Construction and Validation of Gene Knockouts in BCG

Three genes that had not previously been ascribed virulence roles were chosen to test the tranposon-library based predictions. The genes were chosen to fulfil two roles: to confirm the tranposon predictions and to positively identify new virulence factors. We picked pyruvate carboxylase with a well-established function; a cyclopropane-fatty-acyl-phospholipid synthase, with a poorly annotated function and a hypothetical gene with no annotated function. These were ranked 131st, 23rd and 89th most attenuated in vivo in BCG Danish (by fold change) out of 274 attenuating genes. To produce the knockout strains we constructed zeomycin and kanamycin resistant derivatives of pYUB854 (Hyg) (Bardarov et al, 2002), pANE001 (Zeo) and pANE002 (Kan). An inverse PCR using primers pYUB_inv_F and pYUB_inv_R was used to amplify the plasmid backbone of pYUB854 less the hygromycin cassette, and to add Nde1 and Mfe1 restriction ends. Zeomycin and kanamycin cassettes were amplified from plasmids pNCMTB and pMV306 [78 (link)] respectively, using primers, Zeo_casset_F/R and Kan_casset_F/R containing Nde1 and Mfe1 restriction sites at their 3′ ends. The antibiotic cassettes were then cloned into the pYUB854 backbone to give pANE001 and pANE002, and the constructs confirmed by Sanger sequencing. Genomic sequence upstream (LF) and downstream (RF) of the genes to be knocked out were PCR amplified using primers BCG2988_RF_R, BCG2988RF_L, etc. and cloned either side of the antibiotic cassettes of the cosmids pYUB854, pANE001, pANE002 and the apramycin resistant cosmid p0004S (a gift from W. R. Jacobs Jr). Transducing phage were constructed and transduced into BCG using the pHAE159 mycobacteriophage-based method of specialized transduction (Bardarov et al, 2002). The knockouts were confirmed by PCR using primers outside of the upstream and downstream flanking regions both alone and in combination with antibiotic cassette specific primers, such that PCR products would only be obtained if the antibiotic cassettes were in the predicted configuration. Primer sequences are listed in Additional file 1: Table S1.