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This paper investigates periplasmic protein mobility in Shewanella oneidensis using formaldehyde crosslinking to understand extracellular electron transport (EET) mechanisms. The authors demonstrate that FccA (tetraheme flavocytochrome) forms immobile protein complexes with restricted motility, while CctA (small tetraheme cytochrome) remains discrete and mobile. Both proteins contribute to bioelectrochemical nitrate and nitrite reduction. Crosslinking partially inhibits these processes (36–63% activity retained in WT), revealing dual organizational strategies for periplasmic electron relays that maintain EET efficiency despite protein immobilization.

Key insights

  • Mechanism FccA forms high-MW complexes (≥150 kDa) with restricted motility, while CctA remains monomeric (13 kDa) and mobile, with only 25.2% of CctA participating in crosslinking versus extensive FccA crosslinking.

    So what. Design bioelectrochemical systems expecting FccA to function as a stationary electron conduit cluster and CctA as a diffusive shuttle; do not assume all periplasmic cytochromes behave identically.

  • Trade-off Bioelectrochemical nitrate reduction retained 36% of native activity after crosslinking in WT, but nitrite reduction retained 63%, indicating differential dependence on protein mobility between these two reduction pathways.

    So what. When operating MES systems for nitrate vs. nitrite reduction, expect nitrate reduction to be more sensitive to constraints on protein diffusion; consider this when selecting operating potentials or residence times.

  • Mechanism In the ∆cctA knockout strain, nitrate reduction retained 76% and nitrite reduction retained 99% of native activity after crosslinking, compared to 36% and 63% in WT.

    So what. CctA mobility is more critical for nitrate reduction than nitrite reduction; loss of CctA can be partially compensated by FccA-mediated pathways, especially for nitrite.

  • Mechanism Crosslinking efficiency in vitro correlated with protein concentration (5× sample showed rapid accumulation by 5 min vs. 1× sample showing only slight accumulation by 30 min) and inversely with solution viscosity (1 M sucrose reduced crosslinking substantially).

    So what. Protein mobility in bioelectrochemical systems depends on local crowding and viscosity; high cell density or biofilm matrix viscosity will reduce diffusion-limited electron transfer rates.

Opportunity areas6

Specific gaps in this paper, with an experiment that would close each.

  • High No systematic variation of cathode potential during bioelectrochemical nitrate/nitrite reduction to map the operating window where FccA-mediated (immobile) vs. CctA-mediated (mobile) pathways dominate.

    Figure 5 shows cyclic voltammetry between −0.21 and −0.50 V, but only one potential (−0.5 V) is used for activity quantification. The paper does not explore whether FccA and CctA contributions vary with potential, which would be critical for MES design.

    Try. Vary cathode potential from −0.1 to −0.6 V vs. Ag/AgCl in 0.1 V steps; measure current for NO3− and NO2− reduction in WT and ∆cctA strains; correlate activity loss after crosslinking to potential.

  • Medium No measurement of dissolved oxygen, pH, or ionic strength effects on periplasmic protein mobility and crosslinking efficiency.

    In vitro crosslinking was performed at fixed pH 7.4 and 30°C. Real bioelectrochemical systems experience pH gradients (especially at cathodes) and variable ionic strength. The paper does not test whether these conditions alter FccA/CctA mobility or their crosslinking behavior.

    Try. Repeat in vitro crosslinking at pH 6.0–8.5 and ionic strengths 0.1–1.0 M; measure crosslinking efficiency and SEC profiles; correlate to biofilm performance.

  • High No direct measurement of electron transfer rates or kinetics between FccA and its redox partners (MtrA, NapAB, NrfA) under different protein mobility states.

    The paper infers electron transport mechanisms from crosslinking patterns and biofilm activity, but does not measure electron transfer kinetics directly. This limits understanding of how much FccA immobility actually slows electron transfer vs. how much CctA mobility compensates.

    Try. Use pulse-chase or stopped-flow spectroscopy to measure electron transfer rates between FccA and MtrA, NapAB, or NrfA in native vs. crosslinked periplasmic fractions.

  • Medium No exploration of how hydraulic retention time (HRT) or current density affects the balance between FccA-mediated and CctA-mediated pathways in continuous-flow bioelectrochemical systems.

    All experiments are batch or potentiostatic biofilm cultures. MES reactors operate under continuous flow with varying HRT and current density. The paper does not test whether these operating parameters shift the relative contribution of mobile vs. immobile electron relays.

    Try. Operate continuous-flow MES at HRTs of 2, 6, 12, 24 h and current densities of 0.5–5 A/m² for NO3− reduction; measure activity in WT vs. ∆cctA; correlate to periplasmic protein composition via proteomics.

  • Medium No measurement of N2O or NO2− accumulation during bioelectrochemical nitrate reduction under different protein mobility constraints.

    The paper measures total NO3− reduction activity but does not quantify whether crosslinking (which reduces protein mobility) shifts the product distribution toward N2O or NO2− accumulation, which would be critical for MES design targeting complete denitrification.

    Try. Measure NO3−, NO2−, NO, and N2O concentrations in biofilm effluent during potentiostatic reduction at −0.5 V in WT and ∆cctA strains before and after crosslinking; use gas chromatography or ion chromatography.

  • Medium No investigation of how biofilm matrix composition or thickness affects periplasmic protein mobility and the relative contributions of FccA vs. CctA to electron transport.

    Biofilms produce extracellular polymeric substances (EPS) that alter local viscosity and diffusion. The paper does not test whether biofilm age, EPS composition, or thickness modulates protein mobility or shifts the balance between FccA and CctA pathways.

    Try. Grow biofilms for 3, 7, 14, and 21 days; measure EPS composition and biofilm thickness; perform in situ crosslinking and measure FccA/CctA mobility; correlate to NO3−/NO2− reduction rates.

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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

Shewanella oneidensisPeriplasmic spaceShewanellaExtracellularCell biologyElectron transport chain

Identifiers

DOI
10.3390/microorganisms13051144
PubMed
40431315
Journal
Microorganisms
Year
2025
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