Figure 1.

Isolation and confirmation of bb0646::gent^R mutants in B. burgdorferi B31 derivative, ML23. (A) Schematic diagram of the bb0646 insertional mutation of DS102. bb0646 was interrupted using a P_flgB-gent^R cassette at a unique NsiI restriction site within the gene. Constructs were transformed into ML23 and putative positive clones were screened by PCR using the indicated primers, 1–4, in panel B. (B) Putative transformants were screened with 3 sets of primers: DS111F/R (1 and 2), DS111-NdeI-F/gent_int- R (1 and 4), and DS111-NdeI-R/gent_int-F (2 and 3). (see Table 2 for primers used). Values shown on the left represent markers (in bp). (C) Southern blot confirmed the presence of the bb0646::gent^R allele via hybridization with a gent^R probe. DNA from B. burgdorferi strains ML23 and DS102 was digested with EcoRV and RsaI as indicated (restriction enzyme sites shown in panel A). Values shown on the left represent markers (in bp). (D) Western blot analysis demonstrated that putative transformants made no detectable BB0646 protein compared to the isogenic parent strain ML23 pBBE22 when B. burgdorferi lysates were analyzed by immunoblot analysis with polyclonal antibody specific for BB0646. An asterisk (*) denotes a non-specific, cross-reactive band that anti-BB0646 recognizes in all isolates. Values shown on the left represent markers (in kDa).