The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

Abstract

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.

Figure 1.

Xer recombination. (A) Schematic representation of the IMEX arrays found on chr1 of a V. cholerae strain from the 7^th (N16961) and the 6^th (O395) pandemics (top) and of the successive integrations of TLCΦ leading to the formation of those arrays (bottom). The arrays result from successive lysogenic conversion events, a new dif site being generated on the right side of each newly-integrated IMEX. Pink, red, orange blocks: TLCΦ, CTXΦ and RS1 prophages; Coloured disks: difA (grey/black), TLCΦ attP (green/pink), TLCΦ attL1 (grey/pink), dif1 (green/black) and current dif site of the strains (black disk). Numbers indicate the order of integration of the phage, which can be directly deduced from its position relative to the current dif site. (B) V. cholerae difA and dif1, TLCΦ attP and attL1 sequences. Green: XerC-binding site; Light grey: central region and non-canonical bp of difA; Blue: XerD-binding site; Pink: non-canonical bp of attP. The top and bottoms are cleaved by XerC and XerD, respectively. The positions of cleavage are indicated by triangles. (C) Xer recombination pathways. XerC and XerD are shown in light and dark grey, respectively. The central region, XerC and XerD-arms of the recombination sites are shaded in grey, green and blue, respectively. Black arrows: conventional recombination pathway; Red arrows: non-conventional asymmetric recombination pathway. The XerD and XerC cleavage points are depicted by empty and filled disks. (D) TLCΦ integration. Black and pink lines: V. cholerae chr1 and TLCΦ DNA, respectively. Question mark: the low affinity of XerD for the XerD-arm of TLCΦ attP poses questions about the possible formation of integration complexes.

Figure 2.

Parallel monitoring of the integration and excision of Mobile DNA elements. (A) Schematic of classical and NGS based strategies to monitor integration and excision efficiencies. ‘Xer on’: growth in the presence of arabinose. Pink: TLCΦ DNA; Grey: plasmid vector DNA; 6: R6Kγ replication origin; cat: chloramphenicol resistance gene; T: transfer origin; dap: diaminopimelic acid; Cm: chloramphenicol; ara: L-arabinose; Xgal: X-gal; lacZ-dif1 reporter: E. coli lacZ gene containing an internal dif1 site. Plasmid- and lacZ-specific P5, and TLCΦ-specific P7 adaptor primers used for next generation sequencing (NGS) are indicated by black, grey and pink arrows, respectively. (B) Scheme of the V. cholerae reporter strains. White circle: chromosome 1 replication origin; red, blue and black crosses: dif and adjoining IMEX deletion, xerD deletion and V. cholerae lacZ deletion, respectively; black rectangle: E. coli lacZ-dif1 reporter; light and dark grey rectangles: synthetic xerC and xerD operon under the control of the arabinose promoter (Para). Yellow shading depicts the subcellular region and timing of activity of FtsK during the cell cycle. (C) Non-replicative mini-TLCΦ plasmid libraries. Top panel: top strand of the XerD-arm of the degenerate attP motifs. Sequence legend as in Figure 1B. Bottom panel: scheme of XafT⁺ and XafT⁻mini-TLCΦ plasmids. Yellow rectangle: xafT gene; sawed lines: stop mutation.

Figure 3.

Non-replicative mini-TLCΦ plasmid integration. (A) Integration frequencies of mini-TLCΦ plasmids harbouring dif1 or TLCΦ attP, and global integration frequencies of n₈gtg, n₅a₂n₃g and tagn₈ plasmid libraries (). FtsK panel: XafT⁻ plasmids conjugated in a strain harbouring dif1 at its natural locus; XafT panel: XafT⁺ plasmids conjugated in a strain harbouring dif1 at the lacZ locus; FtsK & XafT panel: XafT⁺ plasmids conjugated in a strain harbouring dif1 at its natural locus. Mean and standard deviations of at least 3 independent assays. (B) Combined sequence of the XerD-arm from the n₈gtg, n₅a₂n₃g and tagn₈ integration libraries. The logos show the frequency of each base at the degenerate positions. The number of integrated motifs corresponding to each logo is indicated above. Individual logos are shown in Supplementary Figure S3. (C) Illustration of our naming scheme for bases of the XerD-binding arm.

Figure 4.

2D-maps representation of the integration results. (A) 2D-map representation methodology: Different [x, y] coordinates are assigned to each nucleotide for the 2⁸ possible motifs as follows: the [x, y] coordinates are initially set to [1, 1]. Then, [, ], [, ], [, ] or [, ] are added to [x, y] for each n position of the degenerate motif if the nth base is A, T, G or C, respectively. Relative integration efficiencies are represented with a dark-blue to bright yellow colour scale. Positions that were not found in the integration libraries are shown in black. (B) 2D-maps of the integrated n₈gtg motifs. FtsK: results from the conjugation of the n₈gtg XafT⁻ mini-TLCΦ library in the strain harbouring lacZ-dif1 at the natural dif1 locus; XafT: results from the conjugation of the n₈gtg XafT⁺ mini-TLCΦ library in the strain harbouring lacZ-dif1 at lacZ locus; FtsK & XafT: results from the conjugation of the n₈gtg XafT⁺ mini-TLCΦ library in the strain harbouring lacZ-dif1 at the natural dif1 locus. Below each 2D-maps are shown zooms of the regions corresponding to the 2⁴ motifs that carry the same first innermost bases as the XerD-arm of TLCΦ attP (tagan₄gtg) and dif1 (ttatn₄gtg). The position of the TLCΦ attP XerD motif and of the motif most similar to the XerD-arm of dif1 are highlighted by white and red squares, respectively. Their sequences are indicated below the panels. Bases identical to the XerD-arms of TLCΦ attP and dif1 are written in blue and red, respectively. 2D-maps of the integrated n₅a₂n₃g and tagn₈ motifs are shown in Supplementary Figure S4. (C) Sequence logo of the XerD-arm of the attP sites of the n₈gtg XafT⁺ mini-TLCΦ plasmids that were found integrated in lacZ-dif1 at lacZ locus. The total number of n₈gtg integrated motifs is indicated. 2D-maps of the integrated n₄tn₃gtg, n₄an₃gtg, n₄gn₃gtg and n₄cn₃gtg motifs, as indicated. The number of integrated motifs from each category is indicated. The sequence logo of the XerD-arm of the integrated sites from each category is shown below each 2D-map. Positions in which the bases of the integrated n₄tn₃gtg, n₄an₃gtg, and n₄cn₃gtg XerD-arm motifs are significantly different from the integrated n₄gn₃gtg XerD-arm motif are indicated by a star (chi²-test, P-value < 0.001).

Figure 5.

Butterfly plots showing the relative integration efficiency of attP sites harbouring the different possible n₈gtg XerD-arm motifs. (A) Butterfly plot representation methodology. Δn: number of bases by which an XerD-arm motif deviates from TLCΦ attP. If the motif is more similar to dif1 than TLCΦ attP, its integration frequency is plotted at the +Δn position of the X-axis. If a motif is not closer to dif1 than TLCΦ attP, its integration frequency is plotted at the –Δn position of the X-axis. (B) Butterfly plots of the integrated n₈gtg motifs. The proportion of integrated motifs falling in the (−) group is indicated. Their contribution to the global frequency of integration of the library is indicated between brackets. We coloured the various attP sites leading to significant integration driven by FtsK (FtsK panel) with respect to their integration frequency: from higher than 10⁻⁶, 10⁻⁷, 5 × 10⁻⁸ or lower than 5 × 10⁻⁸ in red, orange, cyan and blue, respectively. The corresponding sites in the XafT and FtsK & XafT panels are shown with the same colour code. Sites which were absent in the FtsK panel (motifs which were not capable of being integrated by FtsK) are shown in grey. The difference between the mean frequency of integration of red motifs and the integration frequency of TLCΦ attP (located in the middle of the graph) is indicated in red. The positions of TLCΦ attP and of the attP sites most similar to dif1 are indicated by squares. (C) Combined sequence logos of the n₈gtg, n₅a₂n₃g and tagn₈ motifs with an integration frequency higher than 10⁻⁶. The logos show the frequency of each base at the degenerate positions. The number of motifs from which the logo was derived is indicated. Individual logos and butterfly plots of the integrated n₅a₂n₃g and tagn₈ motifs are shown in Supplementary Figure S5.

Figure 6.

Retention efficiency of the different possible n₈gtg XafT⁺ mini-TLCΦ libraries. (A) Retention efficiency from the natural dif1 locus after growth in LB in conditions of production of XerCD (Xer⁺) or not (Xer⁻). Bar plots show the proportion of n₈gtg motifs belonging to the red, orange, cyan, blue and grey categories within the integrated motifs. Retention efficiencies of the n₅a₂n₃g and tagn₈ XafT⁺ mini-TLCΦ libraries are shown in Supplementary Figure S6. (B) Excision from the lacZ locus after growth in L-broth in conditions of production of XerCD (Xer⁺) or not (Xer⁻). (C) Anti-FLAG immunoblot of XafT-3xFLAG expression in integrated vs replicative pTLC conditions. The predicted molecular weight of XafT-3xFLAG C-terminal fusion protein (220 aa) is 25.5 kDa (EMBOSS Pepstats). Immunoblot contains normalized whole-cell samples of V. cholerae strain EPV369 over several plasmid conditions. Negative control is EPV369. Positive control is EPV369 containing an (induced) arabinose-inducible XafT-3xFLAG expression plasmid. Subsequent lanes contained cell extracts from either integrated or replicative versions of pTLC synthetic phage plasmids. White and blue illustrated colonies indicate samples derived from integrated (into the lacZ-dif1 reporter cassette) or replicative V. cholerae clones, respectively. Cri⁻ and ΔattP denote pTLC variants which are unable to replicate or unable to integrate, respectively. Supplementary Figure S6C contains uncropped source images of the original SDS-PAGE, transfer, and immunoblot.

Figure 7.

Relative efficiency of XafT- and FtsKγ-driven Xer recombination reactions. (A) Synapse formation between dif1 and TLCΦ attP. Scheme depicting that the non-canonical XerD-arm of TLCΦ attP should limit synapse formation and that FtsK translocation could dismantle and/or inhibit the formation of synapses, thereby preventing recombination. (B) Scheme of the in vitro recombination substrates, the HJ recombination intermediate and the crossover products. Green ball: 3’ Cy3 label; Red ball: 5’ Cy5 label; Grey, Green and Blue rectangles: Central region, XerC-binding and XerD-binding arm, respectively. The DNA strands exchanges by XerC and XerD are depicted as thin and fat lines, respectively. (C) XafT- and FtsKγ-promoted recombination reactions. Left panel: recombination of a short labelled dif1 substrate (S1) and a long non-labelled dif1 substrate (S2); Right panel: recombination of a short labelled dif1 substrate (S1) and a long non-labelled TLCΦ attP substrate (S2). The S2 substrates are depicted above each gel image. +: V. cholerae XerC, V. cholerae XerD or MBP-XafT, as indicated; Dγ: V. cholerae XerD-FtsKγ fusion; −: mock buffer of the corresponding purified proteins. A white star indicates the presence of a faint HJ band.