scholarly journals Bacterial Microcompartments Coupled with Extracellular Electron Transfer Drive the Anaerobic Utilization of Ethanolamine in Listeria monocytogenes

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Zhe Zeng ◽  
Sjef Boeren ◽  
Varaang Bhandula ◽  
Samuel H. Light ◽  
Eddy J. Smid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Listeria Monocytogenes ◽  
Electron Acceptor ◽  
Cell Membranes ◽  
Extracellular Electron Transfer ◽  
Vitamin B ◽  
Content Type ◽  
Redox Imbalance ◽  
Bacterial Microcompartments

ABSTRACT Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from cell membranes. EA catabolism is suggested to occur in a cellular metabolic subsystem called a bacterial microcompartment (BMC), and the activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that the activation of eut is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent in Listeria monocytogenes. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics, and electron microscopy. First, we show vitamin B12-induced activation of the eut operon in L. monocytogenes coupled to the utilization of EA, thereby enabling growth. Next, we demonstrate BMC formation connected with EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP-generating acetate branch causes an apparent redox imbalance due to the reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMCs to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild-type, BMC mutant, and EET mutant strains, we demonstrate an interaction between BMCs and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of BMC-dependent EA catabolism in L. monocytogenes growth in anaerobic environments like the human gastrointestinal tract, with a crucial role for the flavin-based EET system in redox balancing. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness, and as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires the balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

scholarly journals Bacterial microcompartments linked to the flavin-based extracellular electron transfer drives anaerobic ethanolamine utilization in Listeria monocytogenes

2020 ◽  
Author(s):  
Zhe Zeng ◽  
Sjef Boeren ◽  
Varaang Bhandula ◽  
Samuel H. Light ◽  
Eddy J. Smid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Listeria Monocytogenes ◽  
Electron Acceptor ◽  
Cell Membranes ◽  
Anaerobic Growth ◽  
Extracellular Electron Transfer ◽  
Severe Illness ◽  
Redox Imbalance ◽  
Bacterial Microcompartments

AbstractEthanolamine (EA) is a valuable microbial carbon and nitrogen source derived from phospholipids present in cell membranes. EA catabolism is suggested to occur in so-called bacterial microcompartments (BMCs) and activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that activation of eut in Listeria monocytogenes is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics and electron microscopy. First, we show B12-induced activation of the eut operon in L. monocytogenes coupled to uptake and utilization of EA thereby enabling growth. Next, we demonstrate BMC formation in conjunction to EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP generating acetate branch causes an apparent redox imbalance due to reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMC to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild type, a BMC mutant and a EET mutant, we demonstrate an interaction between BMC and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of anaerobic BMC-dependent EA catabolism in the physiology of L. monocytogenes, with a crucial role for the flavin-based EET system in redox balancing.IMPORTANCEListeria monocytogenes is a food-borne pathogen causing severe illness and, as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

scholarly journals Geothrix fermentans Secretes Two Different Redox-Active Compounds To Utilize Electron Acceptors across a Wide Range of Redox Potentials

2012 ◽  
Vol 78 (19) ◽  
pp. 6987-6995 ◽  
Author(s):  
Misha G. Mehta-Kolte ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Redox Potential ◽  
Electron Acceptor ◽  
Extracellular Electron Transfer ◽  
Redox Potentials ◽  
Active Compounds ◽  
Content Type ◽  
Electron Shuttles ◽  
Redox Active ◽  
Geothrix Fermentans

ABSTRACTThe current understanding of dissimilatory metal reduction is based primarily on isolates from the proteobacterial generaGeobacterandShewanella. However, environments undergoing active Fe(III) reduction often harbor less-well-studied phyla that are equally abundant. In this work, electrochemical techniques were used to analyze respiratory electron transfer by the only known Fe(III)-reducing representative of theAcidobacteria,Geothrix fermentans. In contrast to previously characterized metal-reducing bacteria, which typically reach maximal rates of respiration at electron acceptor potentials of 0 V versus standard hydrogen electrode (SHE),G. fermentansrequired potentials as high as 0.55 V to respire at its maximum rate. In addition,G. fermentanssecreted two different soluble redox-active electron shuttles with separate redox potentials (−0.2 V and 0.3 V). The compound with the lower midpoint potential, responsible for 20 to 30% of electron transfer activity, was riboflavin. The behavior of the higher-potential compound was consistent with hydrophilic UV-fluorescent molecules previously found inG. fermentanssupernatants. Both electron shuttles were also produced when cultures were grown with Fe(III), but not when fumarate was the electron acceptor. This study reveals thatGeothrixis able to take advantage of higher-redox-potential environments, demonstrates that secretion of flavin-based shuttles is not confined toShewanella, and points to the existence of high-potential-redox-active compounds involved in extracellular electron transfer. Based on differences between the respiratory strategies ofGeothrixandGeobacter, these two groups of bacteria could exist in distinctive environmental niches defined by redox potential.

scholarly journals A Membrane-Bound Cytochrome Enables Methanosarcina acetivorans To Conserve Energy from Extracellular Electron Transfer

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Dawn E. Holmes ◽  
Toshiyuki Ueki ◽  
Hai-Yan Tang ◽  
Jinjie Zhou ◽  
Jessica A. Smith ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Electron Acceptor ◽  
Methane Production ◽  
Genetic Manipulation ◽  
Methanol Conversion ◽  
Extracellular Electron Transfer ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Content Type ◽  
Expression Of Genes

ABSTRACT Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea. IMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.

scholarly journals Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation

2016 ◽  
Vol 82 (17) ◽  
pp. 5428-5443 ◽  
Author(s):  
Sarah E. Barchinger ◽  
Sahand Pirbadian ◽  
Christine Sambles ◽  
Carol S. Baker ◽  
Kar Man Leung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Shewanella Oneidensis ◽  
Oxygen Limitation ◽  
Extracellular Electron Transfer ◽  
Transport Pathways ◽  
Content Type ◽  
Genes Encoding

ABSTRACTIn limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacteriumShewanella oneidensisMR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates thatS. oneidensisMR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of themtrAandmtrChomologsmtrFandmtrDeither remains unaffected or decreases under these conditions. TheompWgene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream ofmtrCandomcA. The transcriptome and mutant analyses ofS. oneidensisMR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires.IMPORTANCEShewanella oneidensisMR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain whyS. oneidensiscan be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer.Shewanellacoordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.

Impact of Graphitic Structures in Pyrogenic Carbon on Microbial Reduction of Ferrihydrite

2021 ◽  
Author(s):  
wentao yu ◽  
baoliang chen
Keyword(s):  
Electron Transfer ◽  
Pyrolysis Temperature ◽  
Shewanella Oneidensis ◽  
Exchange Capacity ◽  
Microbial Reduction ◽  
Systematic Evaluation ◽  
Extracellular Electron Transfer ◽  
Pyrogenic Carbon ◽  
The Impact

<p>Pyrogenic carbon plays important roles in microbial reduction of ferrihydrite by shuttling electrons in the extracellular electron transfer (EET) processes. Despite its importance, a full assessment on the impact of graphitic structures in pyrogenic carbon on microbial reduction of ferrihydrite has not been conducted. This study is a systematic evaluation of microbial ferrihydrite reduction by Shewanella oneidensis MR-1 in the presence of pyrogenic carbon with various graphitization extents. The results showed that the rates and extents of microbial ferrihydrite reduction were significantly enhanced in the presence of pyrogenic carbon, and increased with increasing pyrolysis temperature. Combined spectroscopic and electrochemical analyses suggested that the rate of microbial ferrihydrite reduction were dependent on the electrical conductivity of pyrogenic carbon (i.e., graphitization extent), rather than the electron exchange capacity. The key role of graphitic structures in pyrogenic carbon in mediating EET was further evidenced by larger microbial electrolysis current with pyrogenic carbon prepared at higher pyrolysis temperatures. This study provides new insights into the electron transfer in the pyrogenic carbon-mediated microbial reduction of ferrihydrite.</p>

scholarly journals Extracellular Electron Transfer to Fe(III) Oxides by the Hyperthermophilic Archaeon Geoglobus ahangari via a Direct Contact Mechanism

2013 ◽  
Vol 79 (15) ◽  
pp. 4694-4700 ◽  
Author(s):  
Michael P. Manzella ◽  
Gemma Reguera ◽  
Kazem Kashefi
Keyword(s):  
Electron Acceptor ◽  
Direct Contact ◽  
Alginate Beads ◽  
Hydrothermal Systems ◽  
Microbial Reduction ◽  
Extracellular Electron Transfer ◽  
Cell Contact ◽  
Content Type ◽  
Chemical Conditions ◽  
Contact Mechanism

ABSTRACTThe microbial reduction of Fe(III) plays an important role in the geochemistry of hydrothermal systems, yet it is poorly understood at the mechanistic level. Here we show that the obligate Fe(III)-reducing archaeonGeoglobus ahangariuses a direct-contact mechanism for the reduction of Fe(III) oxides to magnetite at 85°C. Alleviating the need to directly contact the mineral with the addition of a chelator or the electron shuttle anthraquinone-2,6-disulfonate (AQDS) stimulated Fe(III) reduction. In contrast, entrapment of the oxides within alginate beads to prevent cell contact with the electron acceptor prevented Fe(III) reduction and cell growth unless AQDS was provided. Furthermore, filtered culture supernatant fluids had no effect on Fe(III) reduction, ruling out the secretion of an endogenous mediator too large to permeate the alginate beads. Consistent with a direct contact mechanism, electron micrographs showed cells in intimate association with the Fe(III) mineral particles, which once dissolved revealed abundant curled appendages. The cells also produced several heme-containing proteins. Some of them were detected among proteins sheared from the cell's outer surface and were required for the reduction of insoluble Fe(III) oxides but not for the reduction of the soluble electron acceptor Fe(III) citrate. The results thus support a mechanism in which the cells directly attach and transfer electrons to the Fe(III) oxides using redox-active proteins exposed on the cell surface. This strategy confers onG. ahangaria competitive advantage for accessing and reducing Fe(III) oxides under the extreme physical and chemical conditions of hot ecosystems.

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.

scholarly journals Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Prabhat Ranjan Singh ◽  
Anil Kumar Vijjamarri ◽  
Dibyendu Sarkar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Metabolism ◽  
Electron Acceptor ◽  
Latent Infection ◽  
Critical Role ◽  
Hypoxic Stress ◽  
Content Type ◽  
Metabolic Switching ◽  
Virulence Regulator

ABSTRACT Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. IMPORTANCE M. tuberculosis retains the unique ability to establish an asymptomatic latent infection. To understand the mechanisms involved in hypoxic stress which play a critical role in persistence, we show that the virulence regulator PhoP is linked to hypoxia, the dormancy signal. In keeping with this, phoP was shown to play a major role in M. tuberculosis growth under hypoxia even in the presence of surplus nitrogen, the alternate electron acceptor. Our results showing regulation of hypoxia-responsive genes provide new biological insights into role of the virulence regulator in metabolic switching by sensing hypoxia and integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR.

scholarly journals 6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Rongshui Wang ◽  
Jihong Yi ◽  
Jinmeng Shang ◽  
Wenjun Yu ◽  
Zhifeng Li ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Agrobacterium Tumefaciens ◽  
Electron Transport ◽  
Electron Acceptor ◽  
Aromatic Compounds ◽  
Coenzyme Q ◽  
Content Type ◽  
Electron Transfer Flavoprotein ◽  
Nicotine Degradation

ABSTRACT Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria. IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

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