Analysis of novel zinc-binding proteins in the cell wall of Corynebacterium diphtheriae

Zinc is a critical nutrient for all living organisms, including bacterial pathogens such as Corynebacterium diphtheriae, the causative agent of the severe human respiratory disease diphtheria. As such, zinc acquisition is essential for many pathogens to cause disease. We previously showed that the zinc-regulated ABC transporter encoded by the znu locus is one of several zinc uptake systems that support the growth of C. diphtheriae in zinc-limited medium. In this study, we examine the function and cellular localization of components encoded by the znu gene cluster, which includes the ZnuABC transporter and the novel membrane and cell wall proteins ZnuE, ZnuF, and ZnuG. Deletion of znuE results in reduced growth in zinc-limited media, suggesting a role in zinc uptake. While the growth of znuF and znuG mutants was not affected in zinc-limited medium, deletion of these two genes in a mutant also lacking the znuABC transporter restored growth to wild-type levels, suggesting that ZnuF and ZnuG exert an unusual modulating effect on zinc import. ZnuE, ZnuF, and ZnuG possess a unique motif that is associated with Zn binding as demonstrated by thermal shift assays, targeted mutagenesis, and structural analysis. Although ZnuF and ZnuG are both present in the cell wall, only ZnuG contains a sortase recognition signal that is critical for localizing proteins to the cell wall. Furthermore, ZnuF localization does not require any of the six known sortase enzymes in C. diphtheriae, suggesting a novel localization mechanism.

Zinc is a critical nutrient required by many bacterial pathogens. While the function of multiple zinc importer systems has been previously characterized in Corynebacterium diphtheriae, the transporter encoded by the znu gene cluster includes components not found in other metal transport systems. In this report, we examined the roles of three components of the znu gene cluster, ZnuE, ZnuF, and ZnuG, and show that these proteins all possess a putative zinc-binding domain and have varying effects on growth in zinc-limited medium. Additionally, ZnuF uses a novel mechanism for cell wall localization. This study further expands our understanding of C. diphtheriae zinc import and points to a potentially novel mechanism for the localization of cell wall proteins.