Two ABC Transporters and a Periplasmic Metallochaperone Participate in Zinc Acquisition in Paracoccus denitrificans

Abstract

Bacteria must acquire the essential element zinc from extremely limited environments, and this function is performed largely by ATP binding cassette (ABC) transporters. These systems rely on a periplasmic or extracellular solute binding protein (SBP) to bind zinc specifically with a high affinity and deliver it to the membrane permease for import into the cytoplasm. However, zinc acquisition systems in bacteria may be more complex, involving multiple transporters and other periplasmic or extracellular zinc binding proteins. Here we describe the zinc acquisition functions of two zinc SBPs (ZnuA and AztC) and a novel periplasmic metallochaperone (AztD) in Paracoccus denitrificans. ZnuA was characterized in vitro and demonstrated to bind as many as 5 zinc ions with a high affinity. It does not interact with AztD, in contrast to what has been demonstrated for AztC, which is able to acquire a single zinc ion through associative transfer from AztD. Deletions of the corresponding genes singly and in combination show that either AztC or ZnuA is sufficient and essential for robust growth in zinc-limited media. Although AztD cannot support transport of zinc into the cytoplasm, it likely functions to store zinc in the periplasm for transfer through the AztABCD system.

Figure 1

A simplified model for zinc acquisition and homeostasis through ABC transporters in P. denitrificans. Arrows show the direction of zinc transport. The flexible loops for the SBPs AztC and ZnuA are indicated in the figure, and their sequences are given. Underlined His residues in the AztC loop are required for zinc acquisition from the metallochaperone AztD.

Figure 2

Purification of WT and Δloop ZnuA. (A) SDS-PAGE gel: lane 1, MW standard; lane 2, purified WT ZnuA; lane 3, purified Δloop ZnuA. Size exclusion chromatograms of (B) WT and (C) Δloop ZnuA. Predicted MW refers to that determined from the primary sequence, while calculated MW is determined from elution time in comparison with a set of known standards.

Figure 3

Zinc binding by WT and Δloop ZnuA by MF-2 competition assay. (A) Example of a MF-2 assay containing 0.5 μM MF-2 and 1.0 μM apo-WT ZnuA. Arrows indicate the direction of fluorescence changes upon titration with increasing zinc. Intensity change at 330 nm with increasing zinc in the absence (solid circles) and presence (empty circles) of (B) apo-WT ZnuA and (C) Δloop ZnuA. Titrations containing WT or Δloop ZnuA were performed in triplicate, and error bars represent the standard error between experiments. Fits are shown as solid lines.

Figure 4

Zinc binding by WT and Δloop ZnuA by ITC. ITC isotherms and integrated heats of titration of 30 μM WT ZnuA (A and B), Δloop ZnuA (C and D), or a buffer blank (E and F) titrated with 3.0 mM ZnCl₂. Solid lines represent fits to the data resulting in binding parameters listed in Table 2.

Figure 5

Zinc transfer from AztD to WT apo-ZnuA. (A) Apo-WT ZnuA, (B) holo-AztD, and (C) an equimolar mixture were applied to an ion exchange column, and the protein eluted with increasing [NaCl]. The protein was detected by absorbance at 280 nm and converted to concentration using extinction coefficients (solid line) as described in above. One mL fractions were collected and analyzed for zinc content (filled squares). (D) Fractions from C were run on SDS-PAGE and bands compared with zinc content.

Figure 6

(A, B, and C) Growth curves and (D, E, and F) quantitation of cellular zinc for WT and mutant P. denitrificans strains grown in zinc-repleted (A and D, black bars), zinc-limited (B and E, gray bars), and zinc-chelated (C and F, white bars) media. Growth data are presented as the average values for duplicate experiments with error bars representative of the standard deviation. Zinc quantitation is presented as the average values for triplicate experiments with error bars representative of the standard error between replicates. TukeyHSD test was used to evaluate statistical significance of differences. For growth curves, OD₆₀₀ of each mutant strain was compared to WT. Zinc content was compared between strains grown in a given condition. Significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ND = not detected, indicating that zinc content was below the detection limit, which for these samples is approximately 2 ng/mg dry cell weight.

Figure 7

Quantitation of cellular (A) iron and (B) manganese for WT and mutant P. denitrificans strains in zinc-repleted (black bars), zinc-limited (gray bars) and zinc-chelated (white bars) media. Data are presented as the average values for triplicate experiments with error bars representative of the standard error between replicates. TukeyHSD test was used to evaluate statistical significance of differences in metal content as follows: *p < 0.05, **p < 0.01, ***p < 0.001.