Thrust Area 3:Mutagenesis and Functional Characterization of Shewanella oneidensis Genes Involved in Fe(III) and Mn(IV) Oxide Reduction (LDRD #3)
EMSL Project ID
13994
Abstract
Although microbial metal reduction has been extensively studied from standpoints of ecology, physiology, genetics and biochemistry, little is known about the molecular mechanisms and pathways of dissimilatory metal reduction. Therefore the goal of the proposed work is to investigate the role of multiheme cytochromes and other proteins associated with the outer membrane of Shewanella oneidensis MR-1 in the reduction of Fe(III) and Mn(IV) oxides, especially during the terminal electron transfer steps. Using a genetic system specifically developed in our group, we will generate S. oneidensis mutants deficient in metal reduction including those lacking key metal reduction genes. The resulting strains will be functionally characterized using standard physiological tests in conjunction with state-of-the-art microscopic and spectroscopic approaches to evaluate whole-cell electron transfer rates and controlling factors. The obtained information will be complemented by immunohistochemical studies which will be used to identify the localization and interactions of the specific components of the metal-reducing chain in both wild-type and mutant strains. The proposed work is a part of a multi-investigator EMSL Biogeochemistry Grand Challenge (BGC) project focused on understanding the mechanism of interactions at the microbe-mineral interface.Summary of Expected Results
The proposed study is expected to contribute to our understanding of molecular mechanisms involved in the electron transfer and reduction of insoluble metal electron acceptors in S. oneidensis. Towards this goal, we will generate an extensive library of knockout mutants deficient in outer membrane proteins (MtrB), decaheme cytochromes (MtrA, MtrC, and OmcA) as well as protein trafficking and localization (LolABC and sortase). The role of each gene/protein will be elucidated by conducting physiological and biochemical comparisons between the respective mutant(s) and the wild-type MR-1 strain. In addition, we will introduce point mutations to identify structural/functional motifs and/or domains of these proteins essential for the functioning of the metal-reducing electron transport chain. Finally, we will generate polyclonal (IgG) antibodies generated against surface epitopes of MtrC, MtrA and MtrB proteins. Upon initial specificity and sensitivity tests, the IgGs will be used to study the subcellular localization, association and outer membrane architecture in both wild-type and mutant strains using various immunohistochemical methods. The resources generated in the course of this work including mutant strains and antibodies will be made available to all members of the BGC team.
For the first year of this project, the expected outcomes are:
1. Generation and partial characterization of S. oneidensis knockout mutants lacking one or more genes that encode outer membrane proteins (mtrB and mtrE) and cytochromes (mtrD, mtrF, omcA, mtrC, and mtrA).
2. Generation and partial characterization of S. oneidensis mutants deficient in lipoprotein trafficking and sortase genes.
3. Identification of functional/structural domains in MtrB essential for metal reduction (electron transfer).
4. Generation and evaluation of polyclonal antibodies recognizing surface-exposed epitopes of MtrA, MtrB, and MtrC.
Project Details
Project type
Grand Challenge
Start Date
2005-03-02
End Date
2008-10-01
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members