Periplasmic Protein Mobility for Extracellular Electron Transport in Shewanella oneidensis

Abstract

Extracellular electron transport (EET) supports the survival of specific microorganisms on the Earth's surface by facilitating microbial respiration with diverse electron acceptors. A key aspect of EET is the organization of electron relays, i.e., multi-heme c-type cytochromes (MHCs), within the periplasmic space of microbial cells. In this study, we investigated the mobility of periplasmic electron relays in Shewanella oneidensis MR-1, a model strain capable of EET, using in vivo protein crosslinking to the MHCs. First, we established that crosslinking efficiency correlates with the spatial proximity and diffusion coefficient of protein molecules through in vitro tests. Based on these findings, we identified distinct molecular behaviors of periplasmic MHCs, showing that the tetraheme flavocytochrome FccA, which also serves as a periplasmic fumarate reductase, forms protein complexes with limited motility, while the small tetraheme c-type cytochrome CctA remains discrete and mobile. Both MHCs contribute to EET for bioelectrochemical nitrate and nitrite reduction. These findings reveal dual mechanisms for organizing periplasmic electron relays in EET, advancing our understanding of microbial extracellular respiration.

Keywords: bioelectrochemistry; c-type cytochrome; crosslinking; extracellular electron transport; formaldehyde; microbial respiration.

Figure 1

Diagrams on the research contents of this work. (a) Cartoon showing the proposed dynamic model of periplasmic electron transport through rolling CctA or FccA. Solid arrows indicate the directions of electron transport. Dashed arrows indicate the directions of protein movement. (b) The procedure designed for the in vivo study of periplasmic protein mobility in S. oneidensis.

Figure 2

SDS-PAGE and heme-staining results from a formaldehyde-treated periplasmic fraction (a) under protein concentrations of 4.0 mg/mL (5×) and 0.8 mg/mL (1×), and (b) under different-viscosity solutions (1 M or 0 M sucrose was added). Each lane of gels was loaded with 2 µg protein. CL+: crosslinked; CL−: non crosslinked. The staining intensity of the CL+ lanes was plotted. Rectangles and green arrows mark the produced and vanished bands during crosslinking, respectively. Data are representative of triplicate SDS-PAGE experiments.

Figure 3

Size distribution of protein complexes after in vivo crosslinking. (a) SDS-PAGE of cell lysates of CL− and CL+ (10, 30, and 60 min). Each lane was loaded with 5 µg proteins. Rectangles and green arrows mark the produced and vanished bands during crosslinking, respectively. (b) SDS-PAGE results for periplasmic fractions extracted from CL− and CL+ (30 min) cells. (c) SDS-PAGE results for total soluble proteins extracted from CL− and CL+ (30 min) cells. (d) SEC separation to periplasmic fraction extracted from CL− and CL+ (30 min) cells. Curves are normalized to equal heme content. (e) SDS-PAGE results for CL− subfractions marked in (d). Data are representative of triplicate SDS-PAGE and SEC experiments.

Figure 4

Size distribution of FccA and CctA_6×His in crosslinked samples. (a) Specific activity of fumarate reductases in periplasmic and membrane fractions. (b) Activity of fumarate reductases in subfractions of SEC elutes, Figure 3d. Error bars in (a,b) indicate the standard error of triplicate assays. (c) Concentrations of fumarate reductases in subfractions of SEC elutes, Figure 3d. (d) SEC elutes of CctA_6×His-containing proteins. Duplicate SEC experiments were performed and the results are consistent.

Figure 5

Bioelectrochemical reduction of nitrate and nitrite by crosslinked EET system. (a,b) Cyclic voltammagrams exhibited the nitrate (a) and nitrite (b) reduction activities by the WT biofilm before (CL−) and after crosslinking (CL+). –Acceptor: electron acceptor-free conditions. (c,d) Cyclic voltammagrams exhibited the nitrate (c) and nitrite (d) reduction activities by the ΔcctA biofilm before (CL−) and after crosslinking (CL+). Triplicate biofilms of WT and ΔcctA strains were tested and the representative data are shown.