Use of the Cre-lox recombination system to investigate the lp54 gene requirement in the infectious cycle of Borrelia burgdorferi

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, has a complex genome consisting of a linear chromosome and up to 21 linear and circular plasmids. These plasmids encode numerous proteins critical to the spirochete's infectious cycle and many hypothetical proteins whose functions and requirements are unknown. The conserved linear plasmid lp54 encodes several proteins important for survival in the mouse-tick infectious cycle, but the majority of the proteins are of unknown function and lack homologs outside the borreliae. In this study we adapted the Cre-lox recombination system to create large deletions in the B. burgdorferi genome. Using Cre-lox, we systematically investigated the contribution of 14 adjacent genes on the left arm of lp54 to the overall infectivity of B. burgdorferi. The deletion of the region of lp54 encompassing bba07 to bba14 had no significant effect on the infectious cycle of B. burgdorferi. The deletion of bba01 to bba07 resulted in a slight growth defect but did not significantly affect the ability of B. burgdorferi to complete the infectious cycle. This study demonstrated the utility of the Cre-lox system to efficiently explore gene requirements in B. burgdorferi and surprisingly revealed that a large number of the highly conserved proteins encoded on lp54 are not required to complete the infectious cycle.

FIG. 1.

Schematic diagram illustrating the loxP/Cre-mediated deletion of the gene encoding GFP. The introduction of Cre recombinase into B. burgdorferi containing flaBp-gfp flanked by loxP sites should result in recombination between loxP sites, the excision and loss of gfp from shuttle vector pBSV2G (15) due to the absence of replication factors in this region of the plasmid, and a loss of fluorescence by the cell.

FIG. 2.

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.

FIG. 3.

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).

FIG. 4.

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.

FIG. 5.

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.

FIG. 6.

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.

FIG. 7.

In vitro growth curves. Wild-type B. burgdorferi (A3) and loxP insertion mutants (A3-A07, A3-A01/A07, and A3-A07/A14) (A) or lp54 deletion mutants (A3ΔA1-7 and A3ΔA7-14) and wild-type B. burgdorferi containing Cre on a shuttle vector (A3-Cre) (B) were grown at 35°C in BSKII medium from a starting concentration of 10⁵ spirochetes/ml. Spirochetes were counted at 24-h intervals in Petroff-Hauser chambers, and the mean number of spirochetes per ml was determined from triplicate cultures of each strain, as shown.