Use of the Cre-lox Recombination System To Investigate the lp54 Gene Requirement in the Infectious Cycle of Borrelia burgdorferi

Loss of fluorescence by B. burgdorferi carrying loxP-flanked GFP after introduction of Cre recombinase. Strain B31-A34 was first transformed with shuttle vector pBSV2G-loxP-flaBp-gfp (Fig. 1) and subsequently transformed with the compatible shuttle vector pBSV25 or pBSV25-flgBp-cre, which encodes Cre recombinase. (Top) Presence or absence of GFP fluorescence in these strains as visualized by fluorescence microscopy. (Bottom) Cells were counterstained with the membrane stain FM4-64 to visualize all spirochetes in the same field.

Schematic diagram of the targeted region encompassing bba01 to bba14 of lp54, extending from the left telomere to the ospAB operon. Boxed numbers beneath the diagram indicate previously identified members of paralogous gene families (11, 18). Protein designations above the diagram and gene designations below are based on previous studies and annotations (10, 11, 16, 18, 36, 37).

Insertion of loxP into targeted lp54 loci. (A) Schematic diagram showing how a loxP site (filled arrowheads) and an adjacent selectable marker conferring resistance to streptomycin or kanamycin (aadA and Kan^r, respectively) were introduced by allelic exchange into bba07 and subsequently into either bba01 or bba14 on lp54. The relevant restriction enzyme sites used in cloning allelic exchange constructs are indicated. Small arrows beneath bba07, bba01, and bba14 indicate the positions of oligonucleotides (Table 2) used with PCR to confirm loxP insertions into these loci, as shown below (B). The designations of the resulting strains (Table 1) are shown on the left. (B) PCR amplification of targeted lp54 loci demonstrating an increase in fragment size after loxP insertion. The gene target and PCR primers are indicated above the lanes, and the source of template DNA is shown below the lanes. PCR primer positions and sequences are shown above (A) and in Table 2, respectively. The relative mobility of DNA size standards (kb) is shown on the left.

Deletion of loxP-flanked regions of lp54 after introduction of Cre recombinase. (A and B) Schematic diagrams showing the excision of the intervening DNA between loxP sites (filled arrowheads) present in the lp54 loci bba07 and bba01 (A) or bba07 and bba14 (B) as a result of Cre-mediated recombination. A recombined loxP site and the adjacent resistance cassette are present on a nonreplicating circular DNA fragment. Small arrows beneath bba07, bba01, and bba14 indicate the positions of oligonucleotides (Table 2) used with PCR to confirm the deletion of the intervening sequences in Cre transformants, as shown below (C). The designations of the resulting strains (Table 1) are shown on the left. (C) PCR amplification of targeted lp54 regions in the wild type and deletion mutants. Smaller products spanning the deleted regions were amplified from mutant strains, whereas the larger lp54 fragments were not efficiently amplified from wild-type A3. PCR amplification of the kan and aadA genes demonstrates the presence or absence of these antibiotic resistance cassettes as a consequence of the lp54 deletions. The gene target and PCR primers are indicated above the lanes, and the template DNA is indicated below the lanes. Primer positions are indicated above (A and B), and sequences are shown in Table 2. The relative mobility of DNA size standards (kb) is shown on the left.

Southern blots confirming the deletion of multigene segments of lp54 by loxP/Cre-mediated recombination. Genomic DNAs prepared from the wild type (B31-A3), a strain lacking lp54 (B314), and the lp54 deletion mutants (A3ΔA1-7 and A3ΔA7-14) were subjected to Southern blot analysis with probes specific to bba03 and bba10 to confirm the loss of targeted regions of lp54 containing these loci in the respective loxP/Cre deletion mutants. The source of DNA is identified above the lanes, and the probe is specified below.