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. 2007 Aug 6;75(11):5376–5389. doi: 10.1128/IAI.00238-07

FIG. 7.

FIG. 7.

Complementation of F. tularensis LVS FTL_0706::HimarFT mutant strains in trans using pFTNAT-FTL_0706. (A) Two mutants containing HimarFT insertions within FTL_0706 in opposite orientations were assessed for complementation (locations are indicated by the gene designation and candidate number). LVS FTL_0706-1:: HimarFT contains the groEL promoter (pgro) in the same orientation as FTL_0706. F and R indicate the DNA fragment amplified from the genome, and the EcoRI sites indicate the fragment used for complementation. (B) Western blot analysis of cell lysates from LVS and LVS FTL_0706::HimarFT strains using a mouse monoclonal anti-F. tularensis LPS antibody. The strains analyzed include LVS without (lane 1) or with (lane 2) pFTNAT; LVS FTL_0706-1::HimarFT alone (lane 3), with pFTNAT (lane 4), and with pFTNAT-FTL_0706 (lane 5); and FTL_0706-2::HimarFT alone (lane 6), with pFTNAT (lane 7), and with pFTNAT-FTL_0706 (lane 8). (C) Cytopathic analysis with J774A.1 macrophages using crystal violet staining 2 days after infection at an MOI of 100. The retention of the monolayer after infection is compared to the uninfected control (well 1). Wells 2 and 3 contained wild-type LVS without and with pFTNAT. Wells 4 to 6 contained the F. tularensis LVS FTL_0706::HimarFT mutant candidates alone (wells 4), with pFTNAT (wells 5), and with pFTNAT-FTL_0706 (wells 6).