Characterization of B. anthracis Branched-chain Amino Acid Transporters

DNA corresponding to the sequences of the B. anthracisbrnQ1, brnQ2, brnQ3, brnQ4, brnQ5, and brnQ6 open reading frames was amplified from ANR-1 genomic DNA using PCR with the appropriate oligonucleotides shown in Table S1. To facilitate the detection of proteins encoded by these genes, amplification products carrying a sequence encoding a FLAG tag fused to the 3′ end of the gene were also generated. The purified PCR products were digested with the SalI and BamHI restriction enzymes and finally ligated into the shuttle vector pAW285 such that expression was dependent on the xylose-inducible promoter (70 (link)). As described above, recombinant plasmids were first generated in E. coli TG1 (Table 3) and subsequently transformed into an E. coli strain lacking dam and dcm to obtain unmethylated plasmid DNA for electroporation into B. anthracis. For the expression of the cloned genes, B. anthracis cultures were grown in CA-CO₃ to an OD₆₀₀ of ∼0.3 and induced with 1% xylose. After incubation for 3 h, cells were collected, and cell lysates were prepared as described previously (5 (link)). The production of FLAG-tagged transporter proteins was assessed by Western blotting using anti-FLAG antibody (GenScript, Piscataway, NJ, USA) as described previously (5 (link)).