Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Fevzi Daldal

Abstract

Acquisition, delivery and incorporation of metals to their respective metalloproteins are important cellular processes. These processes are tightly controlled so that cells are not exposed to free metal concentrations that would lead to harmful oxidative damages. Cytochrome c oxidases (Cox) are among these metalloproteins whose assembly and activity involves incorporation of Cu cofactor into their catalytic subunits in addition to the maturation of other subunits. In this study, we focused on the pathways of acquisition of Cu by the facultative phototroph Rhodobacter capsulatus for incorporation into the heme–Cu binuclear center of its cbb3–type Cox (cbb3–Cox). Genetic screens identified a cbb3–Cox defective mutant that requires Cu2+ supplement to produce an active cbb3–Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3–Cox biogenesis in R. capsulatus. In the absence of CcoA, cellular content of Cu decreases, and cbb3–Cox assembly and activity becomes defective. CcoA shows pronounced homology to Major Facilitator Superfamily (MFS) type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein was implicated in cbb3–Cox biogenesis. In order to dissect the mechanism of Cu acquisition in the absence of CcoA, we isolated ΔccoA mutants that were cbb3–Cox defective after addition of Cu. Characterization of these mutants by genetic complementations revealed mutations in cytochrome c maturation (CCM) genes. These mutants were able to grow photosynthetically on the contrary to the usual phenotype of CCM genes deletion mutants. Here we show that these mutations are not directly involved in the Cu trafficking to CcoN but involved in the production of membrane bound cytochrome c subunits of cbb3–Cox. Although this study provides additional information about CCM system in R. capsulatus, the additional pathways of Cu acquisition to cbb3–Cox in the presence of exogenous Cu still remains to be identified. In the future, determination of ccoA bypass mutations will provide novel insights on the maturation and assembly of membrane-integral metalloproteins, and on hitherto unknown function(s) of MFS type transporters in bacterial Cu acquisition.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS