Volume 272, Number 46, Issue of November 14, 1997 pp. 29033-29038
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of the Key Protein for Zinc Uptake in Hemophilus influenzae

(Received for publication, July 17, 1997, and in revised form, August 20, 1997)

Desheng Lu ^§ , Beth Boyd ^§ and Clifford A. Lingwood ^§¶

From the Departments of  Medical Genetics and Microbiology, Biochemistry, and ^¶ Laboratory Medicine and Pathobiology, University of Toronto, and ^§ Division of Microbiology, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

Very little is known about specific mechanisms for zinc accumulation and transport in bacteria. In this study a putative adhesin B in Hemophilus influenzae, the product of gene HI0119, has been identified as a periplasmic zinc-binding protein (PZP1). A pzp1-deficient mutant has been constructed which is defective for growth under aerobic conditions and grows poorly under anaerobic conditions. The growth defect is specifically rescued by supplementing the growth medium with high concentrations of zinc. Subcellular fractionation was used to localize PZP1 to the periplasmic region in a nontypeable H. influenzae strain and in a transfected recombinant Escherichia coli strain (TApzp1). Recombinant PZP1, purified from a periplasmic extract of E. coli strain TApzp1, contained ~two zinc atoms/protein molecule as determined by neutron activation analysis and atomic absorption spectroscopy. The zinc atoms could be removed by incubation with EDTA, and, by further addition of zinc, a total of five zinc atoms/PZP1 could be bound. Direct binding of ⁶⁵Zn to the recombinant protein by Western blot was demonstrated. Taken together, these results provide direct evidence that PZP1 plays a key role in zinc uptake by H. influenzae.

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