FIG. 2.

β-Galactosidase assays. (A) C. diphtheriae C7 (wild type) and C7(β)hm723 (dtxR) carrying the mntA-lacZ fusion on plasmid pPO1 were grown overnight in HIBTW medium with the indicated supplements. +, EDDA, an iron chelator, was added at 30 μM to produce low-iron medium; Fe, 50 μM Fe²⁺ was added to the HIBTW medium containing EDDA; −, no supplement was added to the HIBTW medium (an iron-replete medium). (B) C. diphtheriae C7 carrying mnt-lacZ fusions on plasmids pPO1-pPO6 was grown in HIBTW medium in the presence and absence of 10 μM Mn²⁺. (C) C. diphtheriae C7R (mntR-) and C7A (mntA-) were grown in HIBTW medium containing the indicated supplements. (D) C. diphtheriae strains C7, C7R, and C7(β)hm723 carrying pCMtox (tox-lacZ) were grown overnight in mPGT medium containing the indicated supplements. The addition of 1 μM Fe²⁺ (Fe²⁺ 1 μM) was required for maximum growth of C. diphtheriae strains, and this is considered a low-iron concentration, since maximum derepression of the tox promoter is observed at this iron concentration. LacZ units were determined as described previously (36). Values for all assays are the averages of at least three independent experiments, and results from each experiment did not vary by >15% from the average.