Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding by ZinT, zinc transport and zinc-independent proteins.
Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) 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(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
Alison I
AI
Graham
Stuart
S
Hunt
Sarah L
SL
Stokes
Neil
N
Bramall
Josephine
J
Bunch
Alan G
AG
Cox
Cameron W
CW
McLeod
Robert K
RK
Poole
D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008828:Microbiological Techniques; D020411:Oligonucleotide Array Sequence Analysis; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000172:deficiency / Q000378:metabolism*
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D012269:Ribosomal Proteins; C575805:YkgM protein, E coli; C470844:ZinT protein, E coli; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D015032:Zinc; D016257:Fura-2