Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay.
Understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. Using non-denaturing separations and mass spectrometry identification, in combination with a colorimetric screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins from the D. reducens proteome not previously characterized as iron reductases. Their function was confirmed by heterologous expression in Escherichia coli. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. The proteins identified are NADH : flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase flavin adenine dinucleotide/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble protein fraction, suggesting a type of membrane association, although PSORTb predicts both proteins are cytoplasmic. This study is the first functional proteomic analysis of D. reducens and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.
Department of Microbiology, Cornell University, Ithaca, NY, USA.
Anne Elyse
AE
Otwell
Department of Proteomics and Mass Spectrometry Facility, Cornell University, Ithaca, NY, USA.
Robert W
RW
Sherwood
Department of Proteomics and Mass Spectrometry Facility, Cornell University, Ithaca, NY, USA.
Sheng
S
Zhang
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Ornella D
OD
Nelson
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Zhi
Z
Li
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Hening
H
Lin
Pacific Northwest National Laboratory, Richland, WA, USA.
Stephen J
SJ
Callister
Department of Civil and Environmental Engineering, Cornell University, Ithaca, NY, USA.
Ruth E
RE
Richardson
D045859:Desulfotomaculum / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D038181:FMN Reductase / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D020543:Proteome / Q000378:metabolism; D040901:Proteomics
D005290:Ferric Compounds; D008670:Metals; D020543:Proteome; D009243:NAD; D038181:FMN Reductase; C023903:ferric citrate iron reductase