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Applied and Environmental Microbiology, November 2008, p. 6528-6537, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00780-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

AbiV, a Novel Antiphage Abortive Infection Mechanism on the Chromosome of Lactococcus lactis subsp. cremoris MG1363{triangledown}

Jakob Haaber,1 Sylvain Moineau,2 Louis-Charles Fortier,2,{dagger} and Karin Hammer1*

Center for Systems Microbiology, DTU Biosys, Technical University of Denmark, DK-2800 Lyngby, Denmark,1 Département de biochimie et de microbiologie, Faculté des sciences et de génie, Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, Québec, Canada G1V 0A62

Received 4 April 2008/ Accepted 27 August 2008


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ABSTRACT
 
Insertional mutagenesis with pGhost9::ISS1 resulted in independent insertions in a 350-bp region of the chromosome of Lactococcus lactis subsp. cremoris MG1363 that conferred phage resistance to the integrants. The orientation and location of the insertions suggested that the phage resistance phenotype was caused by a chromosomal gene turned on by a promoter from the inserted construct. Reverse transcription-PCR analysis confirmed that there were higher levels of transcription of a downstream open reading frame (ORF) in the phage-resistant integrants than in the phage-sensitive strain L. lactis MG1363. This gene was also found to confer phage resistance to L. lactis MG1363 when it was cloned into an expression vector. A subsequent frameshift mutation in the ORF completely eliminated the phage resistance phenotype, confirming that the ORF was necessary for phage resistance. This ORF provided resistance against virulent lactococcal phages belonging to the 936 and c2 species with an efficiency of plaquing of 10–4, but it did not protect against members of the P335 species. A high level of expression of the ORF did not affect the cellular growth rate. Assays for phage adsorption, DNA ejection, restriction/modification activity, plaque size, phage DNA replication, and cell survival showed that the ORF encoded an abortive infection (Abi) mechanism. Sequence analysis revealed a deduced protein consisting of 201 amino acids which, in its native state, probably forms a dimer in the cytosol. Similarity searches revealed no homology to other phage resistance mechanisms, and thus, this novel Abi mechanism was designated AbiV. The mode of action of AbiV is unknown, but the activity of AbiV prevented cleavage of the replicated phage DNA of 936-like phages.


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INTRODUCTION
 
Lactococcus lactis starter cultures are used for the production of fermented dairy products worldwide. However, bacteriophage infection of the added starter cultures may lead to fermentation delays or even halt the process. Despite considerable progress over the past few decades that has led to improved phage control measures, this natural phenomenon still remains a significant risk to the dairy industry (2, 14, 55). Three groups of lactococcal phages cause the vast majority of milk fermentation failures, namely, members of the 936 and c2 species, which display great intraspecies homogeneity but are genetically distinct from each other (12, 30, 50), and phages belonging to the P335 species, which exhibit much greater genomic mosaicism (12, 44). To survive infection by these diverse phages, L. lactis strains possess a wide variety of resistance mechanisms. Lactococcal phage defense systems are classified into four general groups depending on the step of the phage lytic cycle that they inhibit (39). The members of two groups either prevent phage adsorption or block DNA ejection (55), and the members of the other two groups are intracellular antiphage hurdles, namely, restriction/modification systems and abortive infection (Abi) mechanisms. The Abi mechanisms have been described by different workers (for reviews, see references 14 and 73) and are arguably the most efficient mechanisms.

To date, 22 lactococcal Abi mechanisms have been isolated, characterized, and designated AbiA through AbiZ (14, 25). Remarkably, most of these mechanisms appear to have a distinct mode of action, although they do share some common features. These features include reduction of the burst size, reduction of the efficiency of plaquing (EOP) (18, 26), and reduction of the efficiency of formation of centers of infection, as well as death of the infected cells (73). The Abi phenotype is usually mediated by a single gene, although in a few cases (AbiE, AbiG, AbiL, AbiT, and AbiU) the system consists of two genes (9, 18, 19, 31, 62). There is some level of sequence similarity between different Abi proteins (14), but in general, these proteins and their genes show little similarity to other proteins and genes in databases. This finding is in agreement with the activity of the proteins against specific phages or phage groups, as well as the particular mechanistic models. Indeed, the Abi mechanisms of wild-type L. lactis strains that have been characterized have been shown to disable members of one, two, or more lactococcal phage groups, although there is a tendency toward broader efficacy against 936 phages (14), which represent the group that causes most dairy fermentation failures. The general effects on the phage lytic cycle have been revealed for most Abi mechanisms, although in most cases much more characterization is required. For example, AbiA, AbiF, AbiK, AbiP, and AbiT were shown to interfere with DNA replication (9, 23, 27, 31, 36), while AbiB, AbiG, and AbiU affected RNA transcription (15, 18, 63). AbiC was shown to cause limited major capsid protein production (59), whereas AbiE, AbiI, and AbiQ affected phage packaging (26). AbiD1 was found to interfere with a phage RuvC-like endonuclease (6, 7), and the presence of AbiZ caused premature lysis of the infected cells (25).

Most Abi systems are encoded on plasmids, some of which are conjugative, thereby enabling lateral transfer of the resistance mechanisms to phage-sensitive L. lactis strains and subsequent industrial use of the phage-resistant derivatives. Usually, Abi mechanisms provide a much stronger phage resistance phenotype when they are plasmid encoded (due to a higher gene copy number) than when they are chromosomally encoded, which may explain why so few lactococcal Abi genes are in bacterial genomes (14, 15, 20, 64, 66).

Natural Abi mechanisms have been used extensively for protection of industrial starter cultures (16, 28). Not surprisingly, the added selective pressure has led to the emergence of phage mutants capable of overcoming the resistance barriers (16, 28). Therefore, isolation of novel phage resistance mechanisms that can be used alone or in combination with other mechanisms to provide increased phage protection to starter cultures is highly desirable (55).

We report here a novel lactococcal Abi system, designated AbiV, which is chromosomally encoded and effective against virulent phages belonging to the 936 and c2 species. This novel system was discovered using insertional mutagenesis of the laboratory workhorse strain L. lactis MG1363. Although insertional mutagenesis is usually used in loss-of-function studies to characterize the effect of a genetic knockout (24, 49, 52), our data indicated that the expression of AbiV was turned on by insertion of pGhost9::ISS1. Microbiological and molecular characterization of AbiV is also described below.


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MATERIALS AND METHODS
 
Bacterial strains, phages, and plasmids.
Bacterial strains used in this study are listed in Table 1, and bacteriophages and plasmids are listed in Table 2. Bacteriophages sk1 and jj50 were kindly provided by F. K. Vogensen (University of Copenhagen). L. lactis strain MB112 was used as the host for selection of phage-resistant integrants. This strain is a derivative of L. lactis subsp. cremoris MG1363 and has a mutation in the upp gene. This mutation makes L. lactis MB112 resistant to fluorouracil, which provided an additional marker to confirm that the phage-resistant integrants were derived from the parental strain (51). Escherichia coli was grown at 37°C in LB medium (70). L. lactis was grown in M17 medium (74) supplemented with 0.5% glucose (GM17). When fluorouracil resistance was tested, L. lactis strains were grown in SA medium supplemented with 0.5% glucose (38). Most lactococci were grown at 30°C; the exceptions were strains containing the thermosensitive vector pGhost9::ISS1, which were grown at 28 and 37°C to allow replication and integration, respectively, of the vector. When L. lactis was grown at temperatures above 30°C, NaCl (1%) was added to M17 medium (41). In phage infection experiments, 10 mM CaCl2 was added to plates or to the medium. When appropriate, antibiotics were added at the following concentrations: for E. coli, 100 µg ml–1 of ampicillin, 10 µg ml–1 of chloramphenicol, and 150 µg ml–1 of erythromycin; and for L. lactis, 3 µg ml–1 of chloramphenicol, 3 µg ml–1 of erythromycin, and 0.3 µg ml–1 fluorouracil.


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  		TABLE 1. Bacterial strains used in this study


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  		TABLE 2. Phages and plasmids used in this study

Insertional mutagenesis with pGhost9::ISS1 and isolation of phage-resistant mutants.
Phage-resistant derivatives of L. lactis MB112 were obtained by insertional mutagenesis using the vector pGhost9::ISS1, essentially as described by Maguin et al. (49). Subsequent cloning of the flanking chromosomal DNA was also performed as described by Maguin et al. (49). To ensure isolation of phage-resistant integrants from distinct events, mutagenesis was performed with three independent cultures of L. lactis MB112 containing pGhost9::ISS1. These cultures were grown overnight at 28°C in GM17 containing erythromycin. The cultures were then diluted 100-fold in GM17 and grown for 2.5 h at 28°C to obtain exponential growth. The cells were incubated at 37°C for 2.5 h to allow plasmid integration. The cultures were diluted again 10,000-fold in 50 ml of GM17 supplemented with erythromycin and incubated overnight at 37°C to allow phenotypic expression. These overnight cultures were inoculated (1%) into fresh GM17 with erythromycin and incubated at 37°C. When the exponential growth phase was reached, aliquots were mixed with the virulent lactococcal phage sk1 (multiplicity of infection [MOI], >1). Following 10 min of incubation at 37°C, the infected cells were plated on GM17 plates containing erythromycin, NaCl (1%), and CaCl2 (10 mM) and incubated for 2 days at 37°C. The frequency of phage-resistant mutants was calculated, and 100 mutants were selected at random from the three independent cultures. The phage-resistant phenotype of these mutants was verified by cross-streaking with virulent phage sk1 at 37°C. Finally, curing the phage-resistant mutants of the pGhost9::ISS1 plasmid, leaving a single copy of ISS1 at the integration site, was performed as described by Maguin et al. (49).

RNA isolation and purification and RT-PCR analysis.
Overnight cultures were diluted 100-fold and grown to an optical density at 600 nm of 0.5 at 37°C. Aliquots (2 ml) were harvested by quick centrifugation (20,000 x g, 30 s), and each pellet was resuspended in a solution of 0.5 M sucrose with 60 mg ml–1 lysozyme. Following incubation (37°C, 15 min), the cells were pelleted and resuspended in 1 ml TRIzol reagent (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. Prior to reverse transcription (RT)-PCR, the RNA samples were treated with a DNase-based TURBO DNA-free kit (Applied Biosystems).

RT-PCR was carried out using a RevertAid First Strand cDNA synthesis kit (Fermentas) as recommended by the manufacturer. As a control, the RT-PCR procedure was carried out without reverse transcriptase to ensure that there was no contaminating DNA in the RNA samples.

Phage assays.
Propagation of phages was performed as described by Emond et al. (27). Determination of the titers of phage lysates (42), the EOP (71), and cross-streaking assays (69) were performed as described previously. Adsorption assays were performed as described elsewhere (71), except that 5 min of incubation was used instead of 15 min. Cell survival (5) was assayed using an MOI of 5. One-step growth curve experiments and center of infection assays (59) were performed using MOIs of 0.2 and 0.5, respectively. The efficiency of formation of centers of infection and the burst size were calculated as previously reported (59).

Phage DNA replication.
Replication of phage DNA was monitored by performing a time course experiment (35). Briefly, total DNA of L. lactis cells was isolated at 10-min intervals from cultures infected with reference phage p2 (MOI, 2), which is closely related to phage sk1. Total DNA was digested with EcoRV and heated (65°C, 10 min) prior to gel electrophoresis, which allowed identification of resolved cos sites that were used to distinguish concatemeric and mature phage DNA.

Phage adsorption and DNA ejection assays using SYBR gold staining.
Visualization of phage DNA by labeling with the fluorescent dye SYBR gold was performed as described by Noble and Fuhrman (61), with the following modifications. The original SYBR gold solution was diluted 1,000-fold. The phage lysate was treated with 1 µg ml–1 DNase and RNase for 30 min at 37°C and then stained with the diluted SYBR gold (final concentration, 2.5% [vol/vol]). The mixture was incubated for at least 12 h at 4°C in the dark. One microliter of the labeled phage lysate was mixed with 1 µl of an exponentially growing cell culture and visualized using a Zeiss Axioplan epifluorescence microscope.

Rescue cloning and sequencing of the flanking chromosomal DNA.
Chromosomal DNA was analyzed for six phage-resistant integrants. The HindIII-digested DNA was purified, diluted to favor self-ligation, and ligated as described by Maguin et al. (49). The ligation mixture was electroporated into E. coli EC1000 (45) and plated on LB agar with erythromycin. Plasmid DNA containing the rescued pGhost9::ISS1 plasmid and the flanking chromosomal DNA was isolated from E. coli cells (strains JH-56 to JH-59). Chromosomal DNA flanking the ISS1 element was sequenced using primer 5'-GAAGAAATGGAACGCTC-3' annealing to the ISS1 sequence. The procedure described above was successful for four of the six integrants.

DNA isolation and manipulation.
Plasmid DNA was isolated from L. lactis using a QIAprep Spin Miniprep kit (Qiagen) according to the manufacturer's recommendations, except that lysozyme (15 mg ml–1) was added to buffer P1 and the lysis solution with the resuspended cells was incubated at 37°C for 30 min before the rest of the protocol was performed. Restriction enzymes, T4 DNA ligase, and the Klenow fragment (Fermentas) were used according to the manufacturer's instructions. Electroporation of L. lactis was performed as described previously (37, 58). The DNA fragment corresponding to nucleotides 1021 to 2320 in the GenBank accession number AF324839 sequence (Fig. 1) was first cloned into the E. coli pCR II-TOPO vector (Invitrogen) using TOP10F' cells prior to cloning into the E. coli-L. lactis shuttle vectors pCI372 and pLC5. For protein purification, the DNA fragment corresponding to nucleotides 1273 to 1875 in the GenBank accession number AF324839 sequence was cloned into the His-tagged vector pQE-70 (Qiagen) as recommended by the manufacturer to create pJH11 in E. coli strain JH-62.


Figure 1
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  		FIG. 1. Localization of orf1 (abiV) on the chromosome of L. lactis MG1363. The numbers indicate nucleotide positions in the GenBank accession number AF324839 sequence. (A) L. lactis strains with the transposon-containing vector pGhost9::ISS1 or cured of pGhost9 and thus containing a solitary ISS1 sequence inserted into the chromosome. The numbers and arrows indicate the positions and directions of the inserts. (B) L. lactis strains with the cloned DNA fragment (bp 1021 to 2320) including orf1. The arrows indicate promoter P59, and the x in JH-24 indicates the position of the frameshift mutation introduced into this strain.

Protein purification and gel filtration.
A C-terminally His-tagged protein was purified according to the manufacturer's instructions (Qiagen). Gel filtration was carried out using a HiPrep 16/60 Sephacryl S-300 high-resolution column from GE Healthcare, 150 µg of AbiV, and a flow rate of 0.2 ml min–1. The column was connected to a Shimadzu high-performance liquid chromatography system. The solvent delivery module used was an LC-10ADvp module, and the detector used was an SPD-M10Avp detector (280 nm). The mobile phase contained 50 mM Tris-HCl and 200 mM NaCl (pH 7.6). A Bio-Rad protein standard (catalog no. 151-1901) was used to determine the molecular weight of the purified protein. Each run lasted 800 min. The molecular weight was estimated by determining the mean of four different trials.

Construction of expression vector pLC5.
The low-copy-number vector pGKV259 (76) was digested with PstI (site located downstream from the P59 promoter), which was followed by gel purification. Two complementary oligonucleotides (5'-TGGATCCAAAGGAGGTCCTGCA-3' and 5'-GGACCTCCTTTGGATCCATGCA-3') were annealed together (70) to create a double-stranded linker with PstI-compatible sticky ends. This linker also contained a unique BamHI site and a ribosome-binding site (RBS) (5'-AGGAGG-3'). The linker was inserted into the PstI site of pGKV259, and the ligation mixture was transformed into E. coli MC1061. Transformants were selected on LB plates containing 20 µg ml–1 of chloramphenicol. Positive clones with the linker inserted in the right direction were identified by colony PCR, and clones were confirmed by sequencing. Upon introduction of the linker into pGKV259, the PstI site on the 5' side of the linker was disrupted, whereas the site on the 3' side was conserved. Thus, a unique PstI site was created 8 bp downstream from the RBS. Cloning of a DNA fragment having an ATG start codon into the PstI site of pLC5 enabled efficient transcription from the P59 promoter and translation from the introduced RBS.

DNA and protein analyses.
Sequence similarity searches in databases were performed using BLAST (3) at the NCBI website (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Searches for helix-turn-helix motifs were performed using the website http://npsa-pbil.ibcp.fr/. The molecular weights and pIs of the investigated proteins were estimated using the Protein Calculator at the website http://www.scripps.edu/~cdputnam/protcalc.html. Searches for transmembrane domains and signal peptide motifs were performed using the predictor websites http://www.cbs.dtu.dk/services/TMHMM-2.0/ and http://www.cbs.dtu.dk/services/SignalP/.


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RESULTS
 
Isolation of phage-resistant mutants of L. lactis subsp. cremoris MG1363 using insertional mutagenesis.
The laboratory strain L. lactis subsp. cremoris MB112 (MG1363{Delta}upp) (51) is sensitive to several virulent phages belonging to the 936 and c2 species. To identify genes involved in the sensitivity of L. lactis MG1363 to 936 phages, mutant cells resistant to phage sk1 were isolated after insertional mutagenesis of the host. Insertional mutagenesis was performed with three cultures, which resulted in isolation of three independent insertion libraries. Selection for resistance to virulent lactococcal phage sk1 was performed for all three libraries (MOI, >1). The frequency of phage-resistant colonies was 6.2 x 10–6 ± 2.6 x 10–6, which is 100-fold higher than the frequency of spontaneous phage-resistant mutants obtained from wild-type cultures (5.9 x 10–8 ± 3.0 x 10–8).

Identification of a chromosomal gene involved in phage resistance.
Six confirmed phage-resistant mutants (two mutants randomly selected from each of the three independent cultures) were chosen for plasmid rescue experiments. In four cases it was possible to isolate the inserted plasmid along with a piece of flanking chromosomal DNA (pJH7 to pJH10). Sequence analysis revealed insertions in the same 350-bp region on the chromosomes of the four phage-resistant mutants. The insertions were located in the intergenic region between two genes (designated orf1 and trans) or in the 3' end of the trans gene (Fig. 1A). The inserts were located at nucleotides 1962 (for phage-resistant mutants JH-32 and JH-46), 2240 (JH-48), and 2296 (JH-47). The nucleotide positions are based on the GenBank accession number AF324839 sequence. Strains JH-32 and JH-46 originated from the same mutation library and may be siblings. Since insertions in the three different phage-resistant mutants were observed both in the trans gene and in the intergenic region between trans and orf1, the phage resistance phenotype could not be caused by a knocked-out trans gene. Furthermore, the lin gene downstream of orf1 is transcribed in the opposite direction. Taken altogether, these results indicated that orf1 is a key player in the phage resistance phenotype.

At least two hypotheses could explain the involvement of orf1 in the phage resistance phenotype. orf1 could be part of an operon transcribed from a promoter upstream of trans and thus terminated by the pGhost9::ISS1 insertions, thereby inactivating transcription of the gene. A second possibility is that the pGhost9::ISS1 insertion provided a promoter upstream of orf1. In the latter scenario, transcription of orf1 would be initiated from a promoter in the ISS1 sequence or from the Emr gene in the pGhost9::ISS1 construct, leading to activation of a phage resistance phenotype. When the insertion mutants were cured of the vector pGhost9::ISS1, leaving a single copy of ISS1 at the integration site, cured L. lactis strains JH-49 to JH-52 (Table 1) lost the phage resistance phenotype, suggesting that a promoter activity originated from the vector, possibly from the Emr gene (Fig. 1A).

To test the hypothesis that orf1 was transcribed from a promoter in the inserted plasmid pGhost9::ISS1, an RT-PCR assay was performed with L. lactis strains with and without the insertion. RNA from exponentially growing cells was used as the template for random RT of total RNA, and primers annealing to an internal region of orf1 were used to amplify the cDNA by PCR. A PCR product of the expected size was observed in the mutant with the insertion (Fig. 2, lane 3) but not in the wild-type strain (Fig. 2, lane 2), indicating that the transcription of orf1 was turned on in the mutant. A negative control experiment was conducted without reverse transcriptase, and it confirmed that there was no contaminating DNA in the samples (Fig. 2B). A positive control experiment using primers for the glycolytic genes gapB and pfk was also performed (data not shown). The positive results of this experiment eliminated the possibility of artifacts due to loss of RNA in the samples.


Figure 2
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  		FIG. 2. RT-PCR with RNA isolated from exponentially growing L. lactis cultures. Lane 1, JH-20(pJH2); lane 2, JH-54(pLC5); lane 3, JH-32 (insertion mutant); lane C, control PCR with L. lactis MB112 chromosomal DNA; lane L, ladder (GeneRuler; 100 to 10,000 bp; Fermentas). (A) Experiment performed with reverse transcriptase. (B) Experiment performed without reverse transcriptase.

A silent phage resistance mechanism is encoded by orf1 on the chromosome of L. lactis subsp. cremoris MG1363.
To test the hypothesis that orf1 encodes a phage resistance mechanism, a DNA fragment (positions 1021 to 2320 in the GenBank accession number AF324839 sequence) comprising only one open reading frame (orf1) and the upstream region including the most distant insertional mutation site at position 2296 was cloned in the promoterless shuttle vector pCI372 (pJH6) and in the expression vector pLC5 (pJH2) (Fig. 1B). The resulting constructs were transformed into L. lactis MB112, and the resulting strains (JH-53 and JH-20, respectively) were tested to determine their phage resistance. L. lactis JH-53 (containing pJH6, with no promoter upstream of orf1) was sensitive to phage sk1, whereas L. lactis JH-20 (containing pJH2, with a strong promoter upstream of orf1) was resistant to phage sk1 (Fig. 1B). In fact, the highest level of expression was observed when orf1 was expressed from the pLC5 vector, which was probably due to the strong P59 promoter (Fig. 2, lanes 1). Furthermore, expression of orf1 (pJH2) in another host (L. lactis subsp. lactis IL1403) also conferred a phage resistance phenotype (data not shown). The results described above indicated that expression of orf1 was responsible for the phage resistance phenotype.

To verify that a protein encoded by orf1 was responsible for the resistance to phage sk1, a frameshift mutation was introduced into orf1 by digestion at a unique ClaI site (codon 36 in orf1) and filling of the ends with the Klenow fragment, followed by ligation, confirmation by sequencing, and transformation of the resulting plasmid, pJH3, into L. lactis MB112. The frameshift mutation eliminated the phage resistance phenotype in the resulting strain, JH-24 (Fig. 1B). We concluded that the protein encoded by orf1 was responsible for the phage resistance phenotype.

Sequence and analysis of orf1.
The 1,300-nucleotide DNA fragment cloned into pJH2 was sequenced and found to be 100% identical to the region including nucleotides 1021 to 2320 in the GenBank accession number AF324839 sequence, as well as positions 697547 to 698846 in the complete genome sequence of L. lactis MG1363 (accession number AM406671). The G+C content of orf1 was 31.7%. No suitable promoter was found upstream of orf1 (bp 1 to 430) using Winseq software (F. G. Hansen, unpublished data). This is in agreement with the experimental results showing that orf1 is silent in wild-type L. lactis strain MG1363. The translation start codon of orf1 was preceded by a suitable RBS (5'-TGAACGGAGAG-3'; the underlined nucleotides match the consensus sequence). DNA sequence analysis did not result in identification of any transcription terminator structures between orf1 and the upstream trans gene, leaving the possibility that orf1 could be part of an operon initiated upstream of trans. However, the negative RT-PCR results for orf1 transcription in the wild-type strain (Fig. 2, lane 2) suggest that orf1, and perhaps trans as well, is not expressed under the conditions tested. This was confirmed by a Northern analysis of the insertion mutant L. lactis JH-32 and L. lactis wild-type strain MG1363, in which transcription of orf1 was observed only in JH-32 (data not shown).

The phage resistance mechanism encoded by orf1 is effective against virulent lactococcal phages belonging to the 936 and c2 species.
Representatives of the three main lactococcal phage species, 936, c2, and P335, were tested to determine their sensitivities to orf1 (Table 3). EOPs of approximately 10–4 were obtained for the four c2-like phages tested and for five of the six members of the 936 species tested (Table 3). Phage 712 (936 species) was not sensitive to orf1 (EOP, 1) (Table 3). The seven phages belonging to the P335 species were also not affected by the presence of orf1 (Table 3). Because the efficacy of orf1 with virulent P335 phages was tested using another L. lactis host (SMQ-86), we needed to rule out the possibility that the insensitivity of P335 phages was due to a mutation in pJH2 present in L. lactis SMQ-86 (L. lactis JH-23). Thus, plasmid pJH2 was isolated from L. lactis JH-23 and reintroduced into L. lactis MB-112. Phage p2 was unable to effectively replicate on this recombinant host (EOP, 10–4), indicating that pJH2 was indeed functional and confirming that the P335 phages were not sensitive to orf1.


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  		TABLE 3. EOP and phages tested

The EOPs of 936- and c2-like phages were also determined at 30 and 37°C and were found to be similar, indicating that the system is stable in this temperature range. EOPs of 1.6 x 10–5 ± 0.2 x 10–5 and 2.7 x 10–4 ± 0.3 x 10–4 were obtained for phages sk1 and p2, respectively, when orf1 was expressed from the chromosome in the insertion mutants. These values are in the range obtained when orf1 was expressed from a strong promoter in the low-copy-number expression vector pLC5. However, when the insertion mutants were tested with phage c2, EOPs of 4.6 x 10–1 ± 0.9 x 10–1 were obtained, indicating that a higher level of expression of orf1 is required with c2. Finally, pJH2 was transformed into L. lactis MG1363 (JH-79), and the EOPs with phages p2, 712, and c2 were determined. EOPs similar to those obtained with MB112 eliminated the possibility that the upp deletion in MB112 influenced the phage resistance phenotype.

The efficiency of orf1 as a phage resistance mechanism was further characterized using phage p2 and L. lactis strains JH-20 (pJH2) and MB112 (Table 4). The efficiency of formation of centers of infection of phage p2 with L. lactis JH-20 was 0.5% ± 0.2%, indicating that only 5 of 1,000 infected cells released at least one virulent phage. One-step growth curve experiments were then performed in the presence or absence of pJH2 (orf1), and the burst size was reduced by 72% (from 38.8 ± 5.7 in MB112 to 11.1 ± 5.2 in JH-20) (Table 4).


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  		TABLE 4. Effect of AbiV on phage p2a

The phage resistance system encoded by orf1 is an Abi mechanism.
A series of experiments were conducted to determine the type of phage resistance mechanism encoded by orf1. Adsorption assays showed that the level of adsorption of phage sk1 to phage-resistant cells was 95.9% ± 10.6%, which is similar to the adsorption level for the wild-type host. Furthermore, fluorescently labeled (SYBR gold-labeled) phage DNA was visualized after sk1 infection of the phage-resistant strain L. lactis JH-20 and the phage-sensitive strain MB112. Immediately following phage infection, a fluorescent halo of adsorbed phages was seen surrounding the host cells (data not shown). Less than 15 min after the beginning of phage infection, the fluorescent signal on the cell surface was reduced, while a bright fluorescent signal was observed in the center of the cell, indicating that the phage DNA had been ejected into the host cell (data not shown). Identical results were obtained for phage-resistant and phage-sensitive strains. The results described above indicated that the phage resistance mechanism was not an adsorption- or ejection-blocking mechanism.

Smaller phage plaques were observed at a frequency of 10–4 with L. lactis JH-20, which contained orf1. These plaques were propagated on L. lactis JH-20 cells, as well as on the phage-sensitive host L. lactis MG1363, and were found to be insensitive to the antiphage mechanism, which demonstrated that this mechanism is not a restriction/modification system. A cell survival assay showed that there was virtually no survival of cells of the sensitive strain or the phage-resistant strain upon phage infection (Table 4). Moreover, the few plaques of phage p2 arising at a frequency of 10–4 were smaller on the phage-resistant strain than on the wild-type sensitive L. lactis strain (Table 4). The characteristics described above are well-documented characteristics of abortive phage infection mechanisms. The low G+C content of orf1 (31.7%) is also typical of Abi mechanisms. Taken altogether, the data show that the phage resistance mechanism encoded by orf1 is an abortive infection mechanism. Accordingly, the gene was designated abiV and the Abi mechanism was designated AbiV.

Analysis of the AbiV protein.
AbiV consists of 201 amino acids and has a calculated molecular mass of 22,692 Da. A His-tagged AbiV protein was overexpressed in E. coli and purified. The native molecular mass of the purified AbiV protein was estimated to be 49 ± 0.3 kDa by gel filtration (data not shown), suggesting that AbiV forms a dimer in its native form. The pI was calculated to be 5.37. The protein does not contain any putative transmembrane or signal peptide motifs, and it is therefore likely to be cytosolic. Although 69% of AbiV consists of {alpha}-helixes, no DNA-binding helix-turn-helix motif was found in AbiV. Similarity searches using several bioinformatics tools did not reveal any similarity to other lactococcal proteins or any other phage resistance mechanism, nor was AbiV found in other sequenced genomes of lactococcal strains. Likewise, no conserved domains were found in the protein.

Expression of AbiV does not affect the cellular growth rate or final biomass.
A cell growth experiment was conducted to test whether the expression of AbiV from a strong plasmid-encoded promoter could influence the cell growth rate or the final biomass. L. lactis JH-20 (Abi+) and MB112 (Abi–) grew exponentially in GM17 at 30°C with growth rates of 1.04 ± 0.08 and 1.00 ± 0.03 h–1, respectively. The final concentrations were 2.49 x 109 ± 0.10 x 109 and 2.45 x 109 ± 0.02 x109 cells ml–1, respectively. Based on these experiments, it was concluded that the expression of AbiV did not affect cellular growth. Moreover, the results showed that the newly constructed expression vector pLC5 did not interfere with cell growth.

AbiV affects phage DNA maturation.
The DNA replication of the cos-type virulent lactococcal phage p2 was determined at time intervals during infection of a resistant L. lactis strain (JH-20) and a sensitive L. lactis strain (JH-54). Phage DNA was analyzed by digesting the total DNA isolated from infected cells with EcoRV and then comparing the resulting fragments with the EcoRV restriction map of phage p2 (Fig. 3). Ten minutes after infection, replication of phage DNA was observed in both strains. In sensitive cells, the concentration of phage DNA decreased around 40 min after infection, coinciding with lysis of the host culture. In comparison, phage DNA persisted in the resistant cells throughout the experiment, which was terminated after 2 h.


Figure 3
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  		FIG. 3. Replication of phage p2 DNA during infection of phage-sensitive strain L. lactis JH-54 (A) and phage-resistant strain L. lactis JH-20 (B). Samples were taken at –10, 0, 10, 20, 30, 40, 50, 60, 90, and 120 min, and total DNA was restricted with EcoRV. The band at 5.3 kb (consisting of the bands at 1.3 and 4 kb for mature resolved DNA) spans the cos sites which mark the extremities of the phage genome. The numbers in panel C indicate the sizes of phage p2 DNA fragments after EcoRV digestion and heat treatment. The ladder used was GeneRuler (100 to 10,000 bp; Fermentas).

Analysis of the EcoRV-digested phage DNA pattern revealed (among other bands) two bands at 1.3 and 4 kb, as well as a 5.3-kb fragment in the phage-sensitive culture. The 5.3-kb DNA fragment spanned the cos site on the phage p2 genome, and the 1.3- and 4-kb fragments represented the mature encapsidated phage DNA. Therefore, both replicative DNA and encapsidated DNA were observed in the phage-sensitive strain due to continuous DNA replication throughout the phage lytic cycle and simultaneous encapsidation of mature DNA. With the resistant cells, only the 5.3-kb fragment was obtained. The absence of the 1.3- and 4-kb bands, as well as the presence of phage DNA throughout the experiment, indicated that phage DNA accumulated in its concatemeric (nonmature) form in the resistant L. lactis cells. Similar results were obtained with the closely related phage sk1 (data not shown). The results described above showed that AbiV prevented cleavage of the replicated phage DNA and thus that it acts at a later stage of the phage infection process.


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DISCUSSION
 
Insertional mutagenesis was used to confer phage resistance to L. lactis subsp. cremoris MG1363. Four phage-resistant mutants obtained in three independent insertional mutagenesis experiments were selected for further analysis, and in all cases the insertion was located in the same 350-bp region of the bacterial chromosome. Insertional mutagenesis with the pGhost9::ISS1 system (48, 49) was previously used to knock out factors involved in phage adsorption (24) and in DNA ejection (47). Such loss-of-function effects of insertional mutagenesis are the usual outcome of experiments like these (24, 67, 75). However, using a similar strategy, Luccini et al. also isolated a phage-resistant mutant of Streptococcus thermophilus in which promoter activity from the insertion turned on a downstream restriction/modification mechanism (47). A similar finding was obtained in this study as integration of pGhost9::ISS1 conferred phage resistance to the cells by activating an abortive infection mechanism.

All the ISS1 inserts were oriented in the same direction as orf1, which made us speculate that orf1 could be transcribed from a promoter within ISS1 or from the Emr gene in pGhost9::ISS1 (49). Promoter activity has previously been reported outward of insertion sequences (29) in both the forward (24) and backward (15) directions, and insertion sequences with a high degree of similarity to ISS1 have also been shown to be in the vicinity of abortive infection mechanisms (4, 15). Searches for promoter sequences in the forward direction of ISS1 were unsuccessful. This observation was supported by the reversion to a phage-sensitive phenotype when mutants were cured of the plasmid while they kept a single copy of ISS1 at the integration site (Fig. 1A). However, the activation hypothesis was supported by the results of subsequent transcription analyses using RT-PCR with mRNA from L. lactis strains with and without the insertion. Transcription of orf1 was observed in the presence of an upstream pGhost9::ISS1 insertion. These results indicated that orf1 was indeed turned on by a promoter in pGhost9::ISS1, which was most likely the promoter of the Erm gene. Cloning of orf1 and its upstream region into the promoterless vector pCI372 failed to confer resistance, while similar cloning into the expression vector pLC5 resulted in a phage resistance phenotype. An active orf1 was later shown to encode the abortive infection protein AbiV, confirming that integration by pGhost9::ISS1 can be used to transcribe silent genes on bacterial chromosomes.

The ISS1 insertion sequence has been reported to integrate randomly in the chromosome of L. lactis (49). In the present study, ISS1 integrated in a 350-bp region in three independently obtained mutants, conferring phage resistance. In a similar study with L. lactis subsp. cremoris Wg2 and L. lactis subsp. lactis IL1403, Dupont et al. (24) obtained phage-resistant mutants that were due to a defect in phage adsorption with frequencies around 5 x 10–6, the same frequency obtained in the present study. We did not obtain any mutants with reduced phage adsorption, which indicates either that the phage receptors on L. lactis MG1363 are more difficult to mutate and perhaps are essential for cell growth or that ISS1 integrates in a nonrandom manner in this strain.

abiV is located in a 59-kb DNA region of the L. lactis MG1363 chromosome (nucleotides 657,000 to 706,000), which was previously referred to as an "integration hot spot" (77) because it contains DNA sequences involved in DNA mobility. For example, this region contains genes and sequences usually found on plasmids, as well as almost 20% of the 71 insertion sequences found in the 2,529,478-bp genome of MG1363. Interestingly, two of these insertion sequences, namely IS946 and IS1297, share 98 and 85% nucleotide similarity with ISS1, respectively. There is only one copy of each of these insertion elements in the genome of L. lactis MG1363. The high frequency of insertion elements in this region suggests that it has features favoring the integration of insertion sequences in general and of ISS1-type sequences in particular. Thus, ISS1 integration may not be completely random in L. lactis MG1363. The location of abiV in the integration hot spot region among remnants of plasmid DNA and insertion sequences also makes it tempting to speculate that abiV originated from a plasmid. This would correspond to the observation that most of the previously described lactococcal Abi systems are encoded on plasmids (2, 14, 28, 73).

abiV conferred phage resistance when it was cloned in an expression vector, but it lost this function upon introduction of a frameshift mutation into the gene. Cells expressing AbiV exhibited typical abortive infection characteristics upon phage infection, such as normal phage adsorption and DNA ejection, no restriction/modification activity, and cell death. Very few infected cells released progeny phages, and the burst size was also reduced. Since database searches revealed no similarity to any other phage resistance mechanism, it was concluded that AbiV is a novel Abi mechanism.

AbiV inhibits proliferation of small isometric phages belonging to the 936 species and of prolate phages belonging to the c2 species, but it has no effect on small isometric phages belonging to the P335 species. This range of efficacy against the three main phage groups has been observed with other lactococcal Abi mechanisms (4, 14, 26, 53, 65, 66), but the lack of similarity with other Abi proteins suggests that there is a different mode of action. Combinations of diverse phage resistance mechanisms are often observed for plasmids isolated from wild-type strains of L. lactis that are highly resistant to phages (2, 21, 25, 32). Since AbiV is a novel Abi mechanism, it may be suitable to use it in combination with other phage resistance mechanisms to confer efficient phage resistance to industrial strains of L. lactis.

Lactococcal Abi mechanisms are often characterized further by identifying the general step in the phage lytic cycle that is inhibited (14). In infected AbiV-containing cells, we observed that phage DNA is replicated but maturation is halted, resulting in accumulation of concatemeric phage DNA. Replication of phage DNA indicated that transcription and translation of early phage genes took place in the presence of AbiV.

It was also observed that the efficiency of AbiV activity (expressed as EOP) against 936 phages was not improved by expressing abiV from the strong lactococcal promoter P59 (76) in a low-copy-number plasmid (68) compared to expression from an internal promoter in pGhost9::ISS1 and expression of a single copy from the chromosome. This suggests that only small amounts of AbiV are needed or that competitive inhibition of a substrate is not part of the mode of action of AbiV. However, the expression level was important for the efficiency of activity against c2 phages. Toxicity of Abi proteins has been demonstrated for AbiD1, AbiK, AbiN, and AbiO (14), and in at least two cases, it was associated with regulation of Abi gene expression (4, 27). The absence of effects on the cellular growth rate during high levels of expression of abiV demonstrates that AbiV is not toxic to the cell. The absence of sequence similarity between Abi proteins (10, 14) makes it desirable to obtain structural data for Abi proteins in order to investigate possible correlations between structure and function. The native multimer state of a protein might be a basic parameter to compare Abi proteins. However, except for motifs in AbiA which are putative multimerization sites (22), AbiV is the only Abi mechanism for which the native state (dimer) has been determined.

In conclusion, AbiV is a novel abortive infection mechanism that was discovered on the chromosome of L. lactis subsp. cremoris MG1363. This discovery was possible due to transcription from a promoter within pGhost9::ISS1, which was integrated in the upstream region. AbiV is effective against 936 and c2 phages, but the mode of action of this 23rd lactococcal Abi system needs to be investigated further as it may involve an early transcribed phage gene or gene product.


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ACKNOWLEDGMENTS
 
This work was funded in part by a graduate scholarship from the Technical University of Denmark to J.H. and a strategic grant from the Natural Sciences and Engineering Research Council of Canada to S.M.


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FOOTNOTES
 
* Corresponding author. Mailing address: Center for Systems Microbiology, DTU Biosys, Matematiktorvet, Bldg. 301, Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: 45 45 25 24 96. Fax: 45 45 93 28 09. E-mail: kha{at}bio.dtu.dk Back

{triangledown} Published ahead of print on 5 September 2008. Back

{dagger} Present address: Département de microbiologie et d'infectiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, Québec, Canada J1H 5N4. Back


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Applied and Environmental Microbiology, November 2008, p. 6528-6537, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00780-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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