Severe Zinc Depletion of Escherichia coli ROLES FOR HIGH AFFINITY ZINC BINDING BY ZinT, ZINC TRANSPORTAND ZINC-INDEPENDENT PROTEINS.

Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn[sup2+] from the environment, making it exceptionally difficult to achieve Zn[sup2+] deficiency, and so a comprehensive understanding of the importance of Zn[sup2+] has not been attained. Reduction of the Zn[sup2+] content of Escherichia coli growth medium to 60 nM or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn[sup2+]-deficient medium had a reduced growth rate and contained up to five times less cellular Zn[sup2+]. To understand global responses to Zn[sup2+] deficiency, microarray analysis was conducted of cells grown under Zn[sup2+]-replete and Zn[sup2+]-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn[sup2+]-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc llptake fegulator). A mutant lacking zin Tdisplayed a growth defect and a 3-fold lowered cellular Zn[sup2+] level under Zn[sup2+] limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn[sup2+] or Cd[sup2+]. A further up-regulated gene, ykgM, is believed to encode a non-Zn[sup2+] fingercontaining paralogue of the Zn[sup2+] finger ribosomal protein L31. The gene encoding the periplasmic Zn[sup2+]-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn[sup2+] than was originally added to the culture, presumably because of leaching from the culture vessel. Zn[sup2+] elimination is shown to be a more precise method of depleting Zn[sup2+] than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. [ABSTRACT FROM AUTHOR]