scholarly journals A New Approach for Evaluating Electron Transfer Dynamics by Using In Situ Resonance Raman Microscopy and Chronoamperometry in Conjunction with a Dynamic Model

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Adolf Krige ◽  
Kerstin Ramser ◽  
Magnus Sjöblom ◽  
Paul Christakopoulos ◽  
Ulrika Rova
Keyword(s):  
Electron Transfer ◽  
Dynamic Model ◽  
Oxidation Rate ◽  
Resonance Raman ◽  
Raman Microscopy ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Current Response ◽  
Content Type ◽  
Raman Response

ABSTRACT Geobacter sulfurreducens is a good candidate as a chassis organism due to its ability to form thick, conductive biofilms, enabling long-distance extracellular electron transfer (EET). Due to the complexity of EET pathways in G. sulfurreducens, a dynamic approach is required to study genetically modified EET rates in the biofilm. By coupling online resonance Raman microscopy with chronoamperometry, we were able to observe the dynamic discharge response in the biofilm’s cytochromes to an increase in anode voltage. Measuring the heme redox state alongside the current allows for the fitting of a dynamic model using the current response and a subsequent validation of the model via the value of a reduced cytochrome c Raman peak. The modeled reduced cytochromes closely fitted the Raman response data from the G. sulfurreducens wild-type strain, showing the oxidation of heme groups in cytochromes until a new steady state was achieved. Furthermore, the use of a dynamic model also allows for the calculation of internal rates, such as acetate and NADH consumption rates. The Raman response of a mutant lacking OmcS showed a higher initial oxidation rate than predicted, followed by an almost linear decrease of the reduced mediators. The increased initial rate could be attributed to an increase in biofilm conductivity, previously observed in biofilms lacking OmcS. One explanation for this is that OmcS acts as a conduit between cytochromes; therefore, deleting the gene restricts the rate of electron transfer to the extracellular matrix. This could, however, be modeled assuming a linear oxidation rate of intercellular mediators. IMPORTANCE Bioelectrochemical systems can fill a vast array of application niches, due to the control of redox reactions that it offers. Although native microorganisms are preferred for applications such as bioremediation, more control is required for applications such as biosensors or biocomputing. The development of a chassis organism, in which the EET is well defined and readily controllable, is therefore essential. The combined approach in this work offers a unique way of monitoring and describing the reaction kinetics of a G. sulfurreducens biofilm, as well as offering a dynamic model that can be used in conjunction with applications such as biosensors.

scholarly journals Adaptive Evolution of Geobacter sulfurreducens in Coculture with Pseudomonas aeruginosa

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Lucie Semenec ◽  
Ismael A. Vergara ◽  
Andrew E. Laloo ◽  
Steve Petrovski ◽  
Philip L. Bond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Adaptive Evolution ◽  
Efflux Pump ◽  
Antibiotic Production ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Content Type ◽  
Interaction Dynamics ◽  
Syntrophic Interaction

ABSTRACT Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa. Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducens fabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction. IMPORTANCE Geobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria.

scholarly journals Extracellular Electron Transfer: Respiratory or Nutrient Homeostasis?

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Lars J. C. Jeuken ◽  
Kiel Hards ◽  
Yoshio Nakatani
Keyword(s):  
Electron Transfer ◽  
Iron Reduction ◽  
Ferric Iron ◽  
Shewanella Oneidensis ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Nutrient Homeostasis

ABSTRACT Exoelectrogens are able to transfer electrons extracellularly, enabling them to respire on insoluble terminal electron acceptors. Extensively studied exoelectrogens, such as Geobacter sulfurreducens and Shewanella oneidensis, are Gram negative. More recently, it has been reported that Gram-positive bacteria, such as Listeria monocytogenes and Enterococcus faecalis, also exhibit the ability to transfer electrons extracellularly, although it is still unclear whether this has a function in respiration or in redox control of the environment, for instance, by reducing ferric iron for iron uptake. In this issue of Journal of Bacteriology, Hederstedt and colleagues report on experiments that directly compare extracellular electron transfer (EET) pathways for ferric iron reduction and respiration and find a clear difference (L. Hederstedt, L. Gorton, and G. Pankratova, J Bacteriol 202:e00725-19, 2020, https://doi.org/10.1128/JB.00725-19), providing further insights and new questions into the function and metabolic pathways of EET in Gram-positive bacteria.

scholarly journals An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Caleb E. Levar ◽  
Chi Ho Chan ◽  
Misha G. Mehta-Kolte ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Redox Potentials ◽  
Inner Membrane ◽  
Content Type ◽  
Transfer Strategies ◽  
Reducing Bacteria ◽  
Metal Reducing Bacteria

ABSTRACTDissimilatory metal-reducing bacteria, such asGeobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption ofimcH, encoding an inner membranec-type cytochrome, eliminated the ability ofG. sulfurreducensto reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but theimcHmutant retained the ability to reduce Fe(III) oxides with potentials of ≤−0.1 V versus SHE. TheimcHmutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to −0.1 V versus SHE triggered exponential growth. At potentials of ≤−0.1 V versus SHE, both the wild type and theimcHmutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not triggerimcHmutant growth. These results demonstrate thatG. sulfurreducenspossesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments whereGeobacteris found.IMPORTANCEInsoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein inGeobacter sulfurreducenswhich is required only for electron transfer to high-potential compounds, and we show thatG. sulfurreducenshas the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell.

scholarly journals Generation of High Current Densities in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor

2021 ◽  
Author(s):  
Toshiyuki Ueki ◽  
David J. F. Walker ◽  
Kelly P. Nevin ◽  
Joy E. Ward ◽  
Trevor L. Woodard ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Geobacter Sulfurreducens ◽  
Metal Corrosion ◽  
Extracellular Electron Transfer ◽  
Renewable Electricity ◽  
Gene Deletions ◽  
Putative Gene ◽  
Content Type ◽  
Soils And Sediments ◽  
Current Densities

Geobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes, such as the reduction of Fe(III) oxides in soils and sediments, bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity, direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments, and metal corrosion. Elucidating the phenotypes associated with gene deletions is an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens .

scholarly journals Generation of High Current Densities by Pure Cultures of Anode-Respiring Geoalkalibacter spp. under Alkaline and Saline Conditions in Microbial Electrochemical Cells

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Jonathan P. Badalamenti ◽  
Rosa Krajmalnik-Brown ◽  
César I. Torres
Keyword(s):  
Electron Transfer ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Electrochemical Cells ◽  
Organic Substrates ◽  
High Current ◽  
Content Type ◽  
Pure Cultures ◽  
Current Densities ◽  
Anode Respiring Bacteria

ABSTRACTAnode-respiring bacteria (ARB) generate electric current in microbial electrochemical cells (MXCs) by channeling electrons from the oxidation of organic substrates to an electrode. Production of high current densities by monocultures in MXCs has resulted almost exclusively from the activity ofGeobacter sulfurreducens, a neutrophilic freshwater Fe(III)-reducing bacterium and the highest-current-producing member documented for theGeobacteraceaefamily of theDeltaproteobacteria. Here we report high current densities generated by haloalkaliphilicGeoalkalibacterspp., thus broadening the capability for high anode respiration rates by including other genera within theGeobacteraceae. In this study, acetate-fed pure cultures of two relatedGeoalkalibacterspp. produced current densities of 5.0 to 8.3 and 2.4 to 3.3 A m−2under alkaline (pH 9.3) and saline (1.7% NaCl) conditions, respectively. Chronoamperometric studies of halophilicGlk. subterraneusDSM 23483 and alkaliphilicGlk. ferrihydriticusDSM 17813 suggested that cells performed long-range electron transfer through electrode-attached biofilms and not through soluble electron shuttles.Glk. ferrihydriticusalso oxidized ethanol directly to produce current, with maximum current densities of 5.7 to 7.1 A m−2and coulombic efficiencies of 84 to 95%. Cyclic voltammetry (CV) elicited a sigmoidal response with characteristic onset, midpoint, and saturation potentials, while CV performed in the absence of an electron donor suggested the involvement of redox molecules in the biofilm that were limited by diffusion. These results matched those previously reported for actively respiringGb. sulfurreducensbiofilms producing similar current densities (~5 to 9 A m−2).IMPORTANCEThis study establishes the highest current densities ever achieved by pure cultures of anode-respiring bacteria (ARB) under alkaline and saline conditions in microbial electrochemical cells (MXCs) and provides the first electrochemical characterization of the genusGeoalkalibacter. Production of high current densities among theGeobacteraceaeis no longer exclusive toGeobacter sulfurreducens, suggesting greater versatility for this family in fundamental and applied microbial electrochemical cell (MXC) research than previously considered. Additionally, this work raises the possibility that different members of theGeobacteraceaehave conserved molecular mechanisms governing respiratory extracellular electron transfer to electrodes. Thus, the capacity for high current generation may exist in other uncultivated members of this family. Advancement of MXC technology for practical uses must rely on an expanded suite of ARB capable of using different electron donors and producing high current densities under various conditions.Geoalkalibacterspp. can potentially broaden the practical capabilities of MXCs to include energy generation and waste treatment under expanded ranges of salinity and pH.

scholarly journals Divergent Nrf Family Proteins and MtrCAB Homologs Facilitate Extracellular Electron Transfer inAeromonas hydrophila

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Bridget E. Conley ◽  
Peter J. Intile ◽  
Daniel R. Bond ◽  
Jeffrey A. Gralnick
Keyword(s):  
Electron Transfer ◽  
Outer Membrane ◽  
Aeromonas Hydrophila ◽  
Shewanella Oneidensis ◽  
Geobacter Sulfurreducens ◽  
Model Systems ◽  
Extracellular Electron Transfer ◽  
Metal Reduction ◽  
Inner Membrane ◽  
Content Type

ABSTRACTExtracellular electron transfer (EET) is a strategy for respiration in which electrons generated from metabolism are moved outside the cell to a terminal electron acceptor, such as iron or manganese oxide. EET has primarily been studied in two model systems,Shewanella oneidensisandGeobacter sulfurreducens. Metal reduction has also been reported in numerous microorganisms, includingAeromonasspp., which are ubiquitousGammaproteobacteriafound in aquatic ecosystems, with some species capable of pathogenesis in humans and fish. Genomic comparisons ofAeromonasspp. revealed a potential outer membrane conduit homologous toS. oneidensisMtrCAB. While the ability to respire metals and mineral oxides is not widespread in the genusAeromonas, 90% of the sequencedAeromonas hydrophilaisolates contain MtrCAB homologs.A. hydrophilaATCC 7966 mutants lackingmtrAare unable to reduce metals. Expression ofA. hydrophila mtrCABin anS. oneidensismutant lacking homologous components restored metal reduction. Although the outer membrane conduits for metal reduction were similar, homologs of theS. oneidensisinner membrane and periplasmic EET components CymA, FccA, and CctA were not found inA. hydrophila. We characterized a cluster of genes predicted to encode components related to a formate-dependent nitrite reductase (NrfBCD), here named NetBCD (forNrf-likeelectrontransfer), and a predicted diheme periplasmic cytochrome, PdsA (periplasmicdihemeshuttle). We present genetic evidence that proteins encoded by this cluster facilitate electron transfer from the cytoplasmic membrane across the periplasm to the MtrCAB conduit and function independently from an authentic NrfABCD system.A. hydrophilamutants lackingpdsAandnetBCDwere unable to reduce metals, while heterologous expression of these genes could restore metal reduction in anS. oneidensismutant background. EET may therefore allowA. hydrophilaand other species ofAeromonasto persist and thrive in iron- or manganese-rich oxygen-limited environments.IMPORTANCEMetal-reducing microorganisms are used for electricity production, bioremediation of toxic compounds, wastewater treatment, and production of valuable compounds. Despite numerous microorganisms being reported to reduce metals, the molecular mechanism has primarily been studied in two model systems,Shewanella oneidensisandGeobacter sulfurreducens. We have characterized the mechanism of extracellular electron transfer inAeromonas hydrophila, which uses the well-studiedShewanellasystem, MtrCAB, to move electrons across the outer membrane; however, mostAeromonasspp. appear to use a novel mechanism to transfer electrons from the inner membrane through the periplasm and to the outer membrane. The conserved use of MtrCAB inShewanellaspp. andAeromonasspp. for metal reduction and conserved genomic architecture of metal reduction genes inAeromonasspp. may serve as genomic markers for identifying metal-reducing microorganisms from genomic or transcriptomic sequencing. Understanding the variety of pathways used to reduce metals can allow for optimization and more efficient design of microorganisms used for practical applications.

Geobacter sulfurreducens adapts to low electrode potential for extracellular electron transfer

2016 ◽  
Vol 191 ◽  
pp. 743-749 ◽  
Author(s):  
Luo Peng ◽  
Xiao-Ting Zhang ◽  
Jie Yin ◽  
Shuo-Yuan Xu ◽  
Yong Zhang ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Electrode Potential ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer

scholarly journals A Hybrid Extracellular Electron Transfer Pathway Enhances the Survival of Vibrio natriegens

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Bridget E. Conley ◽  
Matthew T. Weinstock ◽  
Daniel R. Bond ◽  
Jeffrey A. Gralnick
Keyword(s):  
Electron Transfer ◽  
Shewanella Oneidensis ◽  
Anaerobic Respiration ◽  
Basic Research ◽  
Extracellular Electron Transfer ◽  
Content Type ◽  
Electron Transfer Pathway ◽  
Sedimentary Environments ◽  
Genetic Potential ◽  
Iron And Manganese

ABSTRACT Vibrio natriegens is the fastest-growing microorganism discovered to date, making it a useful model for biotechnology and basic research. While it is recognized for its rapid aerobic metabolism, less is known about anaerobic adaptations in V. natriegens or how the organism survives when oxygen is limited. Here, we describe and characterize extracellular electron transfer (EET) in V. natriegens, a metabolism that requires movement of electrons across protective cellular barriers to reach the extracellular space. V. natriegens performs extracellular electron transfer under fermentative conditions with gluconate, glucosamine, and pyruvate. We characterized a pathway in V. natriegens that requires CymA, PdsA, and MtrCAB for Fe(III) citrate and Fe(III) oxide reduction, which represents a hybrid of strategies previously discovered in Shewanella and Aeromonas. Expression of these V. natriegens genes functionally complemented Shewanella oneidensis mutants. Phylogenetic analysis of the inner membrane quinol dehydrogenases CymA and NapC in gammaproteobacteria suggests that CymA from Shewanella diverged from Vibrionaceae CymA and NapC. Analysis of sequenced Vibrionaceae revealed that the genetic potential to perform EET is conserved in some members of the Harveyi and Vulnificus clades but is more variable in other clades. We provide evidence that EET enhances anaerobic survival of V. natriegens, which may be the primary physiological function for EET in Vibrionaceae. IMPORTANCE Bacteria from the genus Vibrio occupy a variety of marine and brackish niches with fluctuating nutrient and energy sources. When oxygen is limited, fermentation or alternative respiration pathways must be used to conserve energy. In sedimentary environments, insoluble oxide minerals (primarily iron and manganese) are able to serve as electron acceptors for anaerobic respiration by microorganisms capable of extracellular electron transfer, a metabolism that enables the use of these insoluble substrates. Here, we identify the mechanism for extracellular electron transfer in Vibrio natriegens, which uses a combination of strategies previously identified in Shewanella and Aeromonas. We show that extracellular electron transfer enhanced survival of V. natriegens under fermentative conditions, which may be a generalized strategy among Vibrio spp. predicted to have this metabolism.

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