Supplementary Material

Transcription

1 Reverse Transcriptase-Mediated Tropism Switching in Bordetella Bacteriophage Minghsun Liu, Rajendar Deora, Sergei R. Doulatov, Mari Gingery, Frederick A. Eiserling, Andrew Preston, Duncan J. Maskell, Robert W. Simons, Peggy A. Cotter, Julian Parkhill, and Jeff F. Miller Supplementary Material Methods Adsorption assay. BPP-1 phage were added to an excess of bacteria (MOI = 0.1) and incubated at 37 o C. At specific time points, aliquots were removed and passed through sterile 0.20 µm filters to retain bacteria and attached phage. The phage titer in each filtrate was determined. Selection for tropism switch. High titer lysates were generated by inducing lysogens with mitomycin C. The lysates were then plated on permissive B. bronchiseptica host strains to achieve confluent plate lysis. The resulting phage particles were collected by resuspending in SM buffer (50 mm Tris, ph 7.5, 10 mm MgSO4, 0.001% gelatin, 0.1 M NaCl) at 4 C and titered on Bvg + and Bvg - bacteria to isolate tropic variants. Construction of mutant phage strains. In most cases, the product of the first PCR with primers 1 and 2 was used in a second round of PCR with the primer 3 and the final product was cloned into pre112. In some cases, two separate PCR reactions with primers 1,2 and 3,4 were carried out. The vector was transferred into the lysogenized strain by conjugation and allelic exchange was carried out essentially as described in ref. 2. VR1. PCR primers: 1. -AGCTCTAGAATGCGCCCCACTTCGGCG GCGGACATCTATCTGCTAGGCGTCTGTGACCACCTGATTCTTG AGCGGTACCGACAAGAACACGGTCGTTG-3 Internal in-frame deletion of 528 bp of BPP-1 and BMP-1 mtd including VR1 (bp , aa ). brt. PCR primers: 1. -AGCTCTGCAATGGCTCTGCCAACGCTACGG CAAAGCCGGCAATTACGAGCGCGGCACCCACAAGCTGCTGCGCAAGTC AGCGGTACCGCTCGATGGTGCTCAAGTC-3 Internal in-frame deletion of 618 bp of BPP-1 and BMP-1 brt including the predicted RT domain (bp , aa ). Brt YMDD ->Brt SMAA. PCR primers: 1. -AGCGGTACCGTGGCGCCGATCTTCGAGGC CGTCATTGGGCGCGCTCCATGGCCGCCATCGTGGTGCTGGGC AGCTCTAGAGCGATGCCATATTGTTCCTCC-3 Substitution of the underlined base pairs in BPP-1 and BMP-1 results in the amino acid change from Y to S (aa 213), D to A (aa 215), and D to A (aa 216) in the predicted active site of Brt. TR. PCR primers: 1. -CGCTTCTAGAGCAACCGATGGAACCCATCG CCAGAAGCTTCGTTATTTCCCAGCCTGCCC CCAGAAGCTTGCATGGCTCTGCCAACGCTACG CGCTGCATGCGCACAAGGCGGTCTTTGAATTCG-3 In-frame deletion of the entire TR (bp 1-134) in BPP-1 and BMP-1.

2 VR1 complementation [BPP-1 brt(vr1 BMP-1 ), BPP-1 brt(vr1 BIP-1 ), BMP-1 brt(vr1 BPP-1 ) in Table 1]. PCR primers: 1. -AGCTCTAGAATGCGCCCCACTTCGGCG AGCGGTACCGACAAGAACACGGTCGTTG-3 PCR fragments containing VR1 and ~700 bp of flanking DNA (1431 bp total) from BPP-1, BMP-1, and BIP-1 were generated and cloned into pbbr1mcs. Plasmids were transferred into brt lysogens by conjugation and the induced lysates screened for plaque forming activity on Bvg + and Bvg - bacteria. PCR primers used for the restriction enzyme-based variability assay. Different sets of PCR primers were used for each of the three PCR reactions: 1. -CTTTCGTACCGTTCCACAATG-3 and -CGCGTGGAACATCTTCACGTC CGCGGCATGACGCTGGTGGC-3 and -GGGTTCCATCGGTTGCCTTG AGCAAGCTTCCTCGATGGGTTCCATC-3 and -AGCGGATCCGAATACACGGC- CAACAC-3 Bordetella phage genomes. The sequencing and annotation of the Bordetella phage genomes will be reported elsewhere (1). Comparison of phage sequences revealed a second region of variability, VR2, within an open reading frame (orf36) predicted to encode a product with sequence similarity to ice nucleation proteins expressed by Pseudomonas species. Phage with different tropisms that had identical VR2 sequences were observed and deletion of VR2 had no effect on tropism switching. A more detailed analysis of VR2 will be reported in a future publication (1). Several additional open reading frames shown in Fig. 3A and their predicted products are as follows: orf11, lytic murein transglycosylase; orf21, head-to-tail joining protein; orf25, terminase large subunit; bpm, adenine DNA methyltransferase; orf31, bacteriophage lambda Cro homolog; c1, bacteriophage lambda repressor homolog. References 1. M. Liu et al. manuscript in preparation. 2. R. A. Edwards, L. H. Keller, D. M. Schifferli, Gene 207, 149 (1998)

3 Supplemental Figure 1. Relative efficiency of plaquing (EOP) by phage variants of different tropisms. For each phage isolate, the numbers were calculated by dividing the number of plaques on Bvg+ bacteria by the number of plaques on Bvg- bacteria. BPP BMP-1 BMP-2 BMP BIP-1 BIP log 10 (relative EOP Bvg+/Bvg-) Supplemental Figure 2. Derivation of phage variants shown in Fig. 3D and Web fig. 3. BPP-1 BMP-1 BMP-10 BMP-11 BMP-13 BMP-14 BIP-11 BIP-12 BIP-13 BPP-14 BPP-2 BIP-1 BMP-12 BMP-15 BPP-12 BPP-10 BPP-11 BPP-13 BPP-15 BPP-16 BIP-10

4

5 Supplemental Figure 3. Expanded data set from Fig. 3D and E. (A) Phage of different tropisms derived in vivo (see Web fig.2). (B) VR1 sequences selected using the restriction enzyme-based asssay (13). The 15 bp sequence at the 3 end of TR (bracketed) corresponds to an invariant region of VR1 that is always observed to differ from the corresponding sequence in TR. Note that adenine 35 (upward arrow) does not correspond to one of the variable bases in (A) but is found to be variable in (B), suggesting that the corresponding threonine is required for phage infectivity. A B A A A L F G G A W N G T S L S G S R A A L W Y S G P S F S F A F F G A R G V C D H L I L BPP-1 CGCTGCTGCGCTATTCGGCGGCGCCTGGAACGGCACGTCGCTCTCGGGTTCTCGCGCTGCGCTCTGGTACAGCGGGCCGTCGTTCTCGTTCGCGTTCTTCGGGGCGCGCGGCGTCTGTGACCACCTGATTCTTG BPP-10 A TC CT TA TA TT TA AA G TT CT AC A MboII A C TGA BPP-11 A GC AA TG GC TA AG AG GC AG TC G A C TGA BPP-12 A TC TT TA TC TA TA GG TT GG TT A A C TGA BPP-13 A AG AG TA AA CT TA TT G TA TA TA A A C TGA BPP-2 A GC TA AA AA T TA TA TT AA AA CT A A C TGA BPP-14 G AG CA TA AA TA TA AG CC GA AG A A C TGA BPP-15 G TA TC TA GC TA TA GC TA CC TA T A C TGA BPP-16 A AG TT TA TA CT T TA GG G TA AG TT A A C TGA BIP-10 A AG AG AA AA GT TA TA CA TA TA A A C TGA BIP-11 A AG TA TA AA AT AT AG AA CC AA A A C TGA BIP-12 A AG CA TA TA TA TA AG AA CC AA A A C TGA BIP-13 A TC TA TA AA TA TA AG AA CC AA A A C TGA BIP-1 A TC TT TA TA TA AA GC AA CAG AA A A C TGA BMP-10 A AG TC AA AA TA TT TA TA AA TT A A C TGA BMP-11 A AG TC AA AA TA TA TA GC AA TT C A C TGA BMP-12 A AG AG AA AA GC AG TA CA TA TA A A C TGA BMP-13 A TC AG AA TC TA TA TA CT TC TT T A C TGA BMP-14 A GC AG AA AA TA TC TA TA TA CA G A C TGA BMP-15 A AG GC TA AA TA AA GC AA CAG AA A A C TGA BMP-1 G AGCT CA TACACGT AA TA TA AG AA CC AA A A C TGA AluI AflIII AF1-2 G AA GC AG C CA AG GG TA GC CT AG T A C TGA AF1-3 G TT TC AG G TC TT TA TA AA CC AA A A C TGA AF2-2 G TA TT AG A TA TA GG TC AA CT CT A A C TGA AF2-14 A TA GG AG A AG AG AA GT TA AG GT A A C TGA AF4-2 A TA AA TC A TA TA TA AG AA CC AA A A C TGA AL1-1 A AA AG AG A AA TA TA AG AA CC AA A A C TGA AL1-2 A TA GC AG C AA TA TA AG AA CC AA A A C TGA AL1-3 A AC AA TC A AA TA TA AG AA CC AA A A C TGA AL1-4 A AA AA AG T TT TC AC TA AA TA GA T A C TGA AL1-19 T TC AA AA A AC TA TA AA TC CA AG A A C TGA AL2-1 T TC AG AG C TA TA GG GA AA CC AA A A C TGA AL2-2 A AA AA AG G CT AG AC GA TA TA AA A A C TGA AL2-3 G TA AA TA T TA TA TC TA AA CC AA A A C TGA MB1-1 G GC AA GG A CT CT TA AG TA AA CA A A C TGA MB1-2 T GC AA GG A CT AG AG TA AA TT AA A A C TGA TR CGCTGCTGCGCTATTCGGCGGCAACTGGAACAACACGTCGAACTCGGGTTCTCGCGCTGCGAACTGGAACAACGGGCCGTCGAACTCGAACGCGAACATCGGGGCGCGCGGCGTCTGTGCCCATCACCTTCTTG

6 Supplemental Figure 4. Transmission of synonymous substitutions from TR to VR1 during phage tropism switching. Silent substitutions at nonvariable positions were introduced into the TR of BPP-1 (A) creating a TseI restriction site or BMP-1 (B) creating an MfeI restriction site. The variable portion of the parental VR1 and the corresponding TR sequences are shown. Marked TRs are designated TRxT-G (for BPP-1) and TRxC- T (for BMP-1). (A) Transmission of a T to G substitution in BPP-1 derivatives selected by plating lysate on a EYJ6 (Bvg - ) host was determined to be 93% (n = 56) by restriction digestion analysis using TseI. VR1 sequences from three independent isolates are shown, designated BMP-TRx1-3. Of the phage plated on a Bvg + phase host and thus not selected for a tropism switch, none were identified to carry the substitution or any other changes in VR1 (eg. BPP-TRx1). (B) Transmission of a C to T substitution in BMP-1 selected using the AflIII variability assay described in ref. 13 was determined to be 100% (n = 15) by restriction digestion analysis using MfeI. VR1 sequences from three representative clones are shown (BP- TRx1 3). The tropisms of the corresponding phage are unknown. TR markers are transmitted into VR1 with a high frequency, independent of selection for productive phage. A X G A W N G T S L S G S R A A L W Y S G P S F S F A F F G BPP-1...GGCGCCTGGAACGGCACGTCGCTCTCGGGTTCTCGCGCTGCGCTCTGGTACAGCGGGCCGTCGTTCTCGTTCGCGTTCTTCGGC......GGCAACTGGAACAACACGTCGAACTCGGGTTCTCGCGCTGCGAACTGGAACAACGGGCCGTCGAACTCGAACGCGAACATCGGC... B T-G G BPP- 1...GGCGCCTGGAACGGCACGTCGCTCTCGGGTTCTCGCGCTGCGCTCTGGTACAGCGGGCCGTCGTTCTCGTTCGCGTTCTTCGGC... BMP- 1...GGCGCCTGGACCAACACGTCGAACTCGGGTTCTCGCGCGGCGTACTGGACCTACAGGCCGACGTCCTCGGGCGCGTACATCGGC... BMP- 2...GGCGCCTGGACCAGCACGTCGAACTCGGGTTCTCGCGCGGCGTACTGGACCTCCGGGCCGTCGATCTCGGGCGCGTTCATCGGC... BMP- 3...GGCGCCTGGAGCAACACGTCGAACTCGGGTTCTCGCGCGGCGTACTGGAACAACGGGCCGTCGAACTCGTTCGCGTACATCGGC... X G S W H Y T S N S G S R A A Y W Y S G P S N S P A N I G BMP-1...GGCAGCTGGCACTACACGTCGAACTCGGGTTCTCGCGCTGCGTACTGGTACAGCGGGCCGTCGAACTCGCCCGCGAACATCGGC......GGCAACTGGAACAACACGTCGAACTCGGGTTCTCGCGCTGCGAACTGGAACAACGGGCCGTCGAACTCGAACGCGAACATCGGC... C-T T BP- 1...GGCAATTGGAGCACCACGTCGTACTCGGGTTCTCGCGCTGCGTACTGGTACAGCGGGCCGTCGAACTCGCTCGCGACCATCGGC... BP- 2...GGCAGTTGGAGCCGCACGTCGTACTCGGGTTCTCGCGCTGCGTACTGGGACATCGGGCCGTCGAACTCGTTCGCGTACATCGGC... BP- 3...GGCAATTGGAGCACCACGTCGTACTCGGGTTCTCGCGCTGCGTACTGGTACAGCGGGCCGTCGAACTCGCTCGCGACCATCGGC...

7 Supplemental Figure 5. Model of the reverse transcriptase-mediated diversity generating mechanism responsible for tropism switching in Bordetella bacteriophage. A segment of a proposed transcript which includes TR is reverse transcribed by Brt using an unknown priming mechanism. Mutagenesis occurs as a result of Brt-mediated error-prone reverse transcription or by an independent mechanism. The resulting reverse transcript in single- or double stranded form then replaces VR1 in the phage genome. The homing process may involve multiple steps and occur by homologous recombination, endonuclease-mediated splicing, or a combination of both. brt TR VR1 mtd 3 3 TR Brt 3 Transcription Priming/Reverse transcription/mutagensis 3 Homing/Integration brt TR VR1 mtd