Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes

Abstract

Liu et al. recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes. Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.

FIG. 1.

BPP-1 virion morphology. Negative-stain transmission electron micrographs of (A) an intact phage particle, (B) an isolated tail (side view), with partially dissociated tail fibers, and (C, D) isolated tails with tail fibers (top view). (E) Schematic diagram (not to scale) showing general particle morphology and hexagonal symmetry.

FIG. 2.

Phage integration site. Plasmids pML83-B102 and pML83-301 contained attL. attR was identified with PCR primers derived from the phage genome and the B. bronchiseptica genome. The Bordetella phage genome contains a 27-bp sequence exactly identical to the 3′ end of the Bordetella his tRNA gene. This allows phage integration into the his tRNA locus without disrupting gene function.

FIG. 3.

Predicted Bordetella phage coding sequences. (A) Arrows represent predicted coding sequences encoding proteins of more than 7 kDa. Functional assignments for several gene clusters are indicated. See the text and Table 2 for details. (B) Schematic representation of the tropism switch region. brt encodes a reverse transcriptase. VR1 is located at the 3′ end of mtd. TR, also required for tropism switching, is not predicted to be part of a coding region and is located downstream from bbp7. (C) Schematic representation of the lysis/lysogeny region. cI encodes the phage repressor, and bbp31 encodes a putative Cro-like protein. bpm encodes an adenine DNA methylase, and its coding sequence is located immediately downstream of bpb31. Bpm does not appear to play a role in the lysis versus lysogeny decision.

FIG. 4.

Syntenic regions conserved between Bordetella phages and other phages and phage-like elements. The genome of BPP-1 is represented by a solid grey bar or black arrows for BPP-1 genes that have homologs present in a similar order in other genomes. Loci from similarly ordered genomic regions in other phages or phage-related elements are represented below the relevant BPP-1 gene. Similarly ordered genomic regions are found in a Legionella pneumophila defective prophage element, insect endosymbiont phage APSE-1, three Xylella fastidiosa 9a5c cryptic prophages, and Staphylococcus aureus phage φ12.

FIG. 5.

Structure and variability of the VR2 segment of bbp36. (A) Graphic representation of VR2 and bbp36. The 19-bp cassette and the three 5-bp spacers are represented by color-coded bars. (B) VR2 sequence variations. The VR2 sequences are represented by color-coded 5-bp spacers. Derivatives of BPP-1, BMP-1, and BIP-1 are listed below the parental phage. Isolates with different tropisms but identical VR2 sequence are marked with *. +, BPP tropism; −, BMP tropism; i, BIP tropism.

FIG. 6.

High-pressure liquid chromatography analysis of BPP-1 and BIP-1 DNA. (A) Elution profile of BPP-1 nucleosides. (B) Elution profile of BIP-1 nucleosides. The N⁶-methyladenine (Me-A) peak (downward arrow at R_t = 29 min) is significantly smaller in BIP-1 than in BPP-1, after taking into account the different scales. MAU, milli-absorbance units.

FIG. 7.

Colonization of murine respiratory tract by RB30 and RB50Gm. Colonization of the nasal cavity 25 days postinoculation with equal numbers of RB30 (a BPP-1 lysogen) and RB50Gm. The proportion of RB50Gm derivatives (white bars) lysogenized by BPP-1 is indicated by the black portion of the histogram bars. The bottom graph shows the colonization efficiency of the RB30 lysogen strain. The overall quantity of RB50 recovered is lower than that of RB30, presumably due to phage killing.