C-type Cytochromes Research Articles

An improved method for rapid evaluation of enzymatic cis/trans isomerization of C18:1 monounsaturated fatty acids

Cytochrome c-type cis/trans fatty acid isomerase (CTI) is a promising candidate for directly controlling cis/trans fatty acid isomerism in lipids-related food products like partially hydrogenated vegetable oils. In this study, to establish a sophisticated analysis platform for the CTI assay, we constructed the reversed micelle reaction system and improved the processes of methylation and GC-FID analysis of C18:1cis/trans monounsaturated fatty acid (MUFA) isomers. Highly stable AOT/isooctane reversed micelles were formed in the presence of periplasmic fractions of Pseudomonas putida KT2440. Using a mid-content cyanopropyl phase DB-FastFAME column, C18:1cis/trans-MUFAs were analyzed rapidly and resolved with resolution factors over 1.34. Based on the newly established assay, the catalytic activity of the periplasmic fraction was precisely determined, and its kinetic parameters (Vmax and Km) were derived as 0.021 mM·min−1 and 0.68 mM, respectively. The following results can provide practical information for investigating CTIs in the fields of food and lipid chemistry.

Vanadate reducing bacteria and archaea may use different mechanisms to reduce vanadate in vanadium contaminated riverine ecosystems as revealed by the combination of DNA-SIP and metagenomic-binning

Vanadium (V) is a transitional metal that poses health risks to exposed humans. Microorganisms play an important role in remediating V contamination by reducing more toxic and mobile vanadate (V(V)) to less toxic and mobile V(IV). In this study, DNA-stable isotope probing (SIP) coupled with metagenomic-binning was used to identify microorganisms responsible for V(V) reduction and determine potential metabolic mechanisms in cultures inoculated with a V-contaminated river sediment. Anaeromyxobacter and Geobacter spp. were identified as putative V(V)-reducing bacteria, while Methanosarcina spp. were identified as putative V(V)-reducing archaea. The bacteria may use the two nitrate reductases NarG and NapA for respiratory V(V) reduction, as has been demonstrated previously for other species. It is proposed that Methanosarcina spp. may reduce V(V) via anaerobic methane oxidation pathways (AOM-V) rather than via respiratory V(V) reduction performed by their bacterial counterparts, as indicated by the presence of genes associated with anaerobic methane oxidation coupled with metal reduction in the metagenome assembled genome (MAG) of Methanosarcina. Briefly, methane may be oxidized through the "reverse methanogenesis" pathway to produce electrons, which may be further captured by V(V) to promote V(V) reduction. More specially, V(V) reduction by members of Methanosarcina may be driven by electron transport (CoMS-SCoB heterodisulfide reductase (HdrDE), F420H2 dehydrogenases (Fpo), and multi-heme c-type cytochrome (MHC)). The identification of putative V(V)-reducing bacteria and archaea and the prediction of their different pathways for V(V) reduction expand current knowledge regarding the potential fate of V(V) in contaminated sites.

Evidence and Mechanisms of Selenate Reduction to Extracellular Elemental Selenium Nanoparticles on the Biocathode.

Intracellular selenium nanoparticles (SeNPs) production is a roadblock to the recovery of selenium from biological water treatment processes because it is energy intensive to break microbial cells and then separate SeNPs. This study provided evidence of significantly more extracellular SeNP production on the biocathode (97-99%) compared to the conventional reactors (1-90%) using transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy. The cathodic microbial community analysis showed that relative abundance of Azospira oryzae, Desulfovibrio, Stenotrophomonas, and Rhodocyclaceae was <1% in the inoculum but enriched to 10-21% for each group when the bioelectrochemical reactor reached a steady state. These four groups of microorganisms simultaneously produce intracellular and extracellular SeNPs in conventional biofilm reactors per literature review but prefer to produce extracellular SeNPs on the cathode. This observation may be explained by the cellular energetics: by producing extracellular SeNPs on the biocathode, microbes do not need to transfer selenate and the electrons from the cathode into the cells, thereby saving energy. Extracellular SeNP production on the biocathode is feasible since we found high concentrations of C-type cytochrome, which is well known for its ability to transfer electrons from electrodes to microbial cells and reduce selenate to SeNPs on the cell membrane.

Co-fitness analysis identifies a diversity of signal proteins involved in the utilization of specific c-type cytochromes

Purposec-Type cytochromes are essential for extracellular electron transfer (EET) in electroactive microorganisms. The expression of appropriate c-type cytochromes is an important feature of these microorganisms in response to different extracellular electron acceptors. However, how these diverse c-type cytochromes are tightly regulated is still poorly understood.MethodsIn this study, we identified the high co-fitness genes that potentially work with different c-type cytochromes by using genome-wide co-fitness analysis. We also constructed and studied the co-fitness networks that composed of c-type cytochromes and the top 20 high co-fitness genes of them.ResultsWe found that high co-fitness genes of c-type cytochromes were enriched in signal transduction processes in Shewanella oneidensis MR-1 cells. We then checked the top 20 co-fitness proteins for each of the 41 c-type cytochromes and identified the corresponding signal proteins for different c-type cytochromes. In particular, through the analysis of the high co-fitness signal protein for CymA, we further confirmed the cooperation between signal proteins and c-type cytochromes and identified a novel signal protein that is putatively involved in the regulation of CymA. In addition, we showed that these signal proteins form two signal transduction modules.ConclusionTaken together, these findings provide novel insights into the coordinated utilization of different c-type cytochromes under diverse conditions.

Radical Mediated Rapid In Vitro Formation of c-Type Cytochrome.

A cytochrome c552 mutant from Thermus thermophilus HB8 (rC552 C14A) was reported, where the polypeptide with replaced Cys14 by alanine, overexpressed in the cytosol of E. coli. The apo-form of the C14A mutant (apo-C14A) without the original prosthetic group was obtained by simple chemical treatments that retained compact conformation amenable to reconstitution with heme b and zinc(II)-protoporphyrin(IX), gradually followed by spontaneous formation of a covalent bond between the polypeptide and porphyrin ring in the reconstituted apo-C14A. Further analysis suggested that the residual Cys11 and vinyl group of the porphyrin ring linked through the thiol-ene reaction promoted by light under ambient conditions. In this study, we describe the kinetic improvement of the covalent bond formation in accordance with the mechanism of the photoinduced thiol-ene reaction, which involves a thiyl radical as a reaction intermediate. Adding a radical generator to the reconstituted C14A mutant with either heme-b or zinc(II) porphyrin accelerated the bond-forming reaction, which supported the involvement of a radical species in the reaction. Partial observation of the reconstituted C14A in a dimer form and detection of sulfuryl radical by EPR spectroscopy indicated a thiyl radical on Cys11, a unique cysteinyl residue in rC552 C14A. The covalent bond forming mediated by the radical generator was also adaptable to the reconstituted apo-C14A with manganese(II)-protoporphyrin(IX), which also exhibits light-mediated covalent linkage formation. Therefore, the radical generator extends the versatility of producing c-type-like cytochrome starting from a metallo-protoporphyrin(IX) and the apo-C14A instantaneously.

Simultaneous boost of anodic electron transfer and exoelectrogens enrichment by decorating electrospinning carbon nanofibers in microbial fuel cell

Microbial fuel cell (MFC) is a promising technology in wastewater recovery driven by microbial metabolism. However, the low power output resulting from the sluggish extracellular electron transfer (EET) between the anode surface and exoelectrogens dramatically restricted the further application. This study fabricated a high-performance anode by decorating porous and conductive electrospinning carbon nanofibers (CNFs). The maximum power density in MFC modified with 14 wt% polyacrylonitrile CNFs (M-CNF14, 9.6 ± 0.2 W m−3) was 1.9 and 2.7 times higher than carbon black modified MFC (M-CB, 5.1 ± 0.1 W m−3) and the blank (M-BA, 3.6 ± 0.1 W m−3), respectively. Denser biofilm and more microbial nanowires were observed in the M-CNF14 anode than in other conditions. Furthermore, the redox peak current of c-type cytochrome was 1.7–21 times higher in M-CNF14 than in the blank control, verifying the preferable EET activity. Several exoelectrogens like Petrimonas and Comamonas were enriched in M-CNF14 and showed a positive correlation to power generation. Besides, more simplified and modular interrelations among exoelectrogens and other bacteria were obtained in M-CNF14. This study revealed the microbial-related mechanism for simultaneously improving EET and exoelectrogens enrichment by CNFs modified anode, providing guidelines for high-performance wastewater recovery.

Structure of Geobacter OmcZ filaments suggests extracellular cytochrome polymers evolved independently multiple times.

While early genetic and low-resolution structural observations suggested that extracellular conductive filaments on metal-reducing organisms such as Geobacter were composed of type IV pili, it has now been established that bacterial c-type cytochromes can polymerize to form extracellular filaments capable of long-range electron transport. Atomic structures exist for two such cytochrome filaments, formed from the hexaheme cytochrome OmcS and the tetraheme cytochrome OmcE. Due to the highly conserved heme packing within the central OmcS and OmcE cores, and shared pattern of heme coordination between subunits, it has been suggested that these polymers have a common origin. We have now used cryo-electron microscopy (cryo-EM) to determine the structure of a third extracellular filament, formed from the Geobacter sulfurreducens octaheme cytochrome, OmcZ. In contrast to the linear heme chains in OmcS and OmcE from the same organism, the packing of hemes, heme:heme angles, and between-subunit heme coordination is quite different in OmcZ. A branched heme arrangement within OmcZ leads to a highly surface exposed heme in every subunit, which may account for the formation of conductive biofilm networks, and explain the higher measured conductivity of OmcZ filaments. This new structural evidence suggests that conductive cytochrome polymers arose independently on more than one occasion from different ancestral multiheme proteins.

Different outer membrane c-type cytochromes are involved in direct interspecies electron transfer to Geobacter or Methanosarcina species.

Direct interspecies electron transfer (DIET) may be most important in methanogenic environments, but mechanistic studies of DIET to date have primarily focused on cocultures in which fumarate was the terminal electron acceptor. To better understand DIET with methanogens, the transcriptome of Geobacter metallireducens during DIET-based growth with G. sulfurreducens reducing fumarate was compared with G. metallireducens grown in coculture with diverse Methanosarcina. The transcriptome of G. metallireducens cocultured with G. sulfurreducens was significantly different from those with Methanosarcina. Furthermore, the transcriptome of G. metallireducens grown with Methanosarcina barkeri, which lacks outer-surface c-type cytochromes, differed from those of G. metallireducens cocultured with M. acetivorans or M. subterranea, which have an outer-surface c-type cytochrome that serves as an electrical connect for DIET. Differences in G. metallireducens expression patterns for genes involved in extracellular electron transfer were particularly notable. Cocultures with c-type cytochrome deletion mutant strains, ∆Gmet_0930, ∆Gmet_0557 and ∆Gmet_2896, never became established with G. sulfurreducens but adapted to grow with all three Methanosarcina. Two porin-cytochrome complexes, PccF and PccG, were important for DIET; however, PccG was more important for growth with Methanosarcina. Unlike cocultures with G. sulfurreducens and M. acetivorans, electrically conductive pili were not needed for growth with M. barkeri. Shewanella oneidensis, another electroactive microbe with abundant outer-surface c-type cytochromes, did not grow via DIET. The results demonstrate that the presence of outer-surface c-type cytochromes does not necessarily confer the capacity for DIET and emphasize the impact of the electron-accepting partner on the physiology of the electron-donating DIET partner.

Riboflavin-rich agar enhances the rate of extracellular electron transfer from electrogenic bacteria inside a thin-layer system

Numerous bacteria owe extracellular electron transport (EET) ability, and the rate enhancement of EET is critical for the emerging sensor technology to detect metabolically active pathogens. Here, the considerable enhancement of microbial current signal was firstly demonstrated in a thin layer electrolyte sandwiched between an agar substrate (AS) containing high concentration riboflavin (RF) and a screen-printed electrode. Covering cells with this AS showed a sharply current increase from 0.033 µA to 1.59 μA (47.7-folds) in EET-capable bacteria Shewanella oneidensis MR-1. Differential pulse voltammograms using gene-deletion mutant strains of S. oneidensis MR-1 revealed thin electrolyte between RF-loaded AS and electrode enhanced the rate of electron transfer via complexes between riboflavin and outer membrane c-type cytochrome. A similar effect in Streptococcus mutans UA159, a biofilm-forming pathogen, was also explored. Moreover, capturing and quantifying both metabolically active microbes from the dry solid surface are demonstrated with RF-loaded AS successfully. The considerable enhancement of the EET in the thin layer electrolyte provides a new direction for designing whole-cell biosensors and understanding a microbe/electrode interaction in a micro-sized space.

Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivaxgelatinosus and Rhodobactersphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.

Experimental evolution of Vibrio cholerae identifies hypervesiculation as a way to increase motility in the presence of polymyxin B.

Vibrio cholerae includes strains responsible for the cholera disease and is a natural inhabitant of aquatic environments. V. cholerae possesses a unique polar flagellum essential for motility, adhesion, and biofilm formation. In a previous study, we showed that motility and biofilm formation are altered in the presence of subinhibitory concentrations of polymyxin B in V. cholerae O1 and O139. In this study, we performed an experimental evolution to identify the genes restoring the motility in the presence of a subinhibitory concentration of polymyxin B. Mutations in five genes have been identified in three variants derived from two different parental strains A1552 and MO10: ihfA that encodes a subunit of the integration host factor (IHF), vacJ (mlaA) and mlaF, two genes belonging to the maintenance of the lipid asymmetry (Mla) pathway, dacB that encodes a penicillin-binding protein (PBP4) and involved in cell wall synthesis, and ccmH that encodes a c-type cytochrome maturation protein. We further demonstrated that the variants derived from MO10 containing mutations in vacJ, mlaF, and dacB secrete more and larger membrane vesicles that titer the polymyxin B, which increases the bacterial survival and is expected to limit its impact on the bacterial envelope and participate in the flagellum’s retention and motility.

Biomolecular Insights into Extracellular Pollutant Reduction Pathways of Geobacter sulfurreducens Using a Base Editor System.

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.

Insights into the syntrophic microbial electrochemical oxidation of toluene: a combined chemical, electrochemical, taxonomical, functional gene-based, and metaproteomic approach

Biodegradation of aromatic hydrocarbons in anoxic contaminated environments is typically limited by the lack of bioavailable electron acceptors. Microbial electrochemical technologies (METs) are able to provide a virtually inexhaustible electron acceptor in the form of a solid electrode.Recently, we provided first experimental evidence for the syntrophic degradation of toluene in a continuous-flow bioelectrochemical reactor known as the “bioelectric well”. Herein, we further analyzed the structure and function of the electroactive toluene-degrading microbiome using a suite of chemical, electrochemical, phylogenetic, proteomic, and functional gene-based analyses.The bioelectric well removed 83 ± 7 % of the toluene from the influent with a coulombic efficiency of 84 %. Cyclic voltammetry allowed to identify the formal potentials of four putative electron transfer sites, which ranged from −0.2 V to +0.1 V vs. SHE, consistent with outer membrane c-type cytochromes and pili of electroactive Geobacter species. The biofilm colonizing the surface of the anode was indeed highly enriched in Geobacter species. On the other hand, the planktonic communities thriving in the bulk of the reactor harbored aromatic hydrocarbons degraders and fermentative propionate-producing microorganisms, as revealed by phylogenetic and proteomic analyses. Most likely, propionate, acetate or other VFAs produced in the bulk liquid from the degradation of toluene were utilized as substrates by the electroactive biofilm. Interestingly, key-functional genes related to the degradation of toluene were found both in the biofilm and in the planktonic communities. Taken as a whole, the herein reported results highlight the importance of applying a comprehensive suite of techniques to unravel the complex cooperative metabolisms occurring in METs.

Inward-to-outward assembly of amine-functionalized carbon dots and polydopamine to Shewanella oneidensis MR-1 for high-efficiency, microbial-photoreduction of Cr(VI)

A novel photosensitized living biohybrid was fabricated by inward-to-outward assembly of amine-functionalized carbon dots (NCDs) and polydopamine (PDA) to Shewanella oneidensis MR-1 and applied for high-efficiency, microbial-photoreduction of Cr(VI). Within a 72 h test period, biohybrids achieved a pronounced catalytic reduction capacity (100%) for 100 mg/L Cr(VI) under visible illumination, greatly surpassing the poor capacity (only 2.5%) displayed by the wild strain under dark conditions. Modular configurations of NCDs and PDA afforded biohybrids with a large electron flux by harvesting extracellular photoelectrons generated from illuminated NCDs and increasing reducing equivalents released from an enlarged intracellular NADH/NAD+ pool. Further, increased production of intracellular c-type cytochromes and extracellular flavins resulting from the modular configuration enhanced the biohybrid electron transport ability. The enhancement of electron transport was also attributed to more conductive conduits at NCDs-PDA junction interfaces. Moreover, because NCDs are highly reductive, the enhanced Cr(VI) reduction was also attributed to direct reduction by the NCDs and the direct Cr(VI) reduction by sterile NCDs-assembled biohybrid was up to 20% in the dark. Overall, a highly efficient strategy for removal/transformation of Cr(VI) by using NCD-assembled photosensitized biohybrids was proposed in this work, which greatly exceeded the performance of Cr(VI)-remediation strategies based on conventional microbial technologies.

Exploiting Violet-Blue Light to Kill Campylobacter jejuni: Analysis of Global Responses, Modeling of Transcription Factor Activities, and Identification of Protein Targets.

ABSTRACTCampylobacter jejuni is a microaerophilic foodborne zoonotic pathogen of worldwide concern as the leading cause of bacterial gastroenteritis. Many strains are increasingly antibiotic resistant and new methods of control are required to reduce food-chain contamination. One possibility is photodynamic inactivation (PDI) using violet-blue (VB) light, to which C. jejuni is highly susceptible. Here, we show that flavin and protoporphyrin IX are major endogenous photosensitizers and that exposure of cells to VB light increases intracellular reactive oxygen species (ROS) to high levels, as indicated by a dichlorodihydrofluorescein reporter. Unusually for an oxygen-respiring bacterium, C. jejuni employs several ROS-sensitive iron-sulfur cluster enzymes in central metabolic pathways; we show that VB light causes rapid inactivation of both pyruvate and 2-oxoglutarate oxidoreductases, thus interrupting the citric acid cycle. Cells exposed to VB light also lose heme from c-type cytochromes, restricting electron transport, likely due to irreversible oxidation of heme-ligating cysteine residues. Evaluation of global gene expression changes by RNAseq and probabilistic modeling showed a two-stage protein damage/oxidative stress response to VB light, driven by specific regulators, including HspR, PerR, Fur, and RacR. Deletion mutant analysis showed that superoxide dismutase and the cytochrome CccA were particularly important for VB light survival and that abolishing repression of chaperones and oxidative stress resistance genes by HcrA, HspR, or PerR increased tolerance to VB light. Our results explain the high innate sensitivity of C. jejuni to VB light and provide new insights that may be helpful in exploiting PDI for novel food-chain interventions to control this pathogen.IMPORTANCE Campylobacteriosis caused by C. jejuni is one of the most widespread zoonotic enteric diseases worldwide and represents an enormous human health and economic burden, compounded by the emergence of antibiotic-resistant strains. New interventions are urgently needed to reduce food-chain contamination. Although UV light is well known to be bactericidal, it is highly mutagenic and problematic for continuous exposure in food production facilities; in contrast, narrow spectrum violet-blue (VB) light is much safer. We confirmed that C. jejuni is highly susceptible to VB light and then identified some of the global regulatory networks involved in responding to photo-oxidative damage. The identification of damaged cellular components underpins efforts to develop commercial applications of VB light-based technologies.

Genome analysis of the candidate phylum MBNT15 bacterium from a boreal peatland predicted its respiratory versatility and dissimilatory iron metabolism.

Uncultured bacteria of the candidate phylum MBNT15, distantly related to Desulfobacterota, have been identified in a broad range of mostly organic-rich aquatic environments. We assembled a near-complete genome of a member of MBNT15 from a boreal peatland metagenome and used genomic data to analyze the metabolic pathways of this bacterium and its ecological role. This bacterium, designated SHF-111, was predicted to be rod shaped, it lacks flagellar machinery but twitching motility is encoded. Genome-based phylogenetic analysis supported the phylum-level classification of the MBNT15 lineage. Genome annotation and metabolic reconstruction revealed the presence of the Embden–Meyerhof, Entner–Doudoroff and pentose phosphate pathways, as well as the complete tricarboxylic acid (TCA) cycle, and suggested a facultatively anaerobic chemoheterotrophic lifestyle with the ability to ferment peptides, amino acids, fatty acids and simple sugars, and completely oxidize these substrates through aerobic and anaerobic respiration. The SHF-111 genome encodes multiple multiheme c-type cytochromes that probably enable dissimilatory iron reduction. Consistently, the relative abundance of MBNT15 in peatlands positively correlated with iron concentration. Apparently, in the wetland ecosystem, MBNT15 representatives play the role of scavengers, carrying out the complete mineralization of low molecular weight organic substances formed as a result of microbial degradation of complex polymeric substrates. Comparative genome analysis of the MBNT15 phylum revealed that vast majority of its members are capable of aerobic respiration and dissimilatory iron reduction and some species also can reduce sulfur and nitrogen compounds, but not sulfate. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Deferrimicrobium borealis, within a candidate phylum Deferrimicrobiota.

Single-cell metagenomics and metagenomics approaches reveal extracellular electron transfer of psychrophilic electroactive biofilms

Revealing the metabolic functions and the extracellular electron transfer (EET) process of psychrophilic electroactive biofilms (PEB) is important for understanding the functional adaptability of electroactive bacteria (EAB) to low temperatures. In this study, single cell sorting (SCS)-based metagenomics sequencing was used to characterize the composition and function of the PEB. Biofilm microbiome analysis showed that Geobacter, with a relative abundance of 62.64%, dominated the PEB enriched in the bioelectrochemical system (BES) at 4 °C. Both the metagenome (MAG) and single-cell metagenome (SCM) revealed that there were no obvious metabolic differences between the original biofilm and the sorted single cells. The presence of genes associated with type IV pilus, c-type cytochromes, and riboflavin indicated that the EET potential was maintained in the PEB at low temperatures. These results suggested that SCM provides an alternative approach to reconstruct the metabolic functions of uncultured and slow-growing EAB.

Coupling riboflavin de novo biosynthesis and cytochrome expression for improving extracellular electron transfer efficiency in Shewanella oneidensis.

Shewanella oneidensis MR-1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c-type cytochromes (c-Cyts). However, relatively low RF production and inappropriate amount of c-Cyts substantially impede the capacity of improving the EET rate. In this study, coupling of riboflavin de novo biosynthesis and c-Cyts expression was implemented to enhance the efficiency of EET in S. oneidensis. First, the upstream pathway of RF de novo biosynthesis was divided into four modules, and the expression level of 22 genes in above four modules was fine-tuned by employing promoters with different strengths. Among them, genes zwf*, glyA, andybjU which exhibited optimal RF production were combinatorially overexpressed, leading to the enhancement of maximum output power density by 166%. Second, the diverse c-Cyts genes were overexpressed to match high RF production, and omcA was selected for further combination. Third, RF de novo biosynthesis and c-Cyts expression were combined, resulting in 2.34-fold higher power output than the parent strain. This modular and combinatorial manipulation strategy provides a generalized reference to advance versatile practical applications of electroactive microorganisms.

Bio-Pd(0) diverting electron from CoQ-long chain to FDH/Hase-short chain during sulfamethoxazole degradation

Microbial electron output capacity is critical for organic contaminants biodegradation. Herein, original C. freundii JH could oxidate formate in anaerobic respiration, but lack the ability to degrade sulfamethoxazole (SMX). While the incorporation of Pd(0) could effectively improve the electron output via improving the combination between flavins and c-type cytochromes (c-Cyts), increasing the activities of key enzymes (formate dehydrogenase, hydrogenase, F0F1-ATPases), etc. More importantly, the presence of Pd(0) caused the NADH dehydrogenase (complex I) nearly in idle, and triggered the decrease of NADH/NAD+ ratio and increase of H+-efflux transmembrane gradient, eventually resulting in the electrons diverting from CoQ-involved long respiratory chain (decreasing from 91.67% to 36.25%) to FDH/Hases-based hydrogen-producing short chain (increasing from 22.44% to 84.88%), which further intensified the electron output. Above changes effectively launched and guaranteed the high-level SMX degradation by palladized C. freundii JH, alleviating the ecotoxicity of SMX in aquatic and terrestrial environments. These conclusions provided the new view to regulate the microbial electron output behaviors.

Cryo-EM structure of an extracellular Geobacter OmcE cytochrome filament reveals tetrahaem packing.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens were first observed two decades ago, with genetic and biochemical data suggesting that conductive fibres were type IV pili. Recently, an extracellular conductive filament of G. sulfurreducens was found to contain polymerized c-type cytochrome OmcS subunits, not pilin subunits. Here we report that G. sulfurreducens also produces a second, thinner appendage comprised of cytochrome OmcE subunits and solve its structure using cryo-electron microscopy at ~4.3 Å resolution. Although OmcE and OmcS subunits have no overall sequence or structural similarities, upon polymerization both form filaments that share a conserved haem packing arrangement in which haems are coordinated by histidines in adjacent subunits. Unlike OmcS filaments, OmcE filaments are highly glycosylated. In extracellular fractions from G. sulfurreducens, we detected type IV pili comprising PilA-N and -C chains, along with abundant B-DNA. OmcE is the second cytochrome filament to be characterized using structural and biophysical methods. We propose that there is a broad class of conductive bacterial appendages with conserved haem packing (rather than sequence homology) that enable long-distance electron transport to chemicals or other microbial cells.