Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7
- Rupak Mitra†1,
- Gregory J McKenzie†1, 2,
- Liang Yi1, 3,
- Cherline A Lee1, 4 and
- Nancy L Craig1Email author
© Mitra et al; licensee BioMed Central Ltd. 2010
Received: 7 May 2010
Accepted: 23 July 2010
Published: 23 July 2010
The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein.
Tn7 is a very distinctive bacterial transposon that encodes five transposition proteins: Tns A, B, C, D and E . Strikingly, whereas most transposons insert relatively randomly into many different sites, Tn7 transposition is quite specific. TnsD and TnsE are alternative target site-selectors that direct Tn7 transposition into either of two different target DNAs : a very specific chromosomal attachment site or DNAs undergoing DNA replication .
When TnsD is the target selector, Tn7 inserts at high frequency into a specific chromosomal site called an attachment site, attTn7 . Insertion occurs directly downstream of the essential glmS gene, ensuring that it does not disrupt the glmS open reading frame and glmS expression is preserved . Thus, Tn7 can access this highly conserved 'safe haven' insertion site with no obvious fitness costs to the host. Tn7 inserts into attTn7 because TnsD specifically recognizes highly conserved sequences within the protein coding region of glmS , and recruits the rest of the transposition machinery to this site.
The TnsD binding site in Escherichia coli glmS occupies the last 36 bp of the glmS ORF . TnsD also binds the human glmS homologs gfpt-1 and gfpt-2 . GlmS (L-glucosamine--fructose-6-phosphate aminotransferase) is highly conserved and found in a wide variety of organisms from bacteria to humans . The TnsD binding region of glmS encodes the active site region of GlmS, and this amino acid sequence is nearly completely (100% conserved) in all organisms . Indeed, most of the DNA sequence divergence results from variation at the wobble position of each codon (see below). Intriguingly, no particular DNA sequence other than the TnsD binding site is apparently required for attTn7 function, even though the actual point of Tn7 insertion is about 25 bp downstream of the TnsD binding site. Changing this region in E. coli attTn7  does not change Tn7 insertion frequency, and the sequences at the point of Tn7 insertion in attTn7::Tn7 sites in other bacteria are also distinct . Furthermore, the human glmS homologs gfpt-1 and gfpt-2 are efficient targets for Tn7 insertion despite their different sequences downstream of the GlmS ORF . Thus, all the sequence information necessary for Tn7 insertion in attTn7 is apparently conferred by TnsD binding to the end of glmS.
TnsD is a unique, sequence-specific, DNA-binding protein. It has no homologs outside of Tn7-type transposons, about 130 of which are now reported in Genbank. When TnsD binds to attTn7, another Tns protein, TnsC, is recruited , forming a TnsCD-attTn7 complex. DNA footprinting reveals that the TnsCD complex on attTn7 extends from the TnsD binding site to the point of Tn7 insertion that lies about 20 bp downstream of the stop codon of glmS in an intergenic region. Understanding how TnsD works is key to a detailed understanding of how the unique transposon Tn7 functions.
In this paper, we present results that provide insight into TnsD interactions with its attTn7 DNA-binding site and how TnsD functions to recruit its partner attTn7 binding protein TnsC. We determined the nucleotides that are important for TnsD binding in the glmS gene using both in vitro and in vivo assays. Our studies also revealed that in addition to binding to DNA, TnsD interacts with TnsC independently from interactions with attTn7. We also identified key amino acids in TnsD for DNA binding and important regions in TnsD for protein-protein interactions with TnsC. Finally, we characterized dominant-negative mutants of TnsD, which suggest that important interaction domains are distributed throughout the protein. The data show TnsD as a highly complex DNA-binding protein that regulates TnsC activity to activate Tn7 transposition.
Defining important base pairs in the TnsD binding site
The 36 bp TnsD-binding site lies within the 3' end of glmS, which encodes the C-terminus of the protein. This region includes the GlmS active site and is highly conserved at the protein level. We aligned this 36 bp sequence from 25 bacterial glmS genes that have a downstream Tn7 family transposon (GenBank) and the homologous Drosophila, zebrafish and human genes using the Sequence Logo algorithm  to generate a consensus for the attachment site (Figure 1). Not surprisingly for a region that encodes a highly conserved protein, the first two positions at each codon are highly conserved, and variation is seen only at the third 'wobble' position.
TnsD binding to attTn7 in vivo
+23 → +58(wt)
7.4 × 10-5
5.4 × 10-7
5.2 × 10-9
3.6 × 10-8
1.6 × 10-8
5.2 × 10-9
We know from previous work that Tn7 transposition functions well in Salmonella . For the challenge phage experiment, we constructed a P22 phage with the E. coli attTn7 attachment site (+23 → +58) upstream of ant, thus putting the choice of lysogenic vs lytic growth under the control of TnsD binding to attTn7: if TnsD binds, lysogeny should occur. We infected a Salmonella enterica Typhimurium strain containing a plasmid-borne isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible tnsD gene with the challenge phage carrying the E. coli attachment site upstream of ant. The frequency of lysogenization of the attTn7 phage increased more than 100-fold in the presence of TnsD (Table 1). Thus the challenge phage assay can be successfully used to evaluate TnsD binding to attTn7 in vivo. When assayed, the frequency of lysogeny of a phage containing a wild-type attachment site (+23 → +58) was several orders of magnitude higher than the frequency of lysogeny of phages containing any of four mutant attachment sites that bind TnsD poorly in vitro (Table 1). These assays show that TnsD also binds poorly to these mutant sites in vivo, supporting the view that the identity of these positions is important for the site-specific binding activity of TnsD.
attTn7 mutations that block TnsD binding also block Tn7 transposition in vivo
As described above, mutations in attTn7 can block TnsD binding both in vitro and in vivo. We also determined the effect of attTn7 mutations on Tn7 insertion into attTn7 in vivo in E. coli. In these assays, Tn7 transposition into a target plasmid containing attTn7 was evaluated using a 'λ-hop' assay , in which cells containing a plasmid(s) expressing TnsABC+D were infected with a replication- and integration-defective lambda phage derivative carrying a miniTn7 kanamycin resistance cassette (miniTn7-KanR), and the frequency of miniTn7 insertion into the attTn7 plasmid was measured as the fraction of infected cells that became kanamycin-resistant.
We found that mutations that significantly (> 10-fold) decreased TnsD binding to attTn7 also decreased Tn7 transposition in vivo by at least 100-fold (Figure 2C). Thus, attTn7 +31, +33, +42, +43, +45 and +51 are key positions for TnsD-attTn7 interaction. attTn7 sequence changes that had more modest effects on TnsD binding, (reductions of two- to five-fold), also reduced transposition by two- to five-fold. Thus, attTn7 +28, +30, +37, +46, +48 and +54 also make contributions to TnsD-attTn7 interaction.
Crosslinking of attTn7 to TnsD
TnsD contains a zinc-finger motif essential for DNA binding
Targeted mutations to the conserved CCCH residues in TnsD eliminates Tn7 transposition and TnsD-attTn7 binding.
Relative attTn7 binding in vitro
Relative transposition frequency in vivo
TnsD has important DNA-binding determinants throughout the protein
In an attempt to identify directly the domains of TnsD involved in DNA binding, we generated a number of TnsD deletion derivatives, and assessed DNA binding of these mutants in vivo and in vitro. All tested C-terminal deletion derivatives, (TnsD1 to 350, 1 to 399, 1 to 480, 1 to 490 and 1 to 498) were unable to bind DNA as evaluated by in vitro band-shift assays and by in vivo challenge phage assays (data not shown). However, these truncated proteins did retain the ability to interact with TnsC, indicating that they were not simply unfolded (we discuss interaction of TnsD with TnsC below). These observations suggest that in addition to the zinc finger, TnsD contains DNA-binding determinants spread across the entire length of the protein and that several of these are required for attTn7 binding. We were unable to detect interaction of N-terminal TnsD truncations with TnsC or to purify these proteins (data not shown).
Isolation of TnsD dominant-negative mutations
In another approach to identify the functional regions of TnsD, we conducted a screen for mutations in TnsD that have dominant-negative effects on TnsD-dependent transposition. Expected classes of TnsD dominant-negative mutants include those able to bind attTn7 but unable to interact with TnsC, and also mutants unable to bind attTn7 but able to interact with TnsC. We used a promoter capture (papillation) assay as described by Stellwagen and Craig, in which a miniTn7 containing a promoterless lac gene is mobilized from a plasmid donor to chromosomal pseudo-attTn7 sites; attTn7 itself was blocked by another Tn7 element. TnsABC+D were expressed from a pACYC plasmid. Productive hops produce Lac+ cells that form papillae on Lac- colonies on MacConkey lactose plates. To look for dominant-negatives, we used hydroxylamine to mutate a pUC-based TnsD gene, transformed it into the assay strain containing wild-type TnsABC+D, and looked for transformants on MacConkey lactose plates within decreased numbers of papillae. After hydroxylamine mutagenesis of TnsD and screening of 15,000 transformants, we isolated 16 TnsD mutants, 11 of which were missense mutants and five of which were nonsense mutants yielding truncated TnsD; only two mutants (TnsDP147L and TnsDR276C ) were isolated twice (Figure 4, Figure 5, Figure 6).
Dominant-negative mutants of TnsD.
From dominant-negative TnsD screena
Relative transposition in presence of
Relative attTn7 binding in vivod
Relative attTn7 binding in vitro
We also examined the ability of the dominant-negative TnsD mutants to promote transposition in the presence of TnsABC alone to determine if they possess residual TnsD activity. All had substantial defects in TnsD activity, with reductions ranging from at least 2.5-fold to 100-fold compared with wild-type cells.
Using the challenge phage assay, we also evaluated the ability of most of the dominant-negative TnsD missense mutants to bind to attTn7 in vivo, and found that all were significantly reduced in their ability to bind attTn7 (Table 3). Using purified TnsD, we also found that all the missense mutants except C447Y had significantly impaired attTn7 binding in vitro (Table 3).
TnsC increases the binding of TnsD to attTn7
TnsC increases TnsD binding to attTn7 in vivo
< 8.0 × 10-7b
2.4 × 10-3
TnsC + TnsD
2.4 × 10-1
Although the binding of wild-type TnsD to attTn7 is stimulated by TnsC, incubation with TnsC did not 'rescue' the formation of TnsCD-attTn7 complexes with the dominant-negative missense mutant TnsD in vitro (data not shown).
TnsC and TnsD can interact in the absence of attTn7
Yeast 2 hybrid assay shows that TnsC and TnsD can interact in vivo.
TnsC (1 to 555) wt
TnsD (1 to 507) wt
TnsC (1 to 555) wt
TnsD (1 to 436)
TnsC (1 to 555) wt
TnsD (1 to 399)
TnsC (1 to 555) wt
TnsD (1 to 375)
TnsC (1 to 555) wt
TnsD (1 to 350)
TnsC (1 to 555) wt
TnsD (1 to 309)a
TnsC (1 to 555) wt
TnsD (1 to 293)
TnsC (1 to 555) wt
TnsD (8 to 507)
TnsC (1 to 555) wt
TnsD (12 to 507)
TnsC (1 to 555) wt
TnsD (16 to 507)
TnsC (1 to 555) wt
TnsD (22 to 507)
TnsC (1 to 555) wt
TnsD (1 to 507) wt
TnsC (1 to 480)
TnsD (1 to 507) wt
TnsC (1 to 384)
TnsD (1 to 507) wt
TnsC (1 to 332)
TnsD (1 to 507) wt
TnsC (1 to 293)a
TnsD (1 to 507) wt
TnsC (1 to 280)
TnsD (1 to 507) wt
As determined by analysis of a series of C-terminal deletion derivatives, the amino-terminal amino acid sequence (1 to 309) of TnsD retain full binding to TnsC in this assay and thus contains the determinants for TnsD binding to TnsC. The isolation of the TnsD truncation sequence (1 to 340; full-length TnsD is 507 aa) as a dominant negative is consistent with this hypothesis (see below). However, if as few as seven amino acids are removed from the N-terminus of TnsD, the TnsC-TnsD interaction is disrupted (data not shown). The lack of activity of TnsD sequence 1 to 436, even though both longer and shorter constructs are active, may be due to inappropriate folding or to the unmasking of an inhibitory domain.
We also performed deletion analysis of TnsC using the yeast two-hybrid system (Table 5). TnsC-TnsD interaction with TnsD is maintained with deletion of 75 or 171 amino acids from the C terminus of TnsC (full-length is 555 aa), but a deletion of 223 amino acids leads to loss of the interaction with TnsD (Table 5); thus interaction of TnsC with TnsD occurs within the 1 to 293 region of TnsC. The finding that Tn7 inserts at high-frequency in attTn7 using TnsC 86 to 555 (Spencer J and NLC, unpublished observation) suggests that the TnsD interaction domain lies within TnsC 1 to 293.
The sequence-specific binding of TnsD to the 3' end of the coding region of the glmS gene is sufficient to prompt the site - and orientation-specific insertion of Tn7 to a position about 20 bp downstream of glmS, that is, to produce a functional attTn7 site . Previous work in vitro identified the 36 bp span of the TnsD binding site, and demonstrated that this sequence alone is sufficient to direct transposition . In this study, we have further probed the interaction of TnsD with attTn7 and deepened our understanding of Tn7 site-specific insertion. Our results have revealed that determinants of the interaction of TnsD with attTn7 extend throughout the TnsD protein and the TnsD binding site.
The activity of attTn7 is strongly directional: Tn7 always inserts downstream of the glmS termination codon, mediated by activation of a TnsCD-attTn7 complex that promotes orientation-specific insertion of Tn7. Consistent with this asymmetric pattern of insertion, attTn7 does not contain inverted repeats for the binding of an oligomeric TnsD.
Previous studies have shown that a key step in Tn7 insertion into attTn7 is the ability of TnsD to introduce a DNA distortion(s) into attTn7, which we have proposed prompts the binding of TnsC, the regulator of the TnsAB transposase [6, 24]. In addition to providing a higher resolution view of TnsD-attTn7 interaction, we also identified a previously unknown interaction protein-protein interaction between TnsC and TnsD. Determinants for this interaction are located within the N-terminal region of TnsD covering positions 1 to 309 and the N-terminal region of TnsC covering positions 1 to 293. It seems likely that the DNA-mediated and protein-protein interactions are both important to TnsC-mediated activation of transposition.
TnsC also interacts with and activates both subunits of the Tn7 transposase (TnsA and TnsB), which mediate breakage and joining of the ends of Tn7 . TnsA, the transposase subunit that cleaves at the 5' ends of Tn7, and TnsC form a TnsA2C2 complex in solution, mediated by interactions between the N-terminal region of TnsA and the 50 carboxy-terminal amino acids of TnsC . A distinct TnsACD complex has also been observed , which we suggested is the 'target' DNA complex, with which the TnsB transposase subunit mediates breakage and joining at the 3' ends of the transposon . The other transposase subunit TnsB binds specifically to the ends of Tn7, and mediates breakage and joining at the 3' ends . Interactions between TnsC and the C-terminus of TnsB have also been detected . The apposition of the TnsACD-attTn7 and TnsB-Tn7 end complexes results in the assembly and activation of the transposase [28, 31]. Much remains to learned about the mechanism by which TnsD-attTn7 activates TnsC and hence TnsAB.
Functional domains of TnsD
TnsD is a multifunctional protein: it binds specifically to attTn7 and, as we have shown here, interacts with its partner in transpositon, TnsC. In this study, we have made considerable progress in understanding the unique TnsD family of DNA-binding proteins by defining important protein determinants for DNA binding and transposition, and key nucleotides for the TnsD-attTn7 interaction. Although we identified a zinc finger motif in the N-terminal region of TnsD, we were unable to use deletion analysis to identify a functional discrete DNA-binding domain(s). It is possible there are multiple DNA-binding domains throughout the protein, a model consistent with the fact that the region of TnsD interaction with DNA spans about 35 bp. However, we were able to use deletion analysis to localize the TnsC-interaction region of TnsD to its N-terminal amino acids 1 to 309, and the TnsD-interaction region of TnsC to its amino acids 1 to 293. Although our ability to isolate TnsD dominant negatives is consistent with this protein being multifunctional, our inability to further identify crucial subregions within TnsD precludes definitive conclusions of the physical basis of the dominant negatives.
As previously proposed, the ability of TnsD to direct transposition to a defined sequence makes it an attractive candidate for use in targeted delivery of DNA sequences for genetic manipulation in organisms from bacteria to human [7, 13]. Further structure-function analyses of TnsD will not only facilitate a deeper understanding of Tn7 transposition but also the use of Tn7 and TnsD as tools for genomic engineering.
Bacterial strains, plasmids and phages
CAG456 is E. coli (SC122 htpR165) . NLC51 is E. coli F-araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR valR recA56 . BD409 is a derivative of E. coli CW51 with att::promoterless lacZY flanked by Tn7 transposon end sequences sufficient for transposition: 166 bp from the left end of Tn7(Tn7 L) and 90 bp from the right end of (Tn7 R) [4, 34]. Plasmid pMR1 was constructed by PCR amplifying the tnsD gene from pCW4  and cloning it into pCYB1 digested with Nco I and Sap I (New England Biolabs, Ipswich, MA, USA.). pCW4 was used as a source of TnsABCD and pCW15 as a source of TnsABC in the in vivo transposition assays . pCW23 was used for mutating tnsD . Phage KK1, used as the source of Tn7 transposon in the λ-hop assay, is a derivative of 780 (b 2::hisOGD b522 cI857 Pam80 nin5) with hisG9424::Tn10 del 16 del 17::attTn7(-342 to +165)::miniTn7 kanR .
Affinity purification of intein-TnsD
The full length TnsD wild-type and mutant proteins were purified as intein fusions from CAG456 containing plasmid pRM1 or mutant TnsD derivative plasmids. Site-directed mutations in tnsD were generated by PCR (QuickChange Site Directed Mutagenesis System; Agilent Technologies) and verified by direct DNA sequencing. Representative cells were grown at 30°C to an OD600 = 0.5 in Luria broth supplemented with 100 mg/ml carbenicillin, IPTG was added to give 0.4 mM final concentration, and the cells were allowed to grow for an additional 4 h. All subsequent steps were performed at 4°C unless otherwise stated. The cells were separated by centrifugation, and resuspended in the buffer supplied with the kit (Buffer A; 50 mM HEPES pH 8, 500 mM NaCl, 10% v/v glycerol). The cells were then lysed by sonication and separated by centrifugation at 26,000 g for 30 min, the resulting supernatant was then filtered through a 0.45 μm syringe filter (Nalgene, Rochester, NY, USA). The filtrate was applied to pre-equilibrated chitin beads (New England Biolabs) in a 10 ml column and the beads then washed several times (5×) in Buffer A. The washed chitin beads were then treated with Buffer B (50 mM HEPES pH 8, 500 mM NaCl, 10% v/v glycerol, 10 mM MgCl2, 10 mM ATP) for 1 h at room temperature to remove residual GroEL protein. The beads were then washed with several volumes of buffer B at 4°C (2×), and incubated overnight with Buffer C (50 mM HEPES pH 8, 500 mM NaCl, 10% v/v glycerol, 50 mM dithiothreitol (DTT)), which promotes cleavage of TnsD from the intein tag [36, 37]. Full-length TnsD was then eluted from the column using Buffer C without DTT, and peak fractions were pooled, dialyzed against another buffer (500 mM KCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM DTT and 25% v/v glycerol), then stored at -80°C.
Isolation of dominant-negative tnsD mutants
pCW23 (TnsD)  was treated with 1 M hydroxylamine hydrochloride in NaOH at 37°C for 24 h. The mutagen was dialyzed out of the DNA in Tris-EDTA buffer. The DNA was recovered by ethanol precipitation, and transformed into BD409 carrying wild-type tnsABCD on a compatible plasmid, pCW4 . Transformants were plated onto MacConkey lactose plates containing appropriate antibiotics. The plates were incubated at 30°C for 5 days, and transformants were screened for decreased papillation (to Lac+). Plasmid DNA was extracted from these potential mutants and transformed into the same strain background to verify the papillation phenotype. DNA was sequenced to identify the mutations, and recloned for expression.
λ hop assay
The transposition frequency of a miniTn7 kanR element from the integration- and replication-defective phage KK1 into the chromosomal attTn7 site of E. coli strain NLC51 and a plasmid containing the attTn7 site of E. coli strain LA3 was evaluated when Tns proteins were supplied in trans [35, 38] The transposition frequency was the number of kanamycin-resistant colonies per plaque-forming unit.
Binding reactions were performed with wild-type and mutant versions of TnsD proteins and wild-type TnsC protein as described previously .
Protein-DNA UV photo-crosslinking reaction
Protein-DNA crosslinking was performed as described previously .. Oligonucleotides were synthesized (Eurofins MWG Operon, Huntsville, AL, USA) with dT replaced by IdU. Double-stranded (ds)DNAs were then formed by gradual annealing with a complementary oligonucleotide, and then radio-labeled with 5' phosphorylation.
For crosslinking reactions, 25 pmol TnsD and 0.5 pmol attTn7 dsDNA were incubated in solution (25 mM HEPES, 2.5 mM TrisCl, 40 mM DTT, 0.005% BSA and 50 ng/ul herring sperm DNA, pH 7.5) for 20 min at 30°C. Photo-crosslinking was then carried out with UV irradiation at 312 nm (StrataLinker; Agilent Technologies, La Jolla, CA, USA) for 30 min. The reaction was set up so that the top of the open 1.5 ml Eppendorf tube touched the UV tube. After UV crosslinking, the reaction was analyzed by SDS-PAGE.
Yeast two-hybrid assay
The yeast two-hybrid assay was performed according to the manufacturer's protocol (Proquest Two-Hybrid System; Life Technologies Inc., Carlsbad, CA, USA). TnsD was cloned into pDBLeu (Life Technologies Inc) and TnsC into pPC86 (Life Technologies Inc). β-galactosidase assays were performed as described previously .
In vitro transposition reactions
In vitro transposition reactions were performed essentially as described previously . The donor plasmid pEMΔ (5.9 kb) contains a 1.6 kb miniTn7 kanR element. The 3.2 kb target plasmid pRM2  contains a 555 bp attTn7 segment (-342 to +165). Reaction mixtures (100 μl final volume) contained 0.25 nM pEM donor DNA, 2.5 nM pRM2 target plasmid, 28 mM HEPES pH 8.0, 2.2 mM DTT, 4.4 mM Tris pH 7.5, 100 mg/ml tRNA, 50 mg/ml bovine serum albumin (BSA), 0.16 mM EDTA, 0.1 mM MgCl2, 0.1 mM CHAPS detergent, 30 mM NaCl, 21 mM KCl, 1.8% v/v glycerol, 2.0 mM ATP and 15 mM MgOAc unless otherwise indicated. Tns proteins were added as follows: 40 ng TnsA, 25 ng TnsB, 30 ng TnsC and 22 ng TnsD. Reaction mixtures containing all components except donor DNA, TnsA, TnsB and MgOAc were assembled on ice. The assembly reaction mixtures were incubated for 20 min at 30°C; donor DNA, TnsA, TnsB and MgOAc were then added, and the incubation was continued for an additional 20 min. Reactions were stopped by adjusting to 25 mM EDTA, followed by extraction with phenol:chloroform (1:1). The DNA was then precipitated with ethanol, digested with Nde I and separated in a 0.6% agarose gel, with electrophoresis carried out at 50 V for 16 h. The DNAs were transferred to a hybridization membrane (Gene Screen Plus; PerkinElmer, Foster City, CA, USA) and hybridized with a probe specific for miniTn7 kanR. The probe was labeled by random priming with 32P-dCTP and the Klenow fragment of DNA polymerase I (Boehringer Mannheim Biochemicals (BMB), Indianapolis, IN, USA). All blots were analyzed using a phosphorescent imager (PhosphorImager; Molecular Dynamics, Sunnyvale, CA, USA).
Challenge phage assays
Challenge phages were constructed as described in . The wild-type attTn7 attachment site attTn7 (+23 → +58) was cloned in as oligonucleotides (5'-CCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAA-3' and the complementary oligonucleotide into the Sma I site of pPY190, creating pGRG60). All mutant attachment sites were variants of that sequence (as described in the text). The challenge phage assay was carried out as described previously , using plasmid pCYB1-TnsD or mutant variants of that plasmid to express TnsD, selecting for kanamycin-resistant P22 lysogens. Expression of TnsD proteins was induced with 1 mM IPTG. Results are expressed in lysogens/cell. TnsC was expressed constitutively from pGRG63, a plasmid constructed by cutting pCW15 with Sap I and Bs tXI, blunting and religating the plasmid, to eliminate the TnsAB genes.
We thank S. Maloy and J. Gardner for Salmonella strains and P22 phages, and J. Gardner for helpful conversations. This work was supported by an Alberta Heritage Foundation for Medical Research postdoctoral fellowship to G.J.M and NIH grant 5GM076425 to NLC. NLC is an investigator of the Howard Hughes Medical Institute.
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