Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c-type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c-type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

Direct interspecies electron transfer (DIET) has been considered as an effective mechanism for interspecies electron exchange in microbial syntrophy. Understanding DIET-capable syntrophic associations under energy-limited environments is important because these conditions more closely approximate those found in natural subsurface environments than in the batch cultures in the laboratory. This study, investigated the metabolic dynamics and electron transfer mechanisms in DIET-capable syntrophic coculture of Geobacter metallireducens and Geobacter sulfurreducens under electron donor-limited condition. The wild-coculture and the mutant-coculture with a citrate synthase-deficient G. sulfurreducens exhibited similar rates of syntrophic metabolism under ethanol-limited and ethanol-replete conditions. Transcriptomic analyses revealed that, in the mutant-coculture in which interspecies electron exchange was the sole electron source for G. sulfurreducens, the transcription of genes associated with uptake hydrogenase in G. sulfurreducens were significantly repressed and thus DIET tended to be the preferred mode of interspecies electron exchange under electron donor-limited condition. To overcome electron donor limitation, c-type cytochromes in the coculture actively moved from outer membrane to extracellular environment, potentially via increased secretion of outer-membrane vesicles. These results suggested a preferred electron transfer mechanism for DIET-capable syntrophic communities\' survival in the electron donor-limited environments, providing valuable insights into the biogeochemical processes mediated by DIET in natural and engineered environments.

Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.

Dissimilatory metal-reducing bacteria (DMRB) can transfer electrons to extracellular insoluble electron acceptors and play important roles in geochemical cycling, biocorrosion, environmental remediation, and bioenergy generation. c-type cytochromes (c-Cyts) are synthesized by DMRB and usually transported to the cell surface to form modularized electron transport conduits through protein assembly, while some of them are released as extracellularly free-moving electron carriers in growth to promote electron transport. However, the type of these released c-Cyts, the timing of their release, and the functions they perform have not been unrevealed yet. In this work, after characterizing the types of c-Cyts released by Geobacter sulfurreducens under a variety of cultivation conditions, we found that these c-Cyts accumulated up to micromolar concentrations in the surrounding medium and conserved their chemical activities. Further studies demonstrated that the presence of c-Cyts accelerated the process of microbial extracellular electron transfer and mediated long-distance electron transfer. In particular, the presence of c-Cyts promoted the microbial respiration and affected the physiological state of the microbial community. In addition, c-Cyts were observed to be adsorbed on the surface of insoluble electron acceptors and modify electron acceptors. These results reveal the overlooked multiple roles of the released c-Cyts in acting as public goods, delivering electrons, modifying electron acceptors, and even regulating bacterial community structure in natural and artificial environments.

Although it has been established that electron mediators substantially promote extracellular electron transfer (EET), electron shuttling pathways are not fully understood. Here, a new electron shuttling pathway was found in the EET process by Shewanella oneidensis MR-1 with resazurin, a lipophilic electron mediator. With resazurin, the genes encoding outer-membrane cytochromes (mtrCBA and omcA) were downregulated. Although cytochrome deletion substantially reduced biocurrent generation to 1-12% of that of wild-type (WT) cells, the presence of resazurin restored biocurrent generation to 168 μA·cm-2 (ΔmtrA/omcA/mtrC), nearly equivalent to that of WT cells (194 μA·cm-2), indicating that resazurin-mediated electron transfer was not dependent on the Mtr pathway. Biocurrent generation by resazurin was much lower in ΔcymA and ΔmtrA/omcA/mtrC/fccA/cctA mutants (4 and 6 μA·cm-2) than in WT cells, indicating a key role of FccA, CctA, and CymA in this process. The effectiveness of resazurin in EET of Mtr cytochrome mutants is also supported by cyclic voltammetry, resazurin reduction kinetics, and in situ c-type cytochrome spectroscopy results. The findings demonstrated that low molecular weight, lipophilic electron acceptors, such as phenoxazine and phenazine, may facilitate electron transfer directly from periplasmic and inner membrane proteins, thus providing new insight into the roles of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.

The sulfate-reducing microbe Desulfovibrio ferrophilus is of interest due to its relatively rare ability to also grow with Fe(III) oxide as an electron acceptor and its rapid corrosion of metallic iron. Previous studies have suggested multiple agents for D. ferrophilus extracellular electron exchange including a soluble electron shuttle, electrically conductive pili, and outer surface multiheme c-type cytochromes. However, the previous lack of a strategy for genetic manipulation of D. ferrophilus limited mechanistic investigations. We developed an electroporation-mediated transformation method that enabled replacement of D. ferrophilus genes of interest with an antibiotic resistance gene via double-crossover homologous recombination. Genes were identified that are essential for flagellum-based motility and the expression of the two types of D. ferrophilus pili. Disrupting flagellum-based motility or expression of either of the two pili did not inhibit Fe(III) oxide reduction, nor did deleting genes for multiheme c-type cytochromes predicted to be associated with the outer membrane. Although redundancies in cytochrome or pilus function might explain some of these phenotypes, overall, the results are consistent with D. ferrophilus primarily reducing Fe(III) oxide via an electron shuttle. The finding that D. ferrophilus is genetically tractable not only will aid in elucidating further details of its mechanisms for Fe(III) oxide reduction but also provides a new experimental approach for developing a better understanding of some of its other unique features, such as the ability to corrode metallic iron at high rates and accept electrons from negatively poised electrodes. IMPORTANCE Desulfovibrio ferrophilus is an important pure culture model for Fe(III) oxide reduction and the corrosion of iron-containing metals in anaerobic marine environments. This study demonstrates that D. ferrophilus is genetically tractable, an important advance for elucidating the mechanisms by which it interacts with extracellular electron acceptors and donors. The results demonstrate that there is not one specific outer surface multiheme D. ferrophilus c-type cytochrome that is essential for Fe(III) oxide reduction. This finding, coupled with the lack of apparent porin-cytochrome conduits encoded in the D. ferrophilus genome and the finding that deleting genes for pilus and flagellum expression did not inhibit Fe(III) oxide reduction, suggests that D. ferrophilus has adopted strategies for extracellular electron exchange that are different from those of intensively studied electroactive microbes like Shewanella and Geobacter species. Thus, the ability to genetically manipulate D. ferrophilus is likely to lead to new mechanistic concepts in electromicrobiology.

Anode performance has been regarded as a crucial factor determining long-term stability and electricity generation of microbial fuel cells (MFCs), which restricts by the difficult extracellular electron transfer (EET) on the microbe/anode interface. Herein, inspired by biological enzyme systems, this study synthesized the biomimetic nanozymes with Fe-N-S-C active sites as the anode materials of MFCs, which was similar to the hemes of c-type cytochromes (c-Cyts) for boosting EET process. As excepted, an obviously faster start-up and a much higher power density were achieved by the MFCs equipped with Fe-N-S-C nanozymes (startup time, 3.5 d; power density, 2366 ± 34 mW m-2) than that based on traditional carbon cloth (startup time, 5.6 d; power density, 1009 ± 26 mW m-2). Such unique features of Fe-N-S-C nanozymes anode not only greatly favored the bacterial adhesion and the electroactive bacteria enrichment on the anode surface, but also efficiently facilitated the EET process between the electroactive bacteria and anode surface. This study provided a feasible strategy for designing the novel MFC anode materials from the perspective of bionic enzyme.

The interaction of bacteria and archaea with electrodes is a relatively new research field which spans from fundamental to applied research and influences interdisciplinary research in the fields of microbiology, biochemistry, biotechnology as well as process engineering. Although a substantial understanding of electron transfer processes between microbes and anodes and between microbes and cathodes has been achieved in mesophilic organisms, the mechanisms used by microbes under extremophilic conditions are still in the early stages of discovery. Here, we review our current knowledge on the biochemical solutions that evolved for the interaction of extremophilic organisms with electrodes. To this end, the available knowledge on pure cultures of extremophilic microorganisms has been compiled and the study has been extended with the help of bioinformatic analyses on the potential distribution of different electron transfer mechanisms in extremophilic microorganisms.