Figure 2. Appropriate expression of DnaA is required for B. burgdorferi replication.

(A) Growth curve studies were conducted on different B. burgdorferi strains incubated with 0.5 mM IPTG. Three independent cultures of each strain were measured for growth curve analysis. Error bars represent the SEM. The dashed line indicates when the overexpression strain resumed growth. (B) The generation time was determined from the growth curve analyses from three individual cultures of each strain. For the overexpression strain, the growth rate was determined before (early; 1–4 dpi) and after the resumption of growth (late; 4–7 dpi). The resumption of replication in that strain was evidently due to mutation of the overexpressed dnaA gene. The p-values are indicated as follows: **, p ≤ 0.01; ***, p ≤ 0.001; ns, not significant, p > 0.05. (C) IPTG-treated DnaA overexpression spirochetes that resumed growth (conditioned) lost sensitivity to IPTG when passaged to fresh media and did not overproduce 3xFLAG-DnaA as assessed by immunoblot (D), supporting the conclusion that accumulated mutations in the overexpression dnaA permitted replication. (E) Sequenced plasmid from these conditioned spirochetes showed unique mutations (S = substitution; I= Insertion; D = deletion) in the 3xFLAG-dnaA ORF that caused a frameshift and truncation of the full-length protein. The location of the truncation is indicated by *.