Enrichment of functional redox reactive proteins and identification by mass spectrometry results in several terminal Fe(III)-reducing candidate proteins in Shewanella oneidensis MR-1.
Identification of the proteins directly involved in microbial metal-reduction is important to understanding the biochemistry involved in heavy metal-reduction/immobilization and the ultimate cleanup of DOE contaminated sites. Although previous strategies for the identification of these proteins have traditionally required laborious protein purification/characterization of metal-reducing capability, activity is often lost before the final purification step, thus creating a significant knowledge gap. In the current study, subcellular fractions of Shewanella oneidensis MR-1 were enriched for Fe(III)-NTA reducing proteins in a single step using several orthogonal column matrices. The protein content of eluted fractions that demonstrated activity was determined by ultra-high pressure liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A comparison of the proteins identified from active fractions in all separations produced 30 proteins that may act as the terminal electron-accepting protein for Fe(III)-reduction. These include MtrA, MtrB, MtrC and OmcA as well as a number of other proteins not previously associated with Fe(III)-reduction. This is the first report of such an approach where the laborious procedures for protein purification are not required for identification of metal-reducing proteins. Such work provides the basis for a similar approach with other cultured organisms as well as analysis of sediment and groundwater samples from biostimulation efforts at contaminated sites.
Elias
Dwayne A
DA
Department of Biochemistry, University of Missouri-Columbia, United States.
Yang
Feng
F
Mottaz
Heather M
HM
Beliaev
Alexander S
AS
Lipton
Mary S
MS
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D005290:Ferric Compounds / Q000378:metabolism*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010084:Oxidation-Reduction; D020592:Shewanella / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D001425:Bacterial Outer Membrane Proteins; D005290:Ferric Compounds; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate