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Applied and Environmental Microbiology, October 2008, p. 6206-6215, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.01053-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Lactococcal-Prophage-Carried Superinfection Exclusion Genes ^,

Jennifer Mahony,¹ Stephen McGrath,¹ Gerald F. Fitzgerald,^1,2 and Douwe van Sinderen^1,2*

Department of Microbiology,¹ Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland²

Received 10 May 2008/ Accepted 15 August 2008

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Superinfection exclusion (Sie) proteins are prophage-encoded phage resistance systems. In this study, genes encoding Sie systems were identified on the genomes of Lactococcus lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403. These Sie systems are genetically distinct and yet were shown to act specifically against a particular subset of the 936 phage group. Each of the systems allows normal phage adsorption while affecting plasmid transduction and intracellular phage DNA replication, which points to the blocking of phage DNA injection as their common mode of action. Sie-specifying genes found on the MG1363 prophages are also present in various lactococcal strains, whereas the prophage-encoded Sie systems of IL1403 do not appear to be as widely disseminated.

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Lactococcus lactis is an extensively exploited bacterial species in the dairy industry, where it is employed as a starter culture for the production of a variety of cheeses and other fermented products (for a review, see reference 39). Bacteriophages (phages) infecting L. lactis (or other starter cultures) pose a significant economic risk, and therefore considerable efforts have been made to understand the biology of phages aimed at designing rational solutions to combat this problem. Lactococcal phages that are problematic in the dairy industry primarily belong to one of three groups, namely the virulent 936 and c2 types and the P335 type, whose members may be virulent or temperate (23). Various natural phage-derived defense systems against these phages have been identified, which on the basis of their mode of action were classified as abortive infection, adsorption inhibition, injection blocking, or restriction/modification systems (7, 17, 24, 27, 37).

Advances in phage genome sequencing and phage biology have permitted various novel strategies to effect phage resistance (for a review, see reference 35). For example, engineered phage-encoded resistance (so-called "Per") systems, originally derived from lytic lactococcal phages, were developed that act through the titration of essential proteins involved in phage replication (12, 22). Another example of a phage-derived phage resistance system is represented by superinfection exclusion (Sie). Several, mechanistically different Sie systems have been described that are encoded by prophages from gram-negative bacteria (25, 38, 41). For example, the Sie system of the lytic coliphage X174 is not only believed to operate by altering the host's cell surface but is also suspected to interfere with a host protein required for the conversion of the injected single-stranded DNA into the double-stranded replicative form (13).

The first description of a gram-positive Sie system was that of the P335-type temperate lactococcal bacteriophage, Tuc2009, which is present in the lactococcal strain UC509, a starter bacterium used in the production of cheddar cheese (21). The gene encoding this Sie system, named sie₂₀₀₉ (i.e., sie derived from Tuc2009), is located between the integrase- and repressor-encoding genes on the lysogeny module of Tuc2009 (21). The membrane-associated Sie₂₀₀₉ protein was shown to prevent DNA injection of certain members of the 936 phage group without affecting phage adsorption. Interestingly, the ltp gene of the temperate Streptococcus thermophilus phage TP-J34 encodes an Sie that is active against the 936-type lytic lactococcal phage P008, but not against the assayed members of the c2 and P335 lactococcal phage species (36). The ltp gene encodes a membrane-associated lipoprotein that, similar to Sie₂₀₀₉, was shown to interfere with the process of DNA release from the phage head. It was proposed that these systems operate either by masking a component of the cytoplasmic membrane that triggers DNA release or by interacting with the adsorbing phage particle, thereby preventing proper insertion of the phage tail tip into the cytoplasmic membrane (21, 36).

The chromosomes of L. lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403 each harbor six prophages that may also encode Sie systems (6, 40). The present study describes an investigation of the activity of the potential phage-resistance proteins specified by the prophages of these lactococcal strains and their dissemination throughout lactococcal strains used in the dairy industry.

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Phage propagation and plaque assays.
Phages used in this study (Table 1) were propagated on appropriate L. lactis indicator strains, which had been grown to an approximate optical density at 600 nm (OD₆₀₀) of 0.3 in 10 ml M17 broth supplemented with 0.5% glucose. Calcium chloride was added to a final concentration of 10 mM prior to infection of the culture with approximately 10⁸ PFU of the relevant phages, which was then followed by incubation at 30°C until lysis had occurred. The lysates were filtered through a 0.45-µm-pore filter to remove any residual bacterial contamination and stored at 4°C. Plaque assays were carried out as described by Lillehaug (16). These assays were used to determine the efficiency of plaquing (EOP) of the relevant phages on the host expressing the potential Sie systems relevant to that of the sensitive host strain. Where relevant, 0.1% of the cell-free supernatant of the nisin-producing lactococcal strain NZ9700 was added to the plaque assay medium to induce the genes cloned under the control of the nisin-inducible promoter of pNZ8048 (8, 14).

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TABLE 1. Bacteriophages and bacterial strains used in this study

Identification of potential superinfection exclusion genes.
Candidate sie genes were identified based on their location between the repressor- and integrase-encoding genes on the lysogeny module of prophage elements and on the presence of at least one predicted N-terminal transmembrane region in the encoded protein (21). Each of the potential Sie proteins was analyzed for predicted transmembrane regions by in silico analysis using the TMHMM program (http://www.cbs.dtu.dk/services), DAS transmembrane prediction (http://expasy.hcuge.ch), and PROSITE release 18.19 (32). The L. lactis subsp. cremoris MG1363 prophage sequences are located on the L. lactis subsp. cremoris MG1363 genome (42 [GenBank accession no. NC009004]), while the L. lactis subsp. lactis IL1403 prophage bIL285 (NC002666), bIL286 (NC002667), bIL309 (NC002668), and bIL312 (NC002671) sequences are located on the L. lactis subsp. lactis IL1403 genome (6). The putative sie genes located on prophages bIL285, bIL286, and bIL312 of L. lactis IL1403 and T712 and MG-3 of L. lactis MG1363 are adjacent to a putative metalloprotease: therefore, these genes were also analyzed in tandem with the putative sie gene (see below). The genomes of the prophages bIL310 and bIL311 of L. lactis subsp. lactis IL1403 and MG-1, MG-4 and MG-5 of L. lactis subsp. cremoris MG1363 do not appear to possess gene products that possess the criteria set out for superinfection exclusion systems (21).

Plasmid constructions.
Putative sie genes were amplified by PCR using the relevant oligonucleotide primers in which NcoI and XbaI recognition sites were present to facilitate cloning. Where a neighboring putative metalloprotease-encoding gene was identified, the suspected sie gene was amplified individually and in tandem with this metalloprotease-specifying DNA. The generated amplicons were restricted with NcoI and XbaI and cloned into the similarly cut high-copy lactococcal expression vector pNZ44 (20) or pNZ8048 (8), to place them under the control of the constitutive p44 promoter or downstream of the nisin-inducible promoter, respectively. A 1.7-kb fragment encompassing the cos site of phage sk1 was amplified from self-ligated sk1 DNA (representing coordinates 28408 to 1659) (4) and cloned into the AatII and SmaI recognition sites of the low-copy lactococcal vector pPTPL (3) to generate plasmid pPTPL-cos, which was used for transduction assays (see below). All constructs were sequenced by MWG Biotech (Ebersberg, Germany) to verify the integrity of the cloned genes.

Characterization of potential Sie systems.
Adsorption and cell survival assays were performed as described by Garvey et al. (11). Transduction assays were adapted from the protocol set out by McGrath et al. (21). Lysis-in-broth assays were performed by infecting the lactococcal host strain, MG1363, expressing a suspected Sie system from pNZ44 with sk1 at a multiplicity of infection of 1.0, and measuring the OD₆₀₀ at 15-min intervals for a total of 90 min.

Membrane vesicle isolations were performed as described by Otto et al. (26), and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described on gradient (10 to 20%) gels (Lonza, Ltd., Switzerland). Intracellular phage DNA replication was studied as described by McGrath et al. (22). Briefly, the lactococcal host MG1363 harboring pNZ44 or derivatives thereof were infected with sk1 at a multiplicity of infection of 0.5 to 1.0, after which 1-ml samples were taken at intervals of 20 min. Total DNA was isolated from these samples, which was restricted with Sau3AI and subjected to Southern hybridization (33) using the PCR-amplified orf18 of sk1 as a probe.

RNA isolation and cDNA construction.
Cultures were grown to mid-log phase, and those requiring nisin induction had 0.1% of the supernatant of L. lactis NZ9700 added at an OD₆₀₀ of 0.2 to 0.3 and were further incubated until an OD₆₀₀ of approximately 0.5 to 0.6 was reached. Cells were harvested and RNA isolation was performed by a combination of the Macaloid and Roche methods as described by Pool et al. (29). RNA yield was determined using a Nanodrop instrument (Nanodrop Technologies, Inc.). Triplicate RNA samples were isolated for each of the active Sie proteins under the control of pNisA (in pNZ8048) or the constitutive promoter p44 (in pNZ44). To remove contaminating DNA from the RNA preparation, the samples were DNase treated (Ambion) as per the manufacturer's instructions. Five hundred nanograms of a DNase-treated RNA sample was then included in a given reverse transcription-PCR (RT-PCR) (Superscript III reverse transcriptase; Invitrogen) with 200 ng of random nonamers, and the remainder of the protocol was then performed according to the manufacturer's instructions (Invitrogen).

Real-time PCR.
Real-time quantitative RT-PCR was performed using an AbiPrism 7700 sequence detection system (Applied Biosystems, Nieuwekerk-aan-de-IJssel, Netherlands) using the double-stranded DNA intercalating fluorescent agent Sybr green for amplicon detection as described by Marco et al. (19). Data analysis was performed using the formula 2^{(CT target)}/2^{(CT control)}, where C_T represents the threshold of detection, the target is the sie gene of interest, and the control is the housekeeping gene groES. Sie_mg2 and Sie_t712 are present on the genome of MG1363, and therefore the transcription levels of the genes coding for these proteins were used as reference points relative to the situation when these genes were also present on the pNZ plasmid derivatives. For Sie₃₀₉ and Sie₃₁₂, which are encoded by prophages present on the L. lactis subsp. lactis IL1403 genome, the ratio of the transcript levels of the genes under the control of the p44 or pNisA promoters in pNZ44 or pNZ8048, respectively, was determined. All results are the average of three technical replicates of triplicate samples.

Sie survey.
The occurrence of sie genes in lactococcal genomes was determined by a PCR-based assay utilizing oligonucleotides designed to amplify the sie genes identified on the L. lactis MG1363 and IL1403 genomes and the sie gene present on temperate phage Tuc2009 (Table 2). The generated amplicons were purified and then subjected to sequencing (MWG Biotech AG, Ebersberg, Germany), after which the obtained sequences were used for comparative analysis using the SEQMAN and MEGALIGN programs of the DNASTAR software package.

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TABLE 2. Oligonucleotide primers used in this study and database similarities

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Activity range of Sie₂₀₀₉.
The Sie system encoded by the temperate lactococcal phage Tuc2009, Sie₂₀₀₉, had previously been shown to exert activity against the 936-type phages sk1, jj50, and 712 (21). Here, we present an extended study of the activity of Sie₂₀₀₉ against 936-, c2-, and P335-type phages with particular emphasis on the 936-type phages. The obtained data show that four other 936-type phages, p2, 13, jw31, and jw32, are targeted by this system, although the effect of Sie₂₀₀₉ is less dramatic than that observed for sk1, jj50, and 712 (Table 3). Interestingly, Sie₂₀₀₉ was shown to exert complete resistance against p2 when expressed from the lactococcal nisin-inducible expression vector pNZ8048 (induced with 0.1% of the supernatant of the nisin-producing lactococcal strain NZ9700) as compared to a 0.5-log reduction in EOP when cloned in pNZ44 (Table 3).

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TABLE 3. EOP of assayed bacteriophages against the potential superinfection exclusion systems of L. lactis IL1403, MG1363, and UC509

Based on the finding that Sie₂₀₀₉ provides an increased level of protection against p2 using the nisin-inducible system, a study investigating the effect of Sie expression levels on phage resistance was undertaken by varying the nisin levels. As expected, a nisin-dependent increase in phage resistance was observed for Sie₂₀₀₉ against sk1 and p2, which is consistent with the expected increase in expression level of Sie₂₀₀₉ (Table 4). When Sie₂₀₀₉ is expressed from pNZ44 in L. lactis strains W34 and FD13, a low level of phage resistance activity is observed against jw31, jw32, and 13 (Table 3). Since these strains do not carry the nisRK genes, the effect of nisin-induced pNZ8048-Sie₂₀₀₉ was not ascertained.

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TABLE 4. EOP of phages sk1 and p2 on L. lactis NZ9000(pNZ8048-sie2009) induced with various concentrations of nisin

Phage resistance activity of the MG1363 prophage-encoded sie genes.
The sequences of a number of (presumed) temperate lactococcal bacteriophages have become available through whole-genome sequencing (2, 6, 9, 15, 40), which has enabled the identification of several candidate Sie-encoding genes based on their genomic location. Based on their genomic position and the presence of a hydrophobic N terminus in the encoded protein, three potential superinfection exclusion genes were identified on presumed prophages that are present in the L. lactis MG1363 genome (see Fig. S1 in the supplemental material). The orf595 gene of MG-2 (named here as sie_mg2) and orf791 of T712 (designated sie_T712) each encode a protein that possesses a hydrophobic amino terminus with at least one predicted transmembrane domain. A putative metalloprotease-encoding gene is located adjacent to sie_T712 (mp_T712). A similar gene constellation was found on the L. lactis F7/2 prophage, where it was suggested that both genes are required to provide superinfection exclusion (21). An amino-terminal transmembrane domain is not predicted in the case of orf2141 of MG-3; however, this gene is adjacent to a putative metalloprotease gene (mp_mg3), and consequently orf2141was investigated for Sie properties based on its genetic location.

The three putative sie genes were cloned in pNZ44 and pNZ8048, either individually or in combination with their respective putative metalloprotease-encoding genes, where relevant. Each of the pNZ44-based constructs was introduced into a number of lactococcal strains and assayed against a range of 936, c2, and P335 phages (Table 3). In the case of Sie_mg2 and Sie_T712, phage resistance was observed against certain members of the 936-type phages, while no activity was observed for any of the systems against the assayed c2 or P335 phages. When sie_mg2 was expressed using pNZ8048, an increase in conferred phage resistance was observed against two of the phages, presumably due to the higher expression level of the proteins compared to its pNZ44 equivalent (see below).

Sie_T712 was shown to provide complete resistance against sk1, jj50, 712, and p2 when cloned in either vector. MP_T712 alone provides no phage resistance, and no detectable increase in phage resistance or extension of the range of activity of Sie_T712 was observed when cloned in tandem with sie_T712 (data not shown). It is noteworthy that both Sie_mg2 and Sie_T712 exhibit a similar activity spectrum, which in turn is reminiscent of that of Sie₂₀₀₉ (Table 3), despite the lack of any significant sequence similarity among these three systems (data not shown). ORF2141 and/or MP_mg3 did not provide phage resistance against the range of phages assayed in this study (Table 3 and data not shown).

Phage resistance activity of the IL1403 prophage-encoded sie genes.
The six prophages present in the L. lactis subsp. lactis IL1403 genome were analyzed, and four genes with potential as sie genes had been proposed previously (21): orf2 (sie₂₈₅) of bIL285, orf2 of bIL286 (orf2₂₈₆), orf2 (sie₃₀₉) of bIL309, and orf2 (sie₃₁₂) of bIL312. The orf3 gene of bIL285 and the orf3 gene of bIL286 encode putative metalloproteases, while bIL312 possesses two genes between the putative sie gene and the repressor gene, neither of which is a putative metalloprotease gene.

Sie₃₀₉ has an amino acid sequence identical to Sie_IL409 (Table 2), which was previously shown to provide partial resistance against sk1, jj50, and 712 when cloned in pNZ44 (21). In the present study, Sie₃₀₉ was observed to effect a 1,000-fold reduction in EOP against sk1 and jj50, a 100-fold reduction in EOP against p2, and a <10-fold reduction in EOP against 712 when cloned in pNZ44 (Table 3), which is in keeping with previous findings (21). When expressed from the nisin-inducible expression vector pNZ8048, Sie₃₀₉ provided complete phage resistance against sk1 and jj50 and improved the protective effect against 712 and p2 (Table 3).

The putative Sie specified by bIL312, designated here as Sie₃₁₂, bears 94% homology to Sie_mg2 at the amino acid level. Upon expression in L. lactis NZ9000, it was found that, as expected, this system provides the same phage resistance activity as Sie_mg2. The only deviation observed was the effect against 712. It is possible that Sie₃₁₂ and Sie_mg2 may have different interactions with their target, thus resulting in a different level of phage resistance against this phage.

The protein products of sie₂₈₅ and orf3 of bIL285 possess 98% and 95% amino acid identity to Sie_F7/2A and Sie_F7/2B, respectively, and therefore would be expected to exhibit similar activity to Sie_F7/2AB. The latter system has been shown previously to provide a 100-fold reduction in EOP against sk1, jj50, and 712, but apparently only when the genes encoding Sie and putative metalloprotease are both present in the nisin-inducible plasmid pNZ8048 (21).

ORF2₂₈₆ possesses three predicted N-terminal transmembrane domains. However, expression of the encoded protein does not appear to result in any significant phage resistance against the range of phages assayed in the present study when expressed alone or in combination with the downstream metalloprotease (Table 3 and data not shown).

Transcriptional analysis of the identified Sie systems.
Through transcriptional analysis of the lactococcal strain NZ9000 harboring pNZ44 and pNZ8048 and their sie-bearing derivatives, it was shown that the relative abundance of transcripts of the sie genes is increased by 30- to 140-fold when produced under the control of the nisin-inducible promoter, pNisA, relative to the constitutive promoter p44 (Table 5). This data is in keeping with the phenotypic data, from which it is elucidated that Sie_mg2, Sie₃₁₂, and Sie₃₀₉ confer partial resistance against the 936-type phages when cloned in pNZ44 and resistance levels beyond the detection limits of the assay when cloned in pNZ8048. The increase in transcript levels of sie₃₀₉, sie₃₁₂, and sie_mg2 of between 30- and 40-fold in the pNZ8048 clones was significant enough to induce the phenotypic increase in phage resistance. In the case of sie_T712, the increase in transcription level (141-fold) did not matter since full activity of the protein is already observed in the pNZ44 clone. The translation efficiency of the encoded proteins may differ since the transcriptional data do not necessarily correlate with the protein expression data as observed by SDS-PAGE analysis (Fig. 1).

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TABLE 5. Phenotypic characteristics of the potential superinfection exclusion systems using phage sk1, including the ratio of transcript levels of the newly identified Sie proteins under the control of the pNisA and p44 promoters of pNZ8048 and pNZ44, respectivelya

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FIG. 1. Gradient polyacrylamide gel (10 to 20%) of the membrane vesicle (A) and cytoplasmic fractions (B) of L. lactis NZ9000(pNZ8048), NZ9000(pNZ8048-sie309), NZ9000(pNZ8048-sie312), NZ9000(pNZ8048-siemg2), and NZ9000(pNZ8048-sieT712). Lane 1, protein molecular weight marker; lanes 2 to 6, membrane vesicle fractions of the strains listed in the above order; lanes 7 to 11, cytoplasmic fractions of the strains listed in the above order. Each of the expressed Sie proteins is circled in lanes 3 to 6. Panel C represents SDS-PAGE analysis of the cytoplasmic and membrane vesicle fractions of L. lactis NZ9000(pNZ8048) and NZ9000(pNZ8048-sie2009), with the band representing Sie2009 circled in the membrane vesicle fraction in lane 15. Lanes 12 and 13, cytoplasmic fractions of L. lactis NZ9000(pNZ8048) and NZ9000(pNZ8048-sie2009), respectively. Lanes 14 and 15, membrane vesicle fractions of L. lactis NZ9000(pNZ8048) and NZ9000(pNZ8048-sie2009), respectively.

Sie characterization.
Since each of the newly identified Sie systems is predicted to possess at least one transmembrane domain, their possible membrane-association was investigated. For this purpose, membrane and cytoplasmic fractions of each of the Sie-expressing strains were isolated and their protein profiles were analyzed by SDS-PAGE and compared to that of the wild-type host carrying the empty vector. Additionally, the cytoplasmic and membrane fractions of L. lactis NZ9000 expressing Sie₂₀₀₉ were also used as a positive control since Sie₂₀₀₉ was previously shown to be membrane associated (21). In each of the Sie-expressing strains, an additional protein band was observed, which was absent from the L. lactis strain which just carries the empty vector (Fig. 1). Since the deduced molecular weights of each of these membrane-associated proteins correspond well with their expected weights of the expressed proteins (only Sie_T712 runs at a somewhat lower mobility than expected), we can safely assume that Sie₃₀₉, Sie₃₁₂, Sie_mg2, and Sie_T712 are indeed associated with the membrane (Table 5 and Fig. 1). Each system was assayed to determine its effect on phage adsorption. Phage adsorption was normal for each of the four Sie systems assayed (Table 5). All four Sie systems survive phage infection significantly better than the wild type as defined by lysis-in-broth and cell death experiments in which the lactococcal host expressing these systems exhibits continued growth in the presence of the sk1 phage (see Fig. 3a and Table 5). The active systems as determined by lysis-in-broth experiments are Sie_mg2, Sie_T712, Sie₃₁₂, and Sie₃₀₉, while ORF2141and ORF2₂₈₆ behave similarly to the wild-type host and are therefore assumed not to be Sie systems, all of which is consistent with the plaque assay results (Fig. 3b and Table 3).

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FIG. 3. (a) Lysis in broth experiments comparing the lysis profiles of MG1363 harboring pNZ44 in both uninfected cells () and cells infected () with sk1 and its derivatives bearing siemg2 (uninfected, X with short dashes; infected, X with long dashes), sieT712 (uninfected, ; infected ), sie309 (uninfected, •; infected, , and sie312 (uninfected, ; infected ). OD600 readings were taken at 15-min intervals. Each of the active Sie proteins provides resistance against sk1 infection. MG1363 harboring pNZ44 was used as a positive control. Each assay was performed in triplicate. (b) Lysis in broth experiments of L. lactis MG1363(pNZ44) cells both uninfected () and infected () with sk1 and its derivatives bearing the putative Sie genes orf2141mg3 (uninfected, ; infected, ) and orf2286 (uninfected, ; infected, ). OD600 readings were taken at 15-min intervals. These systems appear to provide little or no protection against infection by sk1.

To ascertain the effect of the Sie systems on phage DNA replication within the host, each of the Sie-expressing strains was infected with sk1 to monitor DNA replication of this phage by Southern blotting. Orf2₂₈₆ and Orf2141 have lysis profiles resembling that of the wild-type host (data not shown) and therefore were not examined for their effect on intracellular phage DNA replication. From the obtained results, it is clear that no intracellular replication of sk1 was detected in strains expressing Sie₃₁₂ or Sie_mg2 (Fig. 2). This is expected since the lysis-in-broth experiments with these systems demonstrate that the hosts expressing these systems are essentially immune to infection by sk1. This is also in agreement with the cell death assays, whereby approximately 5% cell death was observed for these systems. Sie₃₀₉ does not confer complete resistance when expressed in pNZ44 (it achieves a 1,000-fold reduction in EOP against sk1), and in agreement with this, we observed a low level of intracellular replication (Table 3 and Fig. 2). Almost 13% cell death was observed in cells expressing Sie₃₀₉ when infected with sk1 (Table 5), which is in line with the background level of DNA replication and the observed incomplete resistance to phage infection. Cells expressing Sie_T712 exhibit complete survival and were shown to completely prevent intracellular phage DNA replication (Table 5). Lysis-in-broth studies also demonstrate the survival of the host strain expressing Sie_T712 after 90 min of incubation with the lytic phage sk1 (Fig. 3a).

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FIG. 2. Southern blot analysis of sk1 intracellular DNA replication. Total cellular DNA isolated at intervals of 20 min after infection over a total period of 60 min, as indicated in the picture. Lanes 1 to 4, MG1363(pNZ44) infected with sk1; lanes 5 to 8, MG1363(pNZ44sie309) infected with sk1; lanes 9 to 12, MG1363(pNZ44sie312) infected with sk1; lanes 13 to 16, MG1363(pNZ44siemg2) infected with sk1; lanes 17 to 20, MG1363(pNZ44sieT712) infected with sk1. DNA was digested with Sau3AI and blotted and probed with the PCR-amplified orf18 gene of sk1.

To examine the effect of these Sie systems on the DNA injection process, transduction assays were performed, whereby the tetracycline-resistant plasmid pPTPL, carrying part of the sk1 genome including its cos site was transformed into the lactococcal host strain. This was infected with sk1, and the resultant lysate was used to transfer pPTPL-cos by means of transduction into L. lactis MG1363 harboring pNZ44 or its derivatives containing the sie genes. The transduction efficiency was calculated as the number of obtained tetracycline-resistant colonies divided by the number of PFU of the transducing lysate on a sensitive host. Each of the tested systems was able to interfere with this process, causing a 10- to 100-fold reduction in transduction efficiency (Table 5). This clearly indicates that these systems are active at the level of DNA injection, similar to the action of Sie₂₀₀₉ (21). While the effect is not as marked as that previously observed with Sie₂₀₀₉ (1,000-fold reduction), it is possible that this is due to the low-copy-number plasmid being used in this study as compared to the system described by McGrath et al. (Table 5). However, the observation that the Sie systems interfere with transduction is in line with previous findings for Sie₂₀₀₉ (21), which suggest that the mode of action of these identified Sie systems, despite their lack of sequence homology, is DNA injection blocking.

Prevalence of Sie-encoding genes among lactococcal strains.
Utilizing oligonucleotide primers specific for sie₂₀₀₉ and those for each of the sie genes identified in the prophages of L. lactis MG1363 and IL1403 (Table 2), a number of lactococcal strains were assayed for the presence of sie genes (see Table S1 in the supplemental material). Altogether, 29 lactococcal strains were surveyed, and of these, no homologs for sie₂₈₅ were identified, while only two strains, L. lactis 1820 and UC503, were found to possess homologues of sie₃₀₉ with an identical nucleotide sequence. Since sie₃₁₂ is nearly identical to sie_mg2, the oligonucleotide primers for sie_mg2 (sequences of which are common to both sie genes) were chosen for the amplification of these sie homologs. Of the MG1363 prophage-encoded sie genes, sie_mg2 and sie_T712 homologs appear to be widely distributed among both L. lactis subsp. lactis and L. lactis subsp. cremoris strains. Homologs of sie_mg2 were identified in L. lactis subsp. cremoris 1934, 1196, IP5, UC503, UC509, UC521 FD13, W34, and 712, as well as in L. lactis subsp. lactis 702, c2, 184, 275, WM1, C10, and 1820. Homologs of sie_T712 were identified in L. lactis subsp. cremoris 1934, 1196, UC503, UC509, UC514, UC521, W34, 712, and 158 and also in L. lactis subsp. lactis 702, 184, 275, WM1, C10, and 1820. In each case, the sequence of each of the amplicons was shown to be identical to that of the sie gene based on which the PCR primers were designed.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Four sie genes were detected on the chromosomes of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363, two of which represent novel Sie systems (Sie_mg2/312 type and Sie_T712 type). Each of these Sie systems possesses a hydrophobic amino terminus, also present in previously identified lactococcal systems (21). Surprisingly, where a putative metalloprotease-encoding gene was observed adjacent to the sie gene in the prophages of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363, no additive or synergistic effects were observed. This finding is inconsistent with a previous finding for the Sie_F7/2AB system in which it was shown that both Sie_F7/2A and Sie_F7/2B are required to effect full phage resistance (21). The apparent synergism may have been due to increased transcription of sie_F7/2A as a result of sequences upstream of the sie gene in the sie_F7/2AB-containing clone that increase the transcription and/or translation of the sie_F7/2A gene (30).

It is known that the sie genes of the prophages of L. lactis subsp. cremoris MG1363 are constitutively expressed in the host (40). This has also been observed for the prototype lactococcal Sie system, Sie₂₀₀₉, encoded by the lactococcal host strain UC509, whose genome carries the temperate phage Tuc2009 (31). These systems, which appear to act specifically against a subset of the 936-type phages, are widespread throughout lactococcal prophages. Surprisingly the lactococcal strains that harbor these prophage-encoded systems remain sensitive to infection by many of the phages which these systems are active against. This may indicate that these systems are needed at high levels in order to fully protect the cells. It may also be a reflection of Sie activity in their natural environment, in which the strains and their resident prophages would not be challenged with as great a multiplicity of infection as they are in these in vitro studies. This may also explain why the prophages of these dairy strains may possess several Sie-encoding genes with the same apparent specificity. It is possible that these systems are synergistically providing a low level of resistance that is difficult to assess without the availability of a prophage-cured derivative of these strains.

The UC5 strain series is the most notable series of strains to possess identical homologues of sie₂₀₀₉. With the exception of 18-16, none of the 936-type phages listed in this study have the ability to infect any of the UC5 strain series or R1, 1814, or C3, which also harbor homologues of sie₂₀₀₉ (data not shown). This may be attributed to one of two reasons: either the specific phage receptor(s) for these phages is not found on these strains or the active Sie system (and possibly other host defense systems) prevents such phages from infecting these specific hosts. However, it is interesting that BLAST searches with Sie₂₀₀₉ resulted in the identification of a protein in Streptococcus thermophilus LMD-9 with 88% amino acid identity, compounding recent suggestions of genetic transfer between S. thermophilus and L. lactis species (1). Indeed, the identification of the ltp system in S. thermophilus phage TPJ-34, which is active against the lactococcal 936-type phage P008, further substantiates this theory (36).

These intriguing systems were shown to be widespread, and though they lack significant homology, they act toward a common purpose of providing resistance against a select group of the 936-type phages. The Sie proteins are alike in being small proteins with a hydrophobic amino terminus, representing one or two transmembrane domains. Furthermore, the Sie proteins possess a strikingly high isoelectric point ranging from 9.5 to 9.8, and a relatively low GC content (27 to 34%) compared to the lactococcal genome (36%) (Table 5) (42).

The specificity of these systems against a limited group of phages may be a reflection of the mode of action of these systems. It is postulated that the phages sensitive to these superinfection exclusion systems require the same host-encoded factor(s) for DNA injection, and it is possible that the superinfection exclusion systems interact with or mask these factors, thereby preventing the phage DNA injection process. It is also possible that the Sie systems interact directly with a structural element of the adsorbed phage. This structural element should be a component in which divergence is observed between the closely related 936-type phages. Analysis of the 936-type phage genomes has revealed that the structural genes of these phages are highly conserved with only a few areas of divergence (18). Most notable among these are the tail tape measure and receptor binding proteins. The receptor binding protein of the 936-type phages is an unlikely target for the Sie systems, since they act at the level of phage adsorption which is not affected by the Sie systems analyzed here. However, the tail tape measure protein represents a much more likely target, particularly since tail tape measure proteins of a number of phages have been implicated in the phage DNA injection process (5, 28). Phylogenetic analysis of these proteins reveals a tripartite subgrouping of the 936-type phages, namely the sk1/jj50, bIL170/P008, and 712 subgroups (10, 18, 34). These apparent phylogenetic subgroups are in keeping with the biological data derived from the present study: the sk1/jj50 subgroup is sensitive to the lactococcal Sie systems, and the bIL170/P008 subgroup is insensitive, while phage 712 exhibits an "intermediate" sensitivity to some of the Sie systems examined here. The possibility that the lactococcal Sie proteins interact directly with the tail tape measure proteins of their phage targets is intriguing and will be the basis of future work. The activity of ltp of TPJ-34 against the 936-type lactococcal phage P008 shows that there are different Sie types that are active against different members of the 936-type phages and perhaps other lactococcal phage species operating by mimicking, masking, or interacting with host- or phage-encoded components required for phage DNA injection. The present work has provided a revealing insight into the prevalence of these systems. Considering that the 936-type phages account for half of all industrial isolates and their detrimental impact, it is of immense importance that an understanding of natural host defense systems is obtained in order to develop starter cultures with increased fitness in their environment.

 
J. Mahony is in receipt of research funding by the Irish Research Council for Science Engineering and Technology under the Embark Initiative. D. van Sinderen is a recipient of a Science Foundation of Ireland Investigatorship award (01/IN1/B198).

We sincerely thank Jytte Josephsen, The Royal Veterinary and Agricultural University, Denmark, for kindly providing lactococcal phages and strains used as part of this study.

 
* Corresponding author. Mailing address: Department of Microbiology, University College Cork, Western Road, Cork, Ireland. Phone: 353 21 4901365. Fax: 353 21 4903101. E-mail: d.vansinderen{at}ucc.ie

Published ahead of print on 22 August 2008.

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

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Applied and Environmental Microbiology, October 2008, p. 6206-6215, Vol. 74, No. 20
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