(A) Bordetella phage DGR cassette undergoes mutagenic homing and facilitates tropism switching. mtd, atd, brt and the two repeats (VR and TR) are indicated. VR and TR are expanded to show the (G/C)₁₄, IMH and IMH* elements. DGR homing leads to VR diversification, resulting in progeny phages with altered Mtd trimers at the distal ends of tail fibers.
(B) PCR-based DGR homing assays. Plasmids pMX-TG1a, b, c are derived from pMX1b, which carries the BPP-1 atd-TR-brt region placed downstream of the BvgAS-regulated fhaB promoter. pMX-TG1a, b, c contain 36 bp inserts (TG1), corresponding to ligated exons of the T4 td group I intron flanked by SalI sites, at TR positions 19 (TG1a), 47 (TG1b) or 84 (TG1c), respectively. Grey and pink arrows represent TR and VR, respectively. Small horizontal arrows indicate primers used for homing assays: P1, sense-strand primer located in mtd; P2, antisense-strand primer downstream of VR; P3 and P4 are sense- and antisense-strand primers, respectively, that anneal to ligated td exons. Cam^R, chloramphenicol resistance gene.
(C) pMX-TG1a, b, c are functional donors for DGR homing. Assays were performed following BPP-1d single-cycle lytic infection of B. bronchiseptica RB50 cells transformed with the indicated donor plasmids. pMX1b (negative control) lacks the TG1 insert. Larger PCR products (*) in lanes 2–4 are Brt-independent PCR artifacts (Figure 3, lanes 1&2 and data not shown). They contain sequences from mtd to TR, have not undergone adenine mutagenesis, and were likely generated by PCR template switching between BPP-1d DNA and contaminating donor plasmids in phage DNA preparations. P1+P2 product levels demonstrate that approximately equal amounts of input DNA were used for PCR homing assays.
(D) Testing the DGR retrohoming hypothesis. The T4 td group I intron (td) and flanking exons (E1, E2) were inserted at position 84 in TR. Following transcription and intron splicing, ligated exons will be retained in a subset of TR-containing transcripts. If homing occurs via an RNA intermediate (retrohoming), some VRs will acquire ligated exons. Primers used for PCR homing assays are indicated by small horizontal arrows: P1 and P2 are described in (B); E1s, sense-strand primer annealing to E1; E2a, antisense-strand primer annealing to E2.
(E) Precisely ligated td exons are transferred to VR during homing. Products from PCR homing assays with indicated primer pairs and donor plasmids are shown. TG1c, pMX-TG1c positive control; td, pMX-td donor plasmid; td/SMAA, RT-deficient pMX-td derivative; P6M3 and ΔP7.1-2a, splicing-defective pMX-td derivatives; td-, pMX1b with the td intron and flanking exons inserted in inverted orientation at TR position 84. *, Brt-independent PCR artifacts.

(A) TR deletion constructs and homing activities. All donor plasmids are derivatives of pMX1b, containing AvrII (Av) and ApaI (Ap) sites in atd and brt, respectively, as well as a sequence tag (TG2). AvrII and ApaI sites were introduced via silent mutations and do not affect DGR homing (Figure S6). FL (full-length) is a positive control with TG2 inserted at position 84. ΔTR1-84, ΔTR11-84, ΔTR23-84, ΔTR33-84, ΔTR33-96 and ΔTR33-113 contain TR deletions with TG2 at deletion junctions. Results from PCR homing assays are summarized: +, homing efficiency similar to the FL positive control; ++, homing activity greater than FL; −, no homing activity detected.
(B) Primers used for homing assays. P5 and P6 are sense- and antisense-strand primers annealing to TG2, respectively; primers P1 and P2 are described in Figure 1.
(C) PCR homing assays with TR deletion constructs. Homing assays were performed following BPP-1d single-cycle lytic infection of RB50 cells transformed with the indicated plasmids. *, Brt-independent PCR artifacts.

DGR homing assays were performed following BPP-1d single-cycle lytic infection of RB50 or RB50ΔrecA cells transformed with the indicated plasmids. TG1c/SMAA, RT-deficient mutant of pMX-TG1c (Figure 1B); other donor plasmids have been described (Figure 1E). Primers used in homing assays are shown below the gel. *, Brt-independent PCR artifacts.

(A) Strategy to identify cDNA integration sites at the 3′ and 5′ ends of VR via marker coconversion. Markers introduced into TR (red Ts) are transferred to VR only if they are located between 3′ cDNA priming and 5′ cDNA integration sites.
(B) Schematic of coconversion experiments. Single C to T markers in donor TRs are indicated. All TRs are tagged with TG1 at position 84.
(C) Homing assays were performed following BPP-1d single-cycle lytic infection of RB50 cells transformed with marked donor plasmids. Results from PCR homing assays are shown in Figure S10 and marker coconversion data are summarized here. Nucleotides at relevant positions in the wild type parental VR are shown in the center. At bottom, the number of progeny VRs with a transferred marker, or no transferred marker at specific sites are shown. Deduced cDNA integration regions at the 3′ and 5′ ends of VR are indicated by brackets.

(A) Experimental design. Donor plasmid pMX-td contains the td group I intron at position 84 in TR, and prophage BPP-1dΔVR1-99 lacks the first 99 bp of VR. cDNA integration at the 3′ or 5′ end of VR was assessed in PCR assays using the same primer pairs described in Figure 1D. (B) VR lacking the first 99 bp supports cDNA integration at the 3′ end, but not the 5′ end. BPP-1dΔVR1-99 (IMH) and BPP-1dΔVR1-99IMH* (IMH*) lysogens transformed with the indicated donor plasmids (described in Figure 1E) were induced with mitomycin C for 2 hrs. Total nucleic acids isolated from induced cultures were assayed for cDNA integration by PCR using primers shown in (A). Pink arrow, cDNA intermediate. *, Brt-independent PCR artifacts.

(A) Strategy to identify requirements for cDNA integration at the 5′ end of VR. Donor plasmid pMX-M50 is a derivative of pMX-ΔTR23-84 (Figure 2A) containing an insert in TR consisting of a 50 bp mtd segment (M50, derived from sequences located upstream of VR). The M50 insert in TR provides homology to the mtd locus in prophage BPP-1dΔVR1-99. Primers are indicated as small horizontal arrows: P7 is a sense-strand primer annealing to mtd; P2, P5 and P6 are described in Figure 1 and Figure 2B. Int., integration.
(B) The M50 insert in TR restores homing into sequences upstream of VR in BPP-1dΔVR1-99. PCR assays were performed on DNA isolated from intact phage particles produced following induction by mitomycin C. RB50 lysogens carried BPP-1d prophage genomes with wild type VR sequences (VRwt) or the VR1-99 deletion (ΔVR1-99). Donor plasmids were as follows: ΔTR23-84, pMX-ΔTR23-84; M50, pMX-M50; M50/SMAA, an RT-deficient mutant of pMX-M50. Product bands 1 and 2 in lane 2 and band 3 in lane 5 are labeled. *, Brt-independent PCR artifacts. (C) Major products derived from bands 1, 2 and 3 in (B) are shown and described in the text. Yellow bars, VR1-99 or portions thereof.

DGR homing occurs via a unique, “copy and replace” pathway that substitutes parental VR sequences with diversified cDNA copies of TR transcripts. cDNA integration at the 3′ end of VR is proposed to occur via a TPRT mechanism, while integration at the 5′ end of VR requires short stretches of TR/VR sequence homology and occurs through template-switching and/or strand displacement. Adenine mutagenesis most likely occurs during minus-strand cDNA synthesis by the DGR-encoded RT. The resulting mismatches in VR DNA strands could be resolved via DNA replication. Pink arrows and bars, cDNAs and cDNA-derived sequences. “N”, any of the four deoxyribonucleotides. Dashed lines, TR flanking sequences. See the text for details.