Engineering Permissive Insertion Sites In The Bacteriophage Phi29 ...

Identification of permissive insertion sites on phi29 TP by transposon mutagenesis

In order to detect permissive insertion sites in the TP sequence we chose a Tn5-based GFP transposition strategy. The GFP was chosen because it provides an easy detection method, it has a well studied autonomous folding and its N and C ends are close to each other [28], so, presumably, the disturbance of the acceptor protein structure would not be too strong. The transposon method we used has been reported before to detect permissive insertions in other proteins [21,29] [30]. The transposon (Fig 1A) has optimized tranposition signals (mosaic end, ME, see S1 File), a GFP coding sequence devoid of transcription and translation, start or stop signals, and a kanamycin resistance gene flanked by NotI sites. As the receptor plasmid, we used the pStrepTP plasmid, a N-terminus double Strep-tag II TP expression vector based on the T7 promoter (S2 File). Once subjected to the reaction with the transposon in the presence of the Tn5 over-active transposase [31], the receptor plasmid could, in theory, acquire the transposon at any point of its sequence compatible with the plasmid replication and the antibiotic resistance function (Fig 1B).

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Fig 1. Construction of the transposition library.

(A) Scheme of the donor plasmid showing the intact transposon and the inserted sequence produced after digestion with NotI and ligation. The transposon comprises two mosaic ends (ME) or transposition signals, the GFP coding sequence and a kanamycin resistance cassette (KmR) flanked by two NotI sites. (B) Scheme of the TP-expressing receptor plasmid and the possible outcomes after the transposition and elimination of the kanamycin cassette (see Materials and Methods for a detailed description).

https://doi.org/10.1371/journal.pone.0164901.g001

To select the transposition events that occurred within the TP sequence we excised the DNA fragment that had a size corresponding to the TP gene plus the transposon and recloned it onto the original vector backbone. After pooling the resulting library, the kanamycin gene and adjacent sequences were eliminated by digestion with NotI and religation (Fig 1B). After that, just the GFP coding sequence, the 19 bp transposon ME ends and two short linkers remained inserted. From this procedure, we obtained a library of about 2000 clones in which the GFP coding sequence was inserted, in principle at any position in the TP gene, and in any of the two possible orientations.

To detect in-frame insertions of the GFP coding sequence into the TP gene (in theory, one out of six events of transposition within the TP gene) we induced the expression of the TP from individual clones from the final transposition library and measured the fluorescence of the cultures in the presence of appropriate controls. Out of 320 individual clones measured, we selected 56 cultures that showed a fluorescence signal clearly above the background and near that of GFP-expressing positive controls. To confirm the presence of GFP insertions on the TP protein in the selected clones, we performed Western blotting with anti-TP antibodies on extracts of the selected cultures and continued to study only those clones that showed a protein band of a size equivalent to TP plus GFP (S1 Fig, panel A, S1 Table).

We selected 11 candidate insertions from the Western positive clones and carried out an assay to detect TP-specific initiation of replication of the phi29 TP-DNA on whole cell-extracts (S1 Table). We observed that, with just three exceptions (S1 Fig, panel B), none of the fusions obtained showed initiation activity. After sequencing, the three replication positive clones corresponded to the same insertion at amino acid 14 in the TP sequence (Fig 2A, GFP14). The rest of the GFP insertions fell on the Nt domain (3 insertions), on the I domain (4 insertions) and 1 insertion at the Ct domain (Fig 2A). In Fig 2B are shown the positions of the insertions on the structure of the TP, for the domains I and Ct, modelled with the software UCSF Chimera [32] and on the reconstructed structure of the Nt domain performed with the program I-Tasser [33]. We considered that the whole GFP protein could be too voluminous to allow the correct folding of the TP and be compatible with its function, and also that, for our purposes, a smaller insertion could be functional and still useful as a probe for permissive sites. So, we eliminated most of the GFP coding sequence by doing PCR with oligonucleotides (see Table 1) that hybridized divergently from the transposition signals and directed the amplification of the plasmid plus the TP gene but excluded the GFP sequence. Each primer also included an EagI site for recircularization of the plasmid. After amplification, EagI cutting and ligation, in each of the clones was left an in-frame insertion of 45 base pairs on the TP gene (S3 File). The corresponding proteins with 15 amino acids insertions were purified with Strep-Tactin columns with the results shown in S2 Fig. The protein concentrations for all the TP variants were quantified by coomasie-staining, and the TP variants were assayed for activity comparing them with the wild-type TP in order to determine a suitable insertion point. To have better standards for comparison, we also purified 2 whole GFP/YFP fusions, one is a YFP fusion at the N-terminus of the TP (YFP TP) that was already assayed in vivo [14] and the other is a GFP fusion at the C-terminus of the TP (TP GFP). This last protein has not been characterized before. We also included two whole GFP insertion mutants in the assays, GFP14 (insertion of the GFP at position 14) and GFP38 (insertion at position 38). GFP14 showed near wild-type activity and GFP38 showed some activity, although very low (S1 Table).

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Fig 2. Localization of the GFP or 15 amino acids insertions on the sequence and tertiary structure of the TP.

(A) The positions of YFP, GFP or 15 amino acid insertions (i) are marked on the sequence of the TP. The different domains of the TP are indicated with colors: N-terminal (blue), Intermediate (green) and C-terminal (yellow). (B) Ribbon diagrams of the 3D structure of the TP. The 3D modeling was performed with the software UCSF Chimera based on the crystallographic data from the file with the Protein Data Bank (PDB) accession number 2EX3 and on a reconstruction of the N-terminal domain using the program I-Tasser. Labels show the positions of the GFP/YFP or the 15 amino acid insertions. The color code for the different domains is the same as in A. Representations of the structure from different angles are shown for better visualization.

https://doi.org/10.1371/journal.pone.0164901.g002