Dissertation/Thesis Abstract

Characterization Of Periplasmic High-Affinity Zinc Uptake Proteins, and Their Mechanisms of Zinc Transfer and Regulation in Paracoccus Denitrificans
by Neupane, Durga, Ph.D., New Mexico State University, 2018, 199; 10980212
Abstract (Summary)

In bacteria, ATP binding cassette transporters (ABC) of cluster A-I exist for the acquisition of an essential trace element zinc from limiting environments. These transporters are crucial for bacterial survival, and the gene knockout strains often demonstrate a growth defect in minimal media and attenuated virulence in many pathogenic bacteria. Thus, such systems are attractive targets for the development of novel antibiotics. This study characterizes the zinc import machinery of Paracoccus denitrificans as a model for highly homologous systems in pathogenic bacteria including the carbapenem resistant Enterobacteriaceae species (CRE) Klebsiella pneumoniae and Citrobacter koseri. The genomes of these bacteria encode two zinc-specific ABC transporters called ZnuABC and AztABCD. Specific and high affinity zinc binding is mediated by solute binding protein (SBP) components of ABC transporters, AztC and ZnuA respectively.

Here, RNA-Seq and real time PCR methods demonstrated that expression of both transporter systems responded to low zinc level. Electrophoretic mobility shift assay (EMSA) suggested that these transporters were regulated by zinc-dependent zinc uptake regulator. A gene deletion knockout experiment demonstrated that either ZnuA or AztC transporter is sufficient and essential for the uptake of zinc from low zinc environment.

The zinc specific SBP transporters are characterized by the presence of a flexible loop rich in His or acidic amino acid residues near zinc binding pocket of the protein. The flexible loop is an unusually long in ZnuA while it is relatively short in AztC. The function of this structure was not clear. Here, we employed different biophysical methods to evaluate the roles of this loop of both SBPs. The results showed that the flexible loop of AztC has no impact on zinc binding affinity, stoichiometry or protein structure, yet it is essential for zinc transfer from the metallochaperone AztD. The roles of specific residues in this process were also evaluated. In ZnuA, the flexible loop provides multiple additional zinc binding sites, possibly fulfilling a zinc storage function. AztD does not transfer zinc to ZnuA. The results demonstrate distinct roles for zinc SBP flexible loops depending on loop structure and context, potentially allowing for acquisition of zinc from different environments.

Indexing (document details)
Advisor: Yukl, Erik T
Commitee: Houston, Kevin D, Mera, Paola E, Nishiguchi, Michele K
School: New Mexico State University
Department: Chemistry And Biochemistry
School Location: United States -- New Mexico
Source: DAI-B 81/10(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry, Molecular biology
Keywords: ABC transporters, Crystal structure, Gene Knockout, Metallochaperone, RNA-Seq, Zinc
Publication Number: 10980212
ISBN: 9781658498302
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