Phage preparations.

Phages were purified using CsCl density gradient centrifugation (47). Where indicated, Tuc2009 was further purified by means of isopycnic centrifugation using a sucrose gradient. The latter procedure was performed by loading CsCl-purified phage onto 20 to 70% sucrose in TBT (100 mM Tris-HCl [pH 7], 100 mM NaCl, 10 mM MgCl₂) gradients, which had been preformed in clear SW41 tubes with a Hoefer SG gradient maker. The phage band was harvested following centrifugation at 35,000 rpm for 1.5 h at 4°C in a SW41 rotor with a Beckman L-60 ultracentrifuge. The virus particles were then suspended in TBT and pelleted by a further centrifugation at 35,000 rpm for 1 h at 4°C. The resultant phage pellet was resuspended in 100 μl of TBT. Plaque assays were performed as described by Lillehaug (31).

Sequence analysis.

Database searches and protein family (pfam) allocations were performed using BLASTN and BLASTP (1) and using conserved domain search programs, respectively, located at the following URL (http://www.ncbi.nlm.nih.gov/). Sequence alignments were performed using the Clustal alignment method and MEGALIGN 3.16 software from a DNASTAR 2002 version 5 software package.

DNA manipulations and sequencing.

PCR amplifications were carried out using an EXPAND long template PCR system (Roche) according to the manufacturer's instructions with a Gene Amp PCR system 2400 thermal cycler (Perkin-Elmer) and the primers described in Table ​Table2.2. Similarly, restriction enzymes, shrimp alkaline phosphatase, and T4 DNA ligase were supplied by Roche and employed as recommended by the manufacturer. Oligonucleotides were manufactured by MWG (Ebersberg, Germany). Purification of plasmids from E. coli was performed using a Wizard Plus SV Miniprep kit (Promega). Plasmid DNA preparations from L. lactis were completed using the protocol of O'Sullivan and Klaenhammer (41). Sequence analysis was performed by MWG (Ebersberg, Germany).

Plasmid construction.

Specific PCR-generated DNA fragments representing sections of tal₂₀₀₉ or the complete lys gene (2) from Tuc2009 were produced. NcoI and BglII sites were incorporated into the relevant synthetic oligonucleotides to insert these PCR products into the E. coli expression vector pQE60 (Table ​(Table1).1). The stop codon that defines the 3′ end of either tal₂₀₀₉ or lys was omitted from complementary oligonucleotides, allowing a translational fusion of the various Tal₂₀₀₉ sections or the Lys protein with the six-His tag encoded by pQE60. In the case in which the tetK gene was inserted into pTal₂₀₀₉-5ΔN to produce pTal₂₀₀₉-5ΔNtet, an NdeI restriction site was included at both ends of the tetK PCR product via incorporation of the NdeI recognition sequence into both oligonucleotides. Otherwise, they were identical to those used by Maguin et al. to amplify the tetK gene with pGhost8 as a template (33). For the construction of pTal₂₀₀₉mut-2ΔN, suitable primers were designed and site-directed mutagenesis was carried out using the PCR-based SOEing technique (22). To check expression of tal₂₀₀₉ in L. lactis, the DNA fragment corresponding to tal₂₀₀₉-2ΔN was restricted from pTal₂₀₀₉-2ΔN by the use of NcoI and HindIII and the smallest fragment generated by this digestion was ligated into the similarly restricted pNZ8048 to produce pNZTal₂₀₀₉-2ΔN. This allowed transfer of the tal₂₀₀₉-2ΔN fragment fused to the six-His codons from pQE60 in pTal₂₀₀₉-2ΔN to a position downstream of the nisin-inducible promoter of the lactococcal expression vector pNZ8048 (15). Electrotransformation of plasmid DNA into E. coli was performed as described by Sambrook et al. (47), while that of L. lactis NZ9800 was performed as described by Wells et al. (54). All DNA cloning steps were initially performed using E. coli as a host. The integrity of all clones was checked by restriction profiling and DNA sequencing.

Protein expression and purification.

Overexpression of target proteins was achieved using the E. coli expression plasmid pQE60 essentially as recommended by Qiagen. Induction was accomplished over a 4-h period and was initiated using isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma) at a final concentration of 1 mM when the culture had reached an optical density at 600 nm of 0.35 to 0.4. Protein expression in L. lactis from pNZ8048 was produced as outlined by de Ruyter et al. (15). Cultures of L. lactis or E. coli (30 ml) were used to express proteins for zymogram assays and autocleavage studies. The cells were harvested by centrifugation in a Hermle Z-323 centrifuge at 4,000 rpm for 20 min. Lysis was carried out by resuspending the pellet in 1 ml of lysis buffer (50 mM NaH₂PO₄ [pH 8.0], 1 M NaCl, 30 mM imidazole) and performing cell disruption in a mini-beadbeater-8 (Biospec products) for 10 min at 4°C.

Whole cells, cellular debris, and insoluble proteins were cleared by centrifugation at 14,000 rpm for 20 min in an Eppendorf benchtop centrifuge. Protein purification of Tal₂₀₀₉-5ΔN involved overexpressing the protein in 300 ml of LB broth. Cells were harvested in a Beckman J2-21 centrifuge at 4,000 rpm for 20 min. The pellet was resuspended in 10 ml of lysis buffer and disrupted in an alcohol ice bath by ultrasonication (Soniprep 150) with 30-s bursts at an amplitude of 10 μm followed by 15-s breaks. This sonication was continued for 10 min. Lysates were cleared as described above, and the overexpressed TalT-5ΔN was purified by immobilized-metal-affinity chromatography. Briefly, this involved passing the lysate through a column containing 4 ml of nickel-nitrilotriacetic acid matrix (Qiagen) which had been preequilibrated with 10 ml of the lysis buffer. Two 10-ml washes were carried out using wash buffer (50 mM NaH₂PO₄ [pH 8.0], 1 M NaCl, 30 mM imidazole, 0.5% Triton X-100, 5 mM β-mercaptoethanol). The protein was then eluted using 10 ml of the elution buffer (50 mM NaH₂PO₄ [pH 8.0], 1 M NaCl, 250 mM imidazole) in approximately 1-ml aliquots. Protein concentrations were determined using a Bio-Rad protein assay in conjunction with a bovine serum albumin standard curve.

SDS-PAGE and zymogram assays.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (27) by the use of a 4% stacking gel and a 12% separating gel. Renaturing SDS-PAGE and zymogram analysis were carried out as outlined by Lepeuple et al. (29) except that whole-cell substrates were produced according to Sheehan et al. (51). When Micrococcus lysodeikticus ATCC4698 cells were employed, however, these were supplied in the form of a lyophilized powder (Sigma). Cells were incorporated into the gels at a final concentration of 0.4% (wt/vol). Protein sizes were compared to that of a prestained protein marker (New England Biolabs).

Western blotting and immunological detection.

Following SDS-PAGE, proteins were transferred onto polyvinylidine difluoride (PVDF) membranes (Immobilon-P; Millipore) with a 0.1 M CAPS (3-[cyclohexylamino]-1-propanesulfonic acid; pH 11)-10% methanol transfer buffer (Mini-protean II; Bio-Rad Laboratories). Tetra-His horseradish peroxidase-conjugated Abs (Qiagen) were used at a 1/1,000 dilution according to the manufacturer's instructions. Detection using polyclonal Abs involved washes (2 × 10 min) of the membranes in TBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl) followed by a 10-min wash in TBS-Tween-Triton (20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 0.05% Tween 20, 0.2% Triton X-100). Membranes were then incubated for 1 h in blocking buffer (TBS, 5% skimmed milk powder, 0.1% Tween 20). The three washes were repeated, after which the primary Ab was allowed to interact with its antigen by incubating the membrane in blocking buffer with an appropriate dilution of the polyclonal rabbit serum. Following the repetition of the three wash steps, the secondary Ab, horse radish peroxidase-conjugated anti-rabbit donkey immunoglobulin G (IgG) (Amersham Biosciences), was incubated with the membrane in the same manner as described for the primary Ab. Further washes (4 × 10 min) in TBS-Tween-Triton were performed, and the antigen-Ab complexes were detected using an ECL Western blotting detection system (Amersham Biosciences) according to the manufacturer's instructions. Where Tetra-His-detecting Abs were employed, protein sizes were determined using a six-His protein marker (Qiagen). To monitor production of Tal₂₀₀₉ in vivo, 100 ml of a Tuc2009 lysate was added to 500 ml of an exponentially growing culture of UC509.9 at an optical density at 600 nm of 0.4 to give a multiplicity of infection (MOI) of approximately 1. Following this, 30-ml samples were taken at 20-min intervals. The cells were harvested and lysed using a bead-beater as described above. Equal concentrations of these protein samples were separated by SDS-PAGE, analyzed by Western blotting, and detected as described above with the polyclonal rabbit Abs directed against Tal₂₀₀₉-5ΔN.

N-terminal sequencing.

Specific protein bands of interest were transferred to PVDF membranes by Western blotting as described above and excised from the membrane following visualization with Coomassie blue R-250. These were N-terminally sequenced by Alta Biosciences, University of Birmingham, England, using Edman degradation.

Cell wall binding.

Assays to determine whether Tal₂₀₀₉ exhibited any cell wall binding capabilities were performed essentially as described by Buist (8). Briefly, 250 μl of exponentially growing UC509.9 cells was resuspended in an equal volume of a cell-free lysate of E. coli M15 cells expressing Tal₂₀₀₉ from pTal₂₀₀₉-1 and incubated for 30 min at 30^°C. Cell-free lysates expressing lys (2) from pLys were similarly incubated with UC509.9 cells as a positive control. These mixtures were centrifuged, and the cell pellets and supernatants were collected. The cells were washed with 250 μl of 0.5× M17 and resuspended in 250 μl of denaturation buffer (3), while 100 μl of the supernatants was dialyzed against distilled water and dried by centrifugation under a vacuum prior to being dissolved in 50 μl of denaturation buffer. Lysin activity was detected by zymogram analysis as described above.

Ab production and neutralization studies.

Polyclonal Abs against purified TalT-5ΔN were raised in rabbits by CN Biosciences (UK) Ltd. An initial immunization with the protein of interest and Freund's complete adjuvant was followed by five subsequent boost injections of TalT-5ΔN. A final serum sample was acquired 11 weeks after the initial immunization. Specific test and control Ab preparations for immunoelectron microscopy were developed as described by Johnsen et al. (24). The glycine elution procedure from Harlow and Lane (21) was performed to specifically obtain only those Abs which bound to Tal₂₀₀₉-5ΔN with a high level of affinity.

To perform Ab neutralization studies, a 5% inoculum from a fresh overnight culture of UC509.9 was set up in 100 μl of double-strength GM17 and left at 30°C. Samples (2 μl) were taken at hourly intervals to count CFU ml⁻¹. CsCl-prepared phage (2 μl) was added to render an MOI of 0.02 for c2 and MG1614 and for Tuc2009 and UC509.9. The cells were made up to a final volume of 200 μl with rabbit serum, which had been dialyzed against TBT and filter sterilized, and 2 μl of 1 M CaCl₂. Further 2-μl samples were taken up to 9 h after the first inoculation. Cell counts at each time point were performed by diluting the cells in Ringers solution, spread plating suitable dilutions on GM17 agar, and incubating the plates at 30°C.

Bacteriophage Tuc2009 contains a structural component with cell wall-degrading activity which can be assigned to Tal₂₀₀₉.

To detect whether Tuc2009 phage particles possess a structural component with lytic activity, zymogram assays were performed to visualize lytic functionality in CsCl-purified phage. A weakly visible lytic activity was observed, corresponding in size to that expected for the protein product of tal₂₀₀₉ (~100 kDa). This result is not shown here due to the poor visibility of this lytic zone, which could be caused by weak enzymatic activity, low concentration, a low level of mobility, or refolding difficulties of this protein. To confirm the predicted lytic activity encoded by tal₂₀₀₉, the complete gene and five progressively smaller portions of the gene were amplified by PCR and individually inserted into the vector pQE60 to generate six different pQE60 derivatives (Table ​(Table1).1). The fragments were chosen to more precisely determine the specific region responsible for lytic activity within the protein. These proteins were overproduced in E. coli M15, and lysates of each of these strains were assayed for lytic abilities by zymogram analysis (Fig. ​(Fig.1).1). All of the fragments of the tal₂₀₀₉ gene, with the exception of the smallest one, were shown to specify a lytic activity. These results located the functional domain of apparent PG-degradative activity to within 166 C-terminal aa of Tal₂₀₀₉, in accordance with what was indicated by bioinformatic analysis. When the zymogram assay was used, Tal₂₀₀₉ was found to be active against cell walls of L. lactis UC509.9 and MG1363 but ineffective against those of Staphylococcus aureus ATCC14458, M. lysodeikticus ATCC4698 (Sigma), and Streptococcus thermophilus CNRZ01205.3 (results not shown).

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FIG. 1.

Zymogram assay to determine lytic activity of Tal₂₀₀₉ in gels containing 0.4% (wt/vol) L. lactis UC509.9 autoclaved cells. Each well was loaded with E. coli M15 lysates with proteins expressed from the following plasmids: lane 1, pQE60; lane 2, pTal₂₀₀₉-1; lane 3, pTal₂₀₀₉-2ΔN; lane 4, pTal₂₀₀₉-3ΔN; lane 5, pTal₂₀₀₉-4ΔN; lane 6, pTal₂₀₀₉-5ΔN; lane 7, pTal₂₀₀₉-6ΔN; lane 8, pTal₂₀₀₉ΔC. Molecular masses are indicated to the left of the gel, and a white arrow denotes the ~30-kDa processed proteins with lytic activity.

Comparative sequence analysis had failed to detect any cell-binding domain in Tal₂₀₀₉. To verify this observation a cell-binding assay was performed using the endolysin of Tuc2009 as a positive control. From work performed by Sheehan et al. (50) Lys, the Tuc2009 endolysin, is known to include a cell wall-recognizing component. As expected, Lys exhibited binding characteristics whereas Tal₂₀₀₉ did not appear to bind the cells of L. lactis UC509.9 (Fig. ​(Fig.2A2A).

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FIG. 2.

(A) Zymogram assay to investigate any possible cell binding capabilities of Tal₂₀₀₉. Lanes 1 and 2 show a zone of lytic activity corresponding to the Tuc2009 endolysin in the supernatant and cell pellet, respectively, following incubation with 509.9 cells. Lane 3 shows that Tal₂₀₀₉ is in the supernatant, while lane 4 shows Tal₂₀₀₉ to be absent from the 509.9 cell pellet. The gels contained 0.4% (wt/vol) L. lactis UC509.9 autoclaved cells. Molecular masses are indicated. (B) Zymogram analysis of the E. coli M15 lysates containing pTal₂₀₀₉-2ΔN or the mutant pTal₂₀₀₉mut-2ΔN in gels containing 0.4% (wt/vol) L. lactis UC509.9 autoclaved cells. Lane 1 contains the negative control pQE60, and lanes 2 and 3 contain pTal₂₀₀₉-2ΔN and pTal₂₀₀₉mut-2ΔN, respectively. Molecular masses are indicated to the left of the gel.

Tal₂₀₀₉ undergoes autoproteolysis at a specific site.

From the zymogram assays it appeared that the three largest fragments of Tal₂₀₀₉ were subjected to a specific cleavage event upon overexpression in E. coli, yielding a smaller lytic moiety of approximately 30 kDa (Fig. ​(Fig.1).1). To determine whether the observed processing was an E. coli-specific event, the second-largest fragment of tal₂₀₀₉, including the six-His tag-encoding region, was restricted from pQE60 and ligated into pNZ8048. Upon electrotransformation into L. lactis NZ9000, production of the resultant protein was induced by nisin and lysates were analyzed for lytic activity by zymogram assay. As seen with E. coli, Tal₂₀₀₉ appeared to undergo processing because the zymogram assays displayed a smaller protein of ~30 kDa exhibiting lytic activity as well as a clear band of the expected size representing the entire protein being produced (data not shown). tal₂₀₀₉-2ΔN was overexpressed in both E. coli and L. lactis and transferred to PVDF membranes by Western blotting before being subjected to N-terminal sequencing. In both cases (expression in E. coli and L. lactis), the ~30-kDa protein possessed the same N-terminal sequence, corresponding to cleavage of Tal₂₀₀₉ at a glycine-rich region GGSSG↓GG, where the arrow indicates the precise point where cleavage occurs. This finding is interesting, because the M37 protein family to which Tal₂₀₀₉ would seem to belong is known to include Gly-Gly endopeptidases. To ensure that this was indeed the cleavage site, gene SOEing was carried out which changed the naturally occurring GGSSGGG amino acid sequence to GRSSRRG by G-to-C transversions in the DNA sequence (mutated amino acid residues are underlined). The product of this gene was expressed in E. coli, and its lysis profile was compared to that of its nonmutated brethren. As expected, only one zone of clearing was observed, corresponding to the unprocessed version of Tal₂₀₀₉ (Fig. ​(Fig.2B2B).

In an effort to discover whether autocatalysis was taking place, a deletion version of tal₂₀₀₉ was cloned into pQE60 to generate pTal₂₀₀₉ΔC. This deletion version of tal₂₀₀₉ is predicted to produce a truncated Tal₂₀₀₉ protein lacking the last C-terminal 66 aa and, as expected, was shown to be lytically inactive, failing to produce a zone of clearing in a zymogram assay (Fig. ​(Fig.1).1). The plasmid containing the smallest lytically active tal₂₀₀₉ fragment was modified by inserting the tetracycline resistance gene tetK into the NdeI restriction site of pQE60 to produce pTal₂₀₀₉-5ΔNtet. This plasmid was then introduced into cells harboring plasmid pTal₂₀₀₉ΔC and maintained by selecting for resistance to tetracycline. These two functional (Tal₂₀₀₉-5ΔN) and nonfunctional (Tal₂₀₀₉ΔC) proteins were then concomitantly expressed, and their interaction was analyzed by immunological detection. Interestingly, we found a discernible autoproteolytic mechanism whereby the larger inactive fragment was cleaved only in the presence of a lytically active segment of Tal₂₀₀₉. This is visualized by the presence of a third fragment reacting to the anti-Tetra-His Abs (Fig. ​(Fig.3).3). The size of this fragment is commensurate to that which would arise if the Tal₂₀₀₉ΔC protein were cleaved at the GGSSGGG region. The second protein fragment produced, corresponding to the N-terminal fraction of Tal₂₀₀₉ΔC, cannot be observed, since the Abs only bind to the C-terminal His-tagged portion of the Tal₂₀₀₉ proteins.

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FIG. 3.

Western blot to detect possible interaction between Tal₂₀₀₉-Δ5N and Tal₂₀₀₉ΔC. The negative control is in lane 2, and the lysates of E. coli cultures overexpressing protein from pTal₂₀₀₉ΔC and pTal₂₀₀₉-5ΔNtet are in lanes 3 and 4, respectively. Lysates resulting from concomitant induction of expression from pTal₂₀₀₉-5ΔNtet and pTal₂₀₀₉ΔC can be observed in lane 5, with a band of ~30 kDa denoted by an arrow. Lane 1 was loaded with a six-His protein ladder which reacts to the commercial Abs, and the molecular masses are indicated.

Identification of posttranslational processing of Tal₂₀₀₉ in vivo and its incorporation into the mature phage virion.

To elucidate the actual expression of Tal₂₀₀₉ in vivo, its production was monitored using polyclonal Abs raised against the purified C-terminal moiety of Tal₂₀₀₉ (Tal₂₀₀₉-5ΔN). As detailed in Materials and Methods, 30-ml samples of a Tuc2009-infected culture were taken at intervals of 20 min for an hour and the harvested cells were disrupted using glass beads. The reaction to the Abs showed the production of two bands, which tally in size to the lytic activities seen upon overexpression of Tal₂₀₀₉ in E. coli and L. lactis (Fig. ​(Fig.4A).4A). These bands were first detected at 20 min following infection. To show that Tal₂₀₀₉ in its entirety is assimilated into the mature phage, Tuc2009 isolated by CsCl gradient was immunologically analyzed. In lanes containing ~10⁹ PFU of Tuc2009 phage particles, a single band was detected of the size expected for the full structural protein (Fig. ​(Fig.4B)4B) and equal to that of the higher-molecular-weight band viewed upon zymogram analysis of the whole phage (results not shown). Determination of the N-terminal sequences of a number of structural proteins of Tuc2009 has indicated that unprocessed Tal₂₀₀₉ is indeed part of the phage structure and that an approximately 60-kDa portion of it inclusive of the N-terminal end is also incorporated into the mature virion (J. Seegers, D. van Sinderen, and G. F. Fitzgerald, unpublished results).

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FIG. 4.

(A) Time point analysis of Tal₂₀₀₉ production in vivo including a lysate of uninfected UC509.9 cells to act as a negative control. The band sizes are indicated, with the lower band appearing slightly stronger than the larger band at 20 min (T₂₀). (B) Western blots of the mature Tuc2009 virion exposed a single band analogous in size to the full Tal₂₀₀₉ protein. Tuc2009 is in the second lane, while a lysate containing Tal₂₀₀₉-5ΔN was run in lane 1 as a positive control. Molecular masses are indicated to the left of the gel.

This work was funded by the Bioresearch Ireland Postgraduate Scheme, Enterprise Ireland (BR/2000/53), the Embark Initiative Postdoctoral Fellowship, and Science Foundation Ireland (02/IN1/B198).

We express our thanks to Horst Neve (Institut für Mikrobiologie, Kiel, Germany) for his advice concerning immunoelectron microscopy and the Centre for Microscopic Analysis (Trinity College, Dublin, Ireland).