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. 2015 Jul 30;290(38):23064–23076. doi: 10.1074/jbc.M115.648642

FIGURE 3.

FIGURE 3.

Construction and in vitro characterization of ΔegtD in M. tuberculosis. A, schematic diagram of the Rv3701c region of the chromosome of M. tuberculosis. Genomic DNA was digested with AflIII, and the blot was probed with a digoxigenin-11-dUTP-labeled DNA fragment containing 314 bp of the egtD 3′-flanking sequence. B, confirmation of the ΔegtD mutant through Southern blotting. AflIII-digested genomic DNA gave rise to the expected 1.77-kbp fragment in wild-type M. tuberculosis (lane 1) and 2.74-kbp fragment in the hygromycin-resistant transductant in which egtD was disrupted with the hyg marker (lane 2). C, PCR analysis of the hygromycin-resistant transductant genomic DNA for the ΔegtD. Left panel, PCR amplification of egtD (966 bp). Right panel, PCR amplification of the hygromycin-resistant cassette (∼700 bp), which replaced egtD in the mutants. D, intracellular EGT levels extracted from wild-type M. tuberculosis, ΔegtD M. tuberculosis, and ΔegtD transformed with pMV261:egtD and quantified by ESI LC-MS/MS. Error bars indicate the means ± S.D. of three independent experiments. Col, colony.