Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background

Abstract

A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen.

FIG. 1.

Experimental outline.

FIG. 2.

Heterogeneous colony morphology of parental B31 MI (P3) with representative examples of A, B, and C clones (arrows).

FIG. 3.

Protein profiles, OspB immunoblotting, and plasmid profiles of B31 MI and representative clonal derivatives. (A) SDS-PAGE of whole-cell lysates stained with silver. A 34-kDa band corresponding to OspB is present in B31 MI and clones B1 and C1 (arrow) but absent in clone A1 and mutant MI-ΔoppAII-7.3. Each lane contains borrelial lysate equivalent to 5 × 10⁷ cells. Molecular masses are indicated on the left. (B) Immunoblotting of whole-cell lysates probed with H4610, a monoclonal antibody that recognizes the amino-terminal segment of OspB. Closed arrow, full-length OspB; open arrow, truncated OspB. Each lane contains borrelial lysates equivalent to 8 × 10⁷ cells. Molecular masses are indicated on the left. (C) Plasmid profiles of B31 MI and clonal derivatives. Total plasmid DNA was separated by electrophoresis through an 0.3% agarose gel and stained with ethidium bromide. Molecular sizes are indicated on the left. Supercoiled (S), linear (L), and nicked (N) forms of cp9 are indicated by arrows on the right.

FIG. 4.

Southern blot analysis of genomic DNA from B31 MI and clonal derivatives. DNAs (500 ng/lane) were separated by electrophoresis and hybridized with probes specific for lp25 (A), lp28-1 (B), or lp28-4 (C). Arrows to the left of each panel indicate the positions of the plasmids. Probes were from BBE17 (lp25), BBF18 (lp28-1), and BBI28 (lp28-4) and were generated with the same primers used to assess plasmid content (Table 1).

FIG. 5.

Growth curves in liquid BSK-II medium of B31 MI and clonal derivatives at 35°C. Each data point represents the arithmetic mean of the number of spirochetes determined in triplicate. Error bars indicate the standard deviation for each triplicate set. Doubling times and standard deviations are indicated to the right. B31 MI and derivatives with faster growth (F) are in the upper panel of the key, and derivatives with slower growth (S) are in the lower panel.

FIG. 6.

Immunoblot of mouse sera probed against recombinant P39. (A) Sera from mice needle inoculated with 5 × 10³ spirochetes. (B) Sera of mice collected 4 weeks after being fed upon by B31 MI- or B1-infected nymphs. Arrows indicate reactivity with p39.

FIG. 7.

Whole-cell lysates of clones A1, B1, and C1 separated by SDS-PAGE and stained with silver. The first two lanes of each panel show lysates derived from cultures grown in vitro at 23°C and after temperature upshift to 35°C, respectively. The third lane of each panel shows lysates derived from spirochetes grown in DMCs. Closed arrow, OspA; open arrow, OspC. Molecular masses are indicated on the left.