Figure 1. ZrlA Is a Peptidase Induced during Zn Starvation.

(A) ZrlA expression was assessed in wild-type (WT) and ΔzrlA (15 μg protein/lane) by immunoblot after growth in lysogeny broth (LB) ± 40 μM TPEN.

(B) Transcriptional changes for zrlA and other predicted d,d-CPases A1S_1248, A1S_2435, and A1S_2479 were assessed by qRT-PCR + 250 μg/ml CP. **p < 0.01 as determined by one-way ANOVA with Tukey multiple comparisons test on three independent experiments, means ± SD.

(C) Modified cadmium-ninhydrin assay was performed on recombinant MBP-ZrlA in the presence of a peptide substrate. *p < 0.05, ***p < 0.001 as determined by one-way ANOVA with Tukey multiple comparisons test on three independent experiments, means ± SD.

(D) ZrlA protein localization was assessed in WT and ΔzrlA (7.5 μg protein/lane) by immunoblot of membrane fractions following growth in LB ± 40 μM TPEN.

(E) Co^II titration, where a tetrahedral or distorted tetrahedral coordination geometry is suggested with a d-d transition of 400 M⁻¹ • cm⁻¹ at 600 nm (Corwin and Koch, 1987). Inset, number of Co^II:ZrlA^42C mol • equivalents corresponding to each spectrum shown; note that all spectra acquired at Co^II:ZrlA^42C overlap, revealing a 1:1 metal binding stoichiometry.

(F) Normalized binding titration of a mixture of ZrlA^42C and competitor quin-2 with Zn in two independent experiments: 17.0 μM ZrlA^42C with 15.2 μM quin-2 (blue filled circles) and 15.1 μM ZrlA^42C with 12.3 μM quin-2 (black filled circles). The continuous lines represent the results of a nonlinear, least-squares fit to a Zn:ZrlA^42C = 1:1 binding model. The global fitting results in K_Zn = 3.6 (±0.4) × 10¹¹ M^{− 1}.

See also Figure S1.