Generation of Mutant Strains.

All mutant strains were generated in the M. tuberculosis Erdman background. Mtb was first transformed with an attL5-integrating plasmid expressing glcB under the control of its native promoter. In this merodiploid strain, the native copy of glcB was replaced with a hygromycin resistance by homologous recombination. This att-site mutant was then used to generate both the glcB-DUC and glcB knockout strains. To generate glcB-DUC, the att-site mutant was transformed with an att-site–integrating plasmid expressing glcB C-terminally tagged with DAS+4 under the control of a Tet-OFF promoter (P750) controlled by the tetracycline repressor T38S38, and also a tweety-site integrating plasmid expressing sspB under the control of a Tet-ON promoter (P1) controlled by TSC10M repressor as described (21). The glcB knockout strain (ΔglcB) was generated from the att-site mutant by replacement transformation of the glcB-expressing plasmid in the attL5 site with a plasmid lacking glcB. The complemented strain was generated by transforming ΔglcB with an attL5-site–integrating plasmid expressing glcB under the control of the native glcB promoter. All vectors used for mutant generation and complementation were constructed using Gateway Cloning Technology (Invitrogen).