Extracellular organic disulfide reduction by Shewanella oneidensis MR-1

Abstract

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

Keywords: DTNB; Shewanella; TNB; cysteine; cystine; geomicrobiology; glutathione; organic sulfur; outer membrane; thiol.

Fig 1

DTNB reduction by the wild-type and mutant strains. (A) Reduction of DTNB to TNB by the wild-type strain in M1 medium with and without lactate. Each data point represents the average of three replicate experiments, and error bars indicate the standard deviation. (B) Overnight cultures grown in M1 lactate medium supplemented with 100 µM DTNB; wild-type (left), D10 mutant (middle), and complemented mutant D10 pdsbD (right). (C) Initial velocity of DTNB reduction for the wild-type, D10, and D10 pdsbD strains. (D) Cell pellets centrifuged; wild-type (left), D10 (middle), and D10 pdsbD (right). (E) Location of transposon insertion. The dsbD gene in yellow and the transposon in red.

Fig 2

DTNB reduction by Mtr mutant strains JG731 (ΔmtrC), JG749 (ΔmtrC/ΔomcA), JG596 (ΔmtrC/ΔomcA/ΔmtrF), and JG1453 (ΔmtrB/ΔmtrE/ΔmtrC/ΔomcA/ΔmtrF/ΔmtrA/ΔmtrD/ΔdmsE/ΔSO4360/ΔcctA). Experiments were performed in triplicate, and error bars represent the standard deviation. Statistically significant differences were observed between the WT and all mutant strains (Student’s t-test, P < 0.01).