GENETICS AND MOLECULAR BIOLOGY

Transduction by φBB-1, a Bacteriophage ofBorrelia burgdorferi

Christian H. Eggers, Betsy J. Kimmel, James L. Bono, Abdallah F. Elias, Patricia Rosa, D. Scott Samuels
DOI: 10.1128/JB.183.16.4771-4778.2001

Article Figures & Data

Figures

  • Fig. 1.
    Fig. 1.

    B. burgdorferi phage particles. Samples were collected from polyethylene glycol-precipitated cell supernatants of an MNNG-treated culture ofB. burgdorferi CA-11.2A and viewed by transmission electron microscopy. Although previously reported as having a simple noncontractile tail (12), subsequent modifications to the purification and preparation protocol (10, 11) reveal that all phage particles observed have intact contractile sheaths, seen here either extended (left) or contracted (right). Phosphotungstic acid stain. Bar, 45 nm.

  • Fig. 2.
    Fig. 2.

    Kanamycin-resistant transformants of B. burgdorferi. (A) pCE210 construct. A 4-kb fragment of phage DNA (solid line) was cloned into the HindIII site of pBluescript SK+ (dashed line). The kan cassette (hatched box) was cloned into a unique NdeI site on the phage fragment 1.1 kb from the SK primer location. The locations of the SK, KS, cp32SK12NdeIF (cp32F), cp32SK12NdeIR (cp32R), and KanR1207F (kanF) oligonucleotides are shown (not drawn to scale). Arrows indicate oligonucleotide site and orientation. (B) Transformed CA-11.2A colonies were screened by PCR using the cp32SK12NdeI primers. PCR products were resolved on a 1% agarose gel and stained with EtBr. Both a small product (no kancassette; black arrow) and a larger product (integrated kancassette; hatched arrow) were amplified from the transformants. (C) Products consistent with a population of cp32s both containing thekan cassette (hatched arrow) and lacking the kancassette (black arrow) were also amplified from phage DNA collected from CA-11.2A/kan R transformants. pCE210 DNA was amplified as a positive (+) control, and wild-type CA-11.2A phage DNA was used as a negative (−) control.

  • Fig. 3.
    Fig. 3.

    Determining the minimum number of cp32 molecules inB. burgdorferi cells and φBB-1 capsids by PCR amplification of VR1. Highly conserved primers that flank a variable region (VR1) of cp32 amplify four different size products fromB. burgdorferi strains CA-11.2A and B31. The two VR1s from the cp32s of B31 at ∼3.3 kb can be resolved with extended electrophoresis times (see Fig. 7). PCR of φBB-1 released from both of these strains amplifies three of the VR1 products (lanes 2 and 4), but not the ∼2.8-kb amplicon (black arrow; see Table 2). Fragments were resolved on a 0.8% agarose gel by FIGE and stained with EtBr. Molecular sizes (in kilobase pairs) are indicated.

  • Fig. 4.
    Fig. 4.

    Analysis of the genomic location of the kanamycin resistance cassette. Total DNA from both parental CA-11.2A (P) and a transformed clone of CA-11.2A/kan R(Tf) was extracted and resolved by two-dimensional gel electrophoresis. The gel was stained with EtBr (A), blotted to nylon, and probed with the cp32-specific probe 4 (B) or pOK12, the source of thekan gene (C). The kan probe has the same hybridization pattern as the cp32-specific probe. The supercoiled form of cp32 is indicated by the black arrow, while the other hybridization sites likely represent the nicked (or lp56) and linearized forms of cp32 generated during DNA isolation. Molecular sizes (in kilobase pairs) are indicated.

  • Fig. 5.
    Fig. 5.

    Analysis of kan cassette packaging by φBB-1. Phage particles were precipitated from both parental (P) and transformed (Tf) CA-11.2A cells. The DNA was extracted and resolved on a 0.5% agarose gel by conventional field electrophoresis. The gel was stained with EtBr (A), then blotted and probed with either probe 4 (cp32-specific; B) or pOK12 (kan; C). Molecular sizes (in kilobase pairs) are indicated.

  • Fig. 6.
    Fig. 6.

    PCR analysis of kanamycin-resistant transductants ofB. burgdorferi. (A) Putative transductants fromB. burgdorferi strains CA-11.2A, B31, and 1A7 were screened by PCR using the KanR1207F and cp32SK12NdeIF primers. pCE210 was amplified as a positive control (+), while the parental cells (P) of each strain served as negative controls (lanes 1, 4, and 6). Lane 2 is the CA-11.2A/kan Rtransformant (Tf). PCR yields a ∼125-bp product from DNA containing the kan cassette (black arrow, lanes 2, 3, 5, and 7). The products were resolved on a 1% agarose gel and stained by EtBr. (B) The kan integration site in each transductant was verified by long-range PCR using the KanR1207F and cp32-VR1R primers.

  • Fig. 7.
    Fig. 7.

    Amplification of variable region VR1 from B. burgdorferi kan transductants. The cp32-VR1 primers were used to amplify the cp32 VR1s of parental CA-11.2A (lane 1), B31 (lane 3), and 1A7 (lane 5) as well as transductants CA-11.2A/TR3 (lane 2), B31/TR1 (lane 4), and 1A7/TR5 (lane 6). The 2,537-bp VR1 (black arrow) was found in all transductants. Amplification products were resolved on 0.8% agarose gels by FIGE and stained with EtBr. Molecular sizes are shown in kilobase pairs.

  • Fig. 8.
    Fig. 8.

    Restriction mapping of the cp32s of B. burgdorferi kan transductants. (A) Comparison of the restriction maps of parental, transformant, and transductant CA-11.2A DNA. Plasmid DNA was extracted from CA-11.2A (lane 1; P), the CA-11.2A/kan R transformant (lane 2; Tf), and CA-11.2A/TR3 (lane 3; Td), digested with EcoRV, and resolved by FIGE. A blot of the gel was probed either with the cp32SK12NdeI PCR product (left panel) or with kan (right panel). A unique ∼6.6-kb fragment of CA-11.2A parental DNA is indicated by the hatched arrow. A ∼8-kb band (black arrow) corresponding to the ∼6.6-kb band plus the 1.3-kb kaninsert is present in both the transformant and the transductant. (B) Comparison of the restriction maps of the cp32s of B. burgdorferi kantransductants. Plasmid DNA from CA-11.2A (lane 1), CA-11.2A/TR3 (lane 2), B31 (lane 3), B31/TR1 (lane 4), 1A7 (lane 5), and 1A7/TR5 (lane 6) was digested with XbaI and resolved by FIGE. A blot of the gel was probed with either the cp32SK12NdeI probe (left panel) or the kanprobe (right panel). A ∼8-kb band (black arrow) is found only in the transductants. The hatched arrow indicates the 6.6-kb fragment from the parental CA-11.2A strain (lacking thekan cassette). Three bands (∗) appear in parental B31 but not B31/TR1. Molecular sizes are shown in kilobase pairs.

Tables

  • Table 1.

    Oligonucleotides generated for this study

    OligonucleotideSequence (5′ to 3′)Application
    cp32-VR1FAAATAAAACTTAGGAGTTGGTTTTGAADiagnostic PCR
    cp32-VR1RTAACTTTCCTAGCGTTAACTTCTGATDiagnostic PCR
    cp32SK12NdeIFACTTTGTTGTAGTGATTATTTGTTCScreening; probe
    cp32SK12NdeIRGGGGAAAGAATTGTTGAAGScreening; probe
    KanR1207FATTACGCTGACTTGACGGG kancassette screening
  • Table 2.

    Predicted fragment sizesa of VR1 of the B31 cp32s

    B. burgdorferi
    B31 plasmid    		Amplicon size (bp)
    cp32-13429 Embedded Image
    cp32-32442
    cp32-43377 Embedded Image
    cp32-62507
    cp32-73575
    cp32-83429 Embedded Image
    cp32-93367 Embedded Image
    1p562796
  • Table 3.

    Amplification of the VR1s of CA-11.2A, B31, and φBB-1a

    Size (bp)Predicted B31 plasmid
    CA-11.2AB31-UM
    Total DNAPhage DNATotal DNAPhage DNA
    3,4263,426cp32-1 or 8
    3,3433,343cp32-9 or 4
    3,0473,047
    2,8192,8281p56
    2,5372,537
    2,484b 2,484cp32-3b
    2,4552,455

    • ↵a Sizes determined by regression using Multi-Analyst software; predictions based on size estimates from ≥3 trials and applicable only to the B31 fragments.

    • ↵b Prediction confirmed by sequencing of VR1.

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