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Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO 3 ), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications., (© 2023 Wiley Periodicals LLC.)

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., (© 2023 Wiley Periodicals LLC.)

Geobacter species are critically involved in elemental biogeochemical cycling and environmental bioremediation processes via extracellular electron transfer (EET), but the underlying biomolecular mechanisms remain elusive due to lack of effective analytical tools to explore into complicated EET networks. Here, a simple and highly efficient cytosine base editor was developed for engineering of the slow-growing Geobacter sulfurreducens (a doubling time of 5 h with acetate as the electron donor and fumarate as the electron acceptor). A single-plasmid cytosine base editor (pYYDT-BE) was constructed in G. sulfurreducens by fusing cytosine deaminase, Cas9 nickase, and a uracil glycosylase inhibitor. This system enabled single-locus editing at 100% efficiency and showed obvious preference at the cytosines in a TC, AC, or CC context than in a GC context. Gene inactivation tests confirmed that it could effectively edit 87.7-93.4% genes of the entire genome in nine model Geobacter species. With the aid of this base editor to construct a series of G. sulfurreducens mutants, we unveiled important roles of both pili and outer membrane c -type cytochromes in long-range EET, thereby providing important evidence to clarify the long-term controversy surrounding their specific roles. Furthermore, we find that pili were also involved in the extracellular reduction of uranium and clarified the key roles of the ExtHIJKL conduit complex and outer membrane c -type cytochromes in the selenite reduction process. This work developed an effective base editor tool for the genetic modification of Geobacter species and provided new insights into the EET network, which lay a basis for a better understanding and engineering of these microbes to favor environmental applications.

Exoelectrogens are distinct from other bacteria owing to their unique extracellular electron transfer (EET) abilities that allow for anaerobic respiration with various external redox-active surfaces, including electrode and metal oxides. Although the EET process is known to trigger diverse extracellular redox reactions, the reverse impact has been long overlooked. Recent evidences show that exoelectrogens can sense the potential changes of external surfaces and alter their EET strategies accordingly, which imparts them remarkable abilities in adapting to diverse and redox-variable environment. This mini-review provides a condensed overview and critical analysis about the recent discoveries on redox-dependent EET pathways of exoelectrogens, with focus on Geobacter sulfurreducens and Shewanella oneidensis. We summarize the detailed responses of various EET components, analyze the drives and mechanisms of such responses, highlight the diversity of EET dynamics among different bacterial species and under integrated effects of redox potential and surface chemistry, and discusses the future research needs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dissimilatory metal reducer Geobacter sulfurreducens can mediate redox processes through extracellular electron transfer and exhibit potential-dependent electrochemical activity in biofilm. Understanding the microbial acclimation to potential is of critical importance for developing robust electrochemically active biofilms and facilitating their environmental, geochemical, and energy applications. In this study, the metabolism and redox conduction behaviors of G. sulfurreducens biofilms developed at different potentials were explored. We found that electrochemical acclimation occurred at the initial hours of polarizing G. sulfurreducens cells to the potentials. Two mechanisms of acclimation were found, depending on the polarizing potential. In the mature biofilms, a low level of biosynthesis and a high level of catabolism were maintained at +0.2 V versus standard hydrogen electrode (SHE). The opposite results were observed at potentials higher than or equal to +0.4 V versus SHE. The potential also regulated the constitution of the electron transfer network by synthesizing more extracellular cytochrome c such as OmcS at 0.0 and +0.2 V and exhibited a better conductivity. These findings provide reasonable explanations for the mechanism governing the electrochemical respiration and activity in G. sulfurreducens biofilms., (© 2019 Wiley Periodicals, Inc.)

Iron reduction mediated by Fe(III)-reducing bacteria (FeRB) occurs in aqueous environments and plays an essential role in removing contaminates in polluted freshwater lakes. Two model FeRB species, Shewanella and Geobacter, have been intensively studied because of their functions in bioremediation, iron reduction, and bioelectricity production. However, the abundance and community diversity of Shewanella and Geobacter in eutrophic freshwater lakes remain largely unknown. In this work, the distribution, abundance and biodiversity of Shewanella, Geobacter and other FeRB in the sediments of a heavily polluted lake, Chaohu Lake, China, across four successive seasons were investigated. Shewanella, Geobacter, and other FeRB were found to be widely distributed in the sediment of this heavily eutrophic lake. Geobacter was abundant with at least one order of magnitude more than Shewanella in cold seasons. Three Shewanella-related operational taxonomic units were detected and sixty one Geobacter-related operational taxonomic units were grouped into three phylogenetic clades. Thiobacillus, Desulfuromonas and Geobacter were identified as the main members of FeRB in the lake sediments. Interestingly, nutrients like carbon, nitrogen, and phosphorus were found to be the key factors governing the abundance and diversity of FeRB. Total FeRB, as well as Geobacter and Shewanella, were more abundant in the heavily eutrophic zone than those in the lightly eutrophic zone. The abundance and diversity of FeRB in the sediments of freshwater lakes were highly related with the degree of eutrophication, which imply that FeRB might have a great potential in alleviating the eutrophication and contamination in aqueous environments.

Azo dyes are a class of recalcitrant organic pollutants causing severe environmental pollution. For their biodecolorization, the azo reductase system was considered as the major molecular basis in bacteria. However, the intracellular localization of azo reductase limits their function for efficient azo dye decolorization. This limitation may be circumvented by electrochemically active bacteria (EAB) which is capable of extracellular respiration. To verify the essential role of extracellular respiration in azo dye decolorization, Geobacter sulfurreducens PCA, a model EAB, was used for the bioreduction of methyl orange (MO), a typical azo dye. G. sulfurreducens PCA efficiently reduced MO into amines. Kinetic results showed that G. sulfurreducens PCA had the highest decolorization efficiency among the currently known MO reducing bacteria. Electrons from acetate oxidization by this strain were transferred by the respiratory chain to MO. The mass and electron balances, fluorescent probing and proteinase K treatment experimental results indicate that the biodecolorization of MO by G. sulfurreducens PCA is an exclusive extracellular process. OmcB, OmcC and OmcE were identified as the key outer-membrane proteins for the extracellular MO reduction. This work deepens our understanding of EAB physiology and is useful for the decontamination of environments polluted with azo dyes. The contribution of extracellular respiration to pollutants reduction will broaden the environmental applications of EAB.