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Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements


Bordetella bacteriophages generate diversity in a gene that specifies host tropism1. This microevolutionary adaptation is produced by a genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats; one serving as a donor template (TR) and the other as a recipient of variable sequence information (VR)1. Here we describe the genetic basis for diversity generation. The directionality of information transfer is determined by a 21-base-pair sequence present at the 3′ end of VR. On the basis of patterns of marker transfer in response to variant selective pressures, we propose that a TR reverse transcript is mutagenized, integrated into VR as a single non-coding strand, and then partially converted to the parental VR sequence. This allows the diversity-generating system to minimize variability to the subset of bases under selection. Using the Bordetella phage cassette as a signature, we have identified numerous related elements in diverse bacteria. These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes.

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Figure 1: Tropism switching by Bordetella bacteriophage.
Figure 2: Multiple substitution experiments.
Figure 3: Mosaic VR sequences result from mutagenic homing.
Figure 4: Diversity-generating retroelements.


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We thank members of the J.F.M. laboratory for constructive input throughout the course of the project. A.H. is a predoctoral trainee recipient of an NIH Microbial Pathogenesis Training Grant. This work was supported by NIH grants to J.F.M.

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Correspondence to Jeff F. Miller.

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Supplementary information

Supplementary Figure 1

This figure shows the organization of the BPP-1 genomic region containing the tropism switching cassette and the results of gene deletion experiments. Deletions in only three genes – brt, atd, and mtd – abolish tropism switching, defining the extent of the cassette. (PDF 17 kb)

Supplementary Figure 2

This figure shows the results of adenine mutagenesis of TR. Addition of an adenine in TR, confers a corresponding site of variability to VR, while removal of TR adenines eliminates variability. Frequencies of variation at adenines are also shown. (PDF 11 kb)

Supplementary Figure 3

This figure shows the results of immunofluorescence experiments with purified BPP-1 Mtd protein and Bordetella bacteria. Mtd binds to Bvg+ and Prn-expressing Bvg- bacteria, but not Bvg- or Bvg+ Δprn bacteria, demonstrating that Mtd binding specificity determines phage tropism. (PDF 33 kb)

Supplementary Figure 4

This figure shows the results of in vitro variability assays with BPP-1 containing deletions in TR and VR. While 3’ VR deletion eliminates variability (Fig. 1c), 5’ deletion (Δ61 phage) does not, indicating that the 5’ boundary of homing is not precisely defined. (PDF 11 kb)

Supplementary Figure 5

This figure shows tropism switching frequencies of BPP- and BMP-MS1, MS2 and MS3 phages (Fig. 2). Deletion of recA has no effect on tropism switching. (PDF 6 kb)

Supplementary Figure 6

This figure shows the TR-VR nucleotide sequence alignments from each DGR. The differences between TRs and VRs suggest that adenine mutagenesis and homing to the 3’ of VR may be conserved mechanisms among DGRs. (PDF 21 kb)

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Doulatov, S., Hodes, A., Dai, L. et al. Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements. Nature 431, 476–481 (2004).

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