control DNA, whereas lanes 2 to 8 represent total DNA isolated at 0, 20,40, 60, 80, 100, and 120 min postinfection, respectively, as indicated above each lane. No lysis was evident with this host during the course of theexperiment.

TABLE 2. Phage resistance profile of L. lactis MG1614 containingAbiE, AbiF, LlaJI, and pNP40 against phage sk1 and sk1.m

(propagated on the LlaJI� host) at 30° C

Strain/plasmid

Phagea

� sk1 � sk1.m

EOP Plaque size(mm) EOP Plaque size

(mm)

MG1614 1 3 1 3MG1614/pPTPi 1 3 1 3MG1614/pPG05

(AbiE)1 2–3 1 2–3

MG1614/pPG23(AbiF)

1 3 1 3

MG1614/pNP40 7 10�4 2, 1, and �1 5 10�3 2, 1, and �1MG1614/pJO-J

(LlaJI)3.3 10�3 3 1 3

VOL. 188, 2006 LACTOCOCCAL PLASMID-ENCODED SAFEGUARD 6637

yielded completely LlaJI-insensitive progeny. These methyl-ated phage (�sk1.m) were therefore expected to be insensitiveto all characterized phage resistance systems present on pNP40and ideal for detection of any remaining unidentified resis-tance mechanism (provided �sk1.m was sensitive to such asystem).

As can be seen from Table 2, �sk1.m formed plaques withequal efficiency on all strains (although slightly tighter plaqueswere formed on the AbiE-containing host), with the exceptionof the host possessing pNP40, confirming the presence of aresidual resistance phenotype against this phage. In addition,accumulation of intracellular �sk1.m DNA was considerablydelayed in the pNP40-containing host compared to that of thesensitive host (Fig. 6). Here, a high intracellular �sk1.m DNAconcentration was detected in the control MG1614 host after40 min, with lysis ensuing. In the pNP40-containing host, anequivalent concentration of intracellular �sk1.m DNA was notdetected until 80 to 100 min postinfection.

A “scan” of pNP40 was performed by the construction of adeletion derivative (pNP40Soe1) and multiple subclones(pORF1, pORF13, pNZ-6.2, pNZ-7, and pNZ-10), essentiallyencompassing the orf11-to-orf35 region, which, when examinedfor any (loss of) associated phage resistance phenotype, failedto reveal the presence of an as-yet-unidentified resistance sys-tem (see Table 1 and Materials and Methods). Therefore, theresidual phage resistance phenotype associated with pNP40could not be ascribed to any of the distinctive genetic deter-minant(s) characterized. This resistance may be attributable tosynergistic enhancement of the characterized resistance sys-tems rather than the presence of a fourth (penetration block-ing) system, particularly since AbiE and AbiF have previouslybeen implicated in such a enhancement phenotype (46). Anexamination of the phage resistance profiles of specific AbiE�

and AbiF� deletion derivatives of pNP40 would be required toverify this suggestion.

Concluding remarks. Analysis of the sequence of the 64.9-kbp pNP40 plasmid has provided the genetic confirmation andlocalization of a number of previously described functions suchas conjugation, cadmium resistance, nisin resistance, bacterio-phage resistance, and replication. In addition, new determi-nants for cold shock resistance and DNA damage repair wereidentified and confirmed phenotypically.

Lactococcal plasmids such as pNP40 appear to endow theirrespective hosts with multiple biologically and biotechnologi-cally important properties, many of which have been geneti-cally characterized (47). The extent to which pNP40 is able tolimit bacteriophage proliferation must surely be a reflection ofthe selective pressure to which this plasmid and associated hosthave been exposed. In conclusion, it would appear that relativeto the sequenced lactococcal plasmids to date (47, 62), themagnitude of the genetic “arsenal” possessed by pNP40 tocope with environmental hazards (some of which are unique tothis plasmid, e.g., recALP, uvrA, abiEi, abiEii, LlaJI, and abiF)is particularly significant.

ACKNOWLEDGMENTS

This study was funded by Science Foundation Ireland (02/IN1/B198).

We thank Paul O’Toole and Stephen McGrath for helpful discus-sions.

REFERENCES

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

2. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid method forisolating large plasmid DNA from lactic streptococci. Appl. Environ. Micro-biol. 46:549–552.

3. Benachour, A., J. Frere, S. Flahaut, G. Novel, and Y. Auffray. 1997. Molec-ular analysis of the replication region of the theta-replicating plasmidpUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC 33315. Mol.Gen. Genet. 255:504–513.

4. Bolotin, A., P. Wincker, S. Mauger, O. Jaillon, K. Malarme, J. Weissenbach,S. D. Ehrlich, and A. Sorokin. 2001. The complete genome sequence of thelactic acid bacterium Lactococcus lactis subsp. lactis IL1403. Genome Res.11:731–753.

5. Boucher, I., E. Emond, M. Parrot, and S. Moineau. 2001. DNA sequenceanalysis of three Lactococcus lactis plasmids encoding phage resistancemechanisms. J. Dairy Sci. 84:1610–1620.

6. Chandler, M., and O. Fayet. 1993. Translational frameshifting in the controlof transposition in bacteria. Mol. Microbiol. 7:497–503.

7. Chandry, P. S., B. E. Davidson, and A. J. Hillier. 1994. Temporal transcrip-tion map of the Lactococcus lactis bacteriophage sk1. Microbiology 140(Pt.9):2251–2261.

8. Cheo, D. L., K. W. Bayles, and R. E. Yasbin. 1991. Cloning and character-ization of DNA damage-inducible promoter regions from Bacillus subtilis. J.Bacteriol. 173:1696–1703.

9. Christie, P. J. 2001. Type IV secretion: intercellular transfer of macromol-ecules by systems ancestrally related to conjugation machines. Mol. Micro-biol. 40:294–305.

10. Cotter, P. D., C. Hill, and R. P. Ross. 2003. A food-grade approach forfunctional analysis and modification of native plasmids in Lactococcus lactis.Appl. Environ. Microbiol. 69:702–706.

11. Davidson, B. E., N. Kordias, M. Dobos, and A. J. Hillier. 1996. Genomicorganization of lactic acid bacteria. Antonie Leeuwenhoek 70:161–183.

12. del Solar, G., R. Giraldo, M. J. Ruiz-Echevarria, M. Espinosa, and R.Diaz-Orejas. 1998. Replication and control of circular bacterial plasmids.Microbiol. Mol. Biol. Rev. 62:434–464.

13. de Ruyter, P. G., O. P. Kuipers, and W. M. de Vos. 1996. Controlled geneexpression systems for Lactococcus lactis with the food-grade inducer nisin.Appl. Environ. Microbiol. 62:3662–3667.

14. Diep, D. B., L. S. Havarstein, and I. F. Nes. 1996. Characterization of thelocus responsible for the bacteriocin production in Lactobacillus plantarumC11. J. Bacteriol. 178:4472–4483.

15. Finn, R. D., J. Mistry, B. Schuster-Bockler, S. Griffiths-Jones, V. Hollich, T.Lassmann, S. Moxon, M. Marshall, A. Khanna, R. Durbin, S. R. Eddy, E. L.Sonnhammer, and A. Bateman. 2006. Pfam: clans, web tools and services.Nucleic Acids Res. 34:D247–D251.

16. Froseth, B. R., R. E. Herman, and L. L. McKay. 1988. Cloning of nisinresistance determinant and replication origin on 7.6-kilobase EcoRI frag-ment of pNP40 from Streptococcus lactis subsp. diacetylactis DRC3. Appl.Environ. Microbiol. 54:2136–2139.

17. Froseth, B. R., and L. L. McKay. 1991. Molecular characterization of thenisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactisDRC3. Appl. Environ. Microbiol. 57:804–811.

18. Furste, J. P., G. Ziegelen, W. Pansegrau, and E. Lanka. 1987. Conjugativetransfer of promiscuous plasmid RP4: plasmid-specified functions essentialfor formation of relaxosomes, p. 553–564. In T. J. Kelly (ed.), Mechanisms ofDNA replication and recombination. Alan R. Liss, New York, N.Y.

19. Garvey, P., G. F. Fitzgerald, and C. Hill. 1995. Cloning and DNA sequenceanalysis of two abortive infection phage resistance determinants from thelactococcal plasmid pNP40. Appl. Environ. Microbiol. 61:4321–4328.

20. Garvey, P., C. Hill, and G. Fitzgerald. 1996. The lactococcal plasmid pNP40encodes a third bacteriophage resistance mechanism, one which affectsphage DNA penetration. Appl. Environ. Microbiol. 62:676–679.

21. Garvey, P., A. Rince, C. Hill, and G. F. Fitzgerald. 1997. Identification of arecA homolog (recALP) on the conjugative lactococcal phage resistanceplasmid pNP40: evidence of a role for chromosomally encoded recALP inabortive infection. Appl. Environ. Microbiol. 63:1244–1251.

22. Gasson, M. J. 1983. Plasmid complements of Streptococcus lactis NCDO 712and other lactic streptococci after protoplast-induced curing. J. Bacteriol.154:1–9.

23. Gomis-Ruth, F. X., G. Moncalian, F. de la Cruz, and M. Coll. 2002. Conju-gative plasmid protein TrwB, an integral membrane type IV secretion systemcoupling protein. Detailed structural features and mapping of the active sitecleft. J. Biol. Chem. 277:7556–7566.

24. Gomis-Ruth, F. X., G. Moncalian, R. Perez-Luque, A. Gonzalez, E. Cabezon,F. de la Cruz, and M. Coll. 2001. The bacterial conjugation protein TrwBresembles ring helicases and F1-ATPase. Nature 409:637–641.

25. Gravesen, A., J. Josephsen, A. von Wright, and F. K. Vogensen. 1995. Char-acterization of the replicon from the lactococcal theta-replicating plasmidpJW563. Plasmid 34:105–118.

6638 O’DRISCOLL ET AL. J. BACTERIOL.

26. Grohmann, E., G. Muth, and M. Espinosa. 2003. Conjugative plasmid trans-fer in gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67:277–301.

27. Harrington, A., and C. Hill. 1991. Construction of a bacteriophage-resistantderivative of Lactococcus lactis subsp. lactis 425A by using the conjugalplasmid pNP40. Appl. Environ. Microbiol. 57:3405–3409.

28. Hill, C., I. J. Massey, and T. R. Klaenhammer. 1991. Rapid method tocharacterize lactococcal bacteriophage genomes. Appl. Environ. Microbiol.57:283–288.

29. Horton, R. M., Z. L. Cai, S. N. Ho, and L. R. Pease. 1990. Gene splicing byoverlap extension: tailor-made genes using the polymerase chain reaction.BioTechniques 8:528–535.

30. Israelsen, H., S. M. Madsen, A. Vrang, E. B. Hansen, and E. Johansen. 1995.Cloning and partial characterization of regulated promoters from Lactococ-cus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80.Appl. Environ. Microbiol. 61:2540–2547.

31. Kearney, K., G. F. Fitzgerald, and J. F. Seegers. 2000. Identification andcharacterization of an active plasmid partition mechanism for the novelLactococcus lactis plasmid pCI2000. J. Bacteriol. 182:30–37.

32. Kenyon, C. J., and G. C. Walker. 1981. Expression of the Escherichia coliuvrA gene is inducible. Nature 289:808–810.

33. Kiewiet, R., S. Bron, K. de Jonge, G. Venema, and J. F. Seegers. 1993. Thetareplication of the lactococcal plasmid pWVO2. Mol. Microbiol. 10:319–327.

34. Kok, J., G. Buist, A. L. Zomer, S. A. van Hijum, and O. P. Kuipers. 2005.Comparative and functional genomics of lactococci. FEMS Microbiol. Rev.29:411–433.

35. Kornberg, A. B. T. 1992. DNA replication. W. H. Freeman Co., New York,N.Y.

36. Lanka, E., and B. M. Wilkins. 1995. DNA processing reactions in bacterialconjugation. Annu. Rev. Biochem. 64:141–169.

37. Law, J., G. Buist, A. Haandrikman, J. Kok, G. Venema, and K. Leenhouts.1995. A system to generate chromosomal mutations in Lactococcus lactiswhich allows fast analysis of targeted genes. J. Bacteriol. 177:7011–7018.

38. Leenhouts, K., G. Buist, A. Bolhuis, A. ten Berge, J. Kiel, I. Mierau, M.Dabrowska, G. Venema, and J. Kok. 1996. A general system for generatingunlabeled gene replacements in bacterial chromosomes. Mol. Gen. Genet.253:217–224.

39. Lessl, M., D. Balzer, K. Weyrauch, and E. Lanka. 1993. The mating pairformation system of plasmid RP4 defined by RSF1010 mobilization anddonor-specific phage propagation. J. Bacteriol. 175:6415–6425.

40. Letunic, I., R. R. Copley, B. Pils, S. Pinkert, J. Schultz, and P. Bork. 2006.SMART 5: domains in the context of genomes and networks. Nucleic AcidsRes. 34:D257–D260.

41. Lillehaug, D. 1997. An improved plaque assay for poor plaque-producingtemperate lactococcal bacteriophages. J. Appl. Microbiol. 83:85–90.

42. Maguin, E., P. Duwat, T. Hege, D. Ehrlich, and A. Gruss. 1992. New thermo-sensitive plasmid for gram-positive bacteria. J. Bacteriol. 174:5633–5638.

43. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol.Biol. Rev. 62:725–774.

44. McGrath, S., G. F. Fitzgerald, and D. van Sinderen. 2001. Improvement andoptimization of two engineered phage resistance mechanisms in Lactococcuslactis. Appl. Environ. Microbiol. 67:608–616.

45. McKay, L. L., and K. A. Baldwin. 1984. Conjugative 40-megadalton plasmidin Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistanceto nisin and bacteriophage. Appl. Environ. Microbiol. 47:68–74.

46. McLandsborough, L. A., L. Sechaud, and L. L. McKay. 1998. Synergisticeffects of abiE or abiF from pNP40 when cloned in combination with abiDfrom pBF61. J. Dairy Sci. 81:362–368.

47. Mills, S., O. E. McAuliffe, A. Coffey, G. F. Fitzgerald, and R. P. Ross. 2006.Plasmids of lactococci: genetic accessories or genetic necessities? FEMSMicrobiol. Rev. 30:243–273.

48. Moineau, S. 1999. Applications of phage resistance in lactic acid bacteria.Antonie Leeuwenhoek 76:377–382.

49. Moineau, S., S. Pandian, and T. R. Klaenhammer. 1994. Evolution of a lyticbacteriophage via DNA acquisition from the Lactococcus lactis chromosome.Appl. Environ. Microbiol. 60:1832–1841.

50. O’Driscoll, J., F. Glynn, O. Cahalane, M. O’Connell-Motherway, G. F.Fitzgerald, and D. Van Sinderen. 2004. Lactococcal plasmid pNP40 encodesa novel, temperature-sensitive restriction-modification system. Appl. Envi-ron. Microbiol. 70:5546–5556.

51. O’Sullivan, D., R. P. Ross, D. P. Twomey, G. F. Fitzgerald, C. Hill, and A.Coffey. 2001. Naturally occurring lactococcal plasmid pAH90 links bacterio-phage resistance and mobility functions to a food-grade selectable marker.Appl. Environ. Microbiol. 67:929–937.

52. O’Sullivan, D., and T. Klaenhammer. 1993. Rapid mini-prep isolation ofhigh-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl.Environ. Microbiol. 59:2730–2733.

53. Pei, J., and N. V. Grishin. 2001. Type II CAAX prenyl endopeptidasesbelong to a novel superfamily of putative membrane-bound metalloproteases.Trends Biochem. Sci. 26:275–277.

54. Pham, P., J. G. Bertram, M. O’Donnell, R. Woodgate, and M. F. Goodman.2001. A model for SOS-lesion-targeted mutations in Escherichia coli. Nature409:366–370.

55. Reuven, N. B., G. Arad, A. Maor-Shoshani, and Z. Livneh. 1999. The mu-tagenesis protein UmuC is a DNA polymerase activated by UmuD�, RecA,and SSB and is specialized for translesion replication. J. Biol. Chem. 274:31763–31766.

56. Rivera, E., L. Vila, and J. Barbe. 1997. Expression of the Pseudomonasaeruginosa uvrA gene is constitutive. Mutat. Res. 377:149–155.

57. Romero, D. A., and T. R. Klaenhammer. 1993. Transposable elements inlactococci: a review. J. Dairy Sci. 76:1–19.

58. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

59. Savijoki, K., H. Ingmer, D. Frees, F. K. Vogensen, A. Palva, and P.Varmanen. 2003. Heat and DNA damage induction of the LexA-like regu-lator HdiR from Lactococcus lactis is mediated by RecA and ClpP. Mol.Microbiol. 50:609–621.

60. Schirawski, J., W. Hagens, G. F. Fitzgerald, and D. Van Sinderen. 2002.Molecular characterization of cadmium resistance in Streptococcus ther-mophilus strain 4134: an example of lateral gene transfer. Appl. Environ.Microbiol. 68:5508–5516.

61. Schroder, G., S. Krause, E. L. Zechner, B. Traxler, H. J. Yeo, R. Lurz, G.Waksman, and E. Lanka. 2002. TraG-like proteins of DNA transfer systemsand of the Helicobacter pylori type IV secretion system: inner membrane gatefor exported substrates? J. Bacteriol. 184:2767–2779.

62. Siezen, R. J., B. Renckens, I. van Swam, S. Peters, R. van Kranenburg, M.Kleerebezem, and W. M. de Vos. 2005. Complete sequences of four plasmidsof Lactococcus lactis subsp. cremoris SK11 reveal extensive adaptation to thedairy environment. Appl. Environ. Microbiol. 71:8371–8382.

63. Tanaka, T., and M. Ogura. 1998. A novel Bacillus natto plasmid pLS32capable of replication in Bacillus subtilis. FEBS Lett. 422:243–246.

64. Tang, M., X. Shen, E. G. Frank, M. O’Donnell, R. Woodgate, and M. F.Goodman. 1999. UmuD�(2)C is an error-prone DNA polymerase, Esche-richia coli pol V. Proc. Natl. Acad. Sci. USA 96:8919–8924.

65. Trotter, M., S. Mills, R. P. Ross, G. F. Fitzgerald, and A. Coffey. 2001. Theuse of cadmium resistance on the phage-resistance plasmid pNP40 facilitatesselection for its horizontal transfer to industrial dairy starter lactococci. Lett.Appl. Microbiol. 33:409–414.

66. Van Houten, B., D. L. Croteau, M. J. Dellavecchia, H. Wang, and C. Kisker.2005. “Close-fitting sleeves”: DNA damage recognition by the UvrABCnuclease system. Mutat. Res. 577:92–117.

67. Walker, S. A., and T. R. Klaenhammer. 2003. The genetics of bacteriophageresistance in lactic acid bacteria, p. 291–315. In B. J. B. Wood and P. J.Warner (ed.), Genetics of lactic acid bacteria, vol. 3. Kluwer Academic/Plenum Publishers, Dordrecht, The Netherlands.

68. Wang, Z. 2001. Translesion synthesis by the UmuC family of DNA poly-merases. Mutat. Res. 486:59–70.

69. Wells, J. M., P. W. Wilson, and R. W. Le Page. 1993. Improved cloningvectors and transformation procedure for Lactococcus lactis. J. Appl. Bac-teriol. 74:629–636.

70. Wilkins, B. M. 2002. Plasmid promiscuity: meeting the challenge of DNAimmigration control. Environ. Microbiol. 4:495–500.

71. Wouters, J. A., H. Frenkiel, W. M. de Vos, O. P. Kuipers, and T. Abee. 2001.Cold shock proteins of Lactococcus lactis MG1363 are involved in cryopro-tection and in the production of cold-induced proteins. Appl. Environ. Mi-crobiol. 67:5171–5178.

72. Wouters, J. A., B. Jeynov, F. M. Rombouts, W. M. de Vos, O. P. Kuipers, andT. Abee. 1999. Analysis of the role of 7-kDa cold-shock proteins of Lacto-coccus lactis MG1363 in cryoprotection. Microbiology 145(Pt. 11):3185–3194.

73. Wouters, J. A., M. Mailhes, F. M. Rombouts, W. M. de Vos, O. P. Kuipers,and T. Abee. 2000. Physiological and regulatory effects of controlled over-production of five cold shock proteins of Lactococcus lactis MG1363. Appl.Environ. Microbiol. 66:3756–3763.

VOL. 188, 2006 LACTOCOCCAL PLASMID-ENCODED SAFEGUARD 6639