Outer-surface C-type Cytochromes Research Articles

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

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

Electrobiocorrosion by microbes without outer-surface cytochromes.

Anaerobic microbial corrosion of iron-containing metals causes extensive economic damage. Some microbes are capable of direct metal-to-microbe electron transfer (electrobiocorrosion), but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation. Previous studies have suggested that respiration with 316L stainless steel as the electron donor is indicative of electrobiocorrosion, because, unlike pure Fe0, 316L stainless steel does not abiotically generate H2 as an intermediary electron carrier. Here, we report that all of the methanogens (Methanosarcina vacuolata, Methanothrix soehngenii, and Methanobacterium strain IM1) and acetogens (Sporomusa ovata and Clostridium ljungdahlii) evaluated respired with pure Fe0 as the electron donor, but only M. vacuolata, Mx. soehngenii, and S. ovata were capable of stainless steel electrobiocorrosion. The electrobiocorrosive methanogens required acetate as an additional energy source in order to produce methane from stainless steel. Cocultures of S. ovata and Mx. soehngenii demonstrated how acetogens can provide acetate to methanogens during corrosion. Not only was Methanobacterium strain IM1 not capable of electrobiocorrosion, but it also did not accept electrons from Geobacter metallireducens, an effective electron-donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe0. The finding that M. vacuolata, Mx. soehngenii, and S. ovata are capable of electrobiocorrosion, despite a lack of the outer-surface c-type cytochromes previously found to be important in other electrobiocorrosive microbes, demonstrates that there are multiple microbial strategies for making electrical contact with Fe0.

Electrically conductive nanowires controlled one pivotal route in energy harvest and microbial corrosion via direct metal-microbe electron transfer

Extracellular electron transfer (EET) plays a critical role in bioelectrochemical processes, allowing coupling between microorganisms and extracellular solid-state electrodes, metals, or other cells in energy metabolism. Previous studies have suggested a role for outer-surface c-type cytochromes in direct metal-to-microbe electron transfer by Geobacter sulfurreducens, a model electroactive bacterium. Here, we examined the possibility of other microbially produced electrical contacts by deleting the gene for PilA, the protein monomer that G. sulfurreducens assembles into electrically conductive protein nanowires (e-pili). Deleting pilA gene inhibited electron extraction from pure iron and 316L stainless steel up to 31% and 81%, respectively more than deleting the gene for the outer-surface cytochrome OmcS. This PilA-deficient phenotype, and the observation that relatively thick biofilms (21.7 μm) grew on the metal surfaces at multi-cell distances from the metal surfaces suggest that e-pili contributed significantly to microbial corrosion via direct metal-to-microbe electron transfer. These results have implications for the fundamental understanding of electron harvest via e-pili by electroactive microbes, their uses in bioenergy production, as well as in monitoring and mitigation of metal biocorrosion.

Cytochrome-mediated direct electron uptake from metallic iron by Methanosarcina acetivorans.

Methane-producing microorganisms accelerate the corrosion of iron-containing metals. Previous studies have inferred that some methanogens might directly accept electrons from Fe(0), but when this possibility was more intensively investigated, H2 was shown to be an intermediary electron carrier between Fe(0) and methanogens. Here, we report that Methanosarcina acetivorans catalyzes direct metal-to-microbe electron transfer to support methane production. Deletion of the gene for the multiheme, outer-surface c-type cytochrome MmcA eliminated methane production from Fe(0), consistent with the key role of MmcA in other forms of extracellular electron exchange. These findings, coupled with the previous demonstration that outer-surface c-type cytochromes are also electrical contacts for electron uptake from Fe(0) by Geobacter and Shewanella species, suggest that the presence of multiheme c-type cytochromes on corrosion surfaces might be diagnostic for direct metal-to-microbe electron transfer and that interfering with cytochrome function might be a strategy to mitigate corrosion.

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.

The Role of Exopolysaccharides in Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) is an effective mechanism for microbial species to exchange electrons cooperatively during syntrophic metabolism. It is generally accepted that DIET is mainly mediated by electrically conductive pili and outer surface c-type cytochromes (c-Cyts). However, as an extracellular matrix is ubiquitous and abundant on the surface of microorganisms, the effect and mechanism of exopolysaccharides on DIET are still unclear. This study constructed a co-culture of exopolysaccharides-deficient Geobacter sulfurreducens with Geobacter metallireducens to explore the role of exopolysaccharides in DIET. Results revealed that the deficiency of exopolysaccharides extended the metabolic period of the co-culture by 44.4% and changed the proportions of each species in the co-culture. The exopolysaccharides-deficient co-culture failed to form large, tight spherical aggregates and the expression of c-Cyts and pili was decreased. The addition of magnetite and granular activated carbon (GAC), respectively, might compensate for the functions of c-Cyts and pili in the first generation of co-culture, but the stimulatory effect on the metabolic stable period co-culture was fairly limited. These findings demonstrate that non-conductive exopolysaccharides are an important component of DIET aggregates and an extracellular matrix for DIET-required c-Cyts.

Flagella act as Geobacter biofilm scaffolds to stabilize biofilm and facilitate extracellular electron transfer

Flagella are widely expressed in electroactive biofilms; however, their actual role is unknown. To understand the role of flagella, two Geobacter sulfurreducens strains (KN400 and PCA, with and without flagella, respectively) were selected. We restored flagellum expression in trans in strain PCA and prevented flagellum expression in strain KN400. Electrochemical results showed that flagellum restoration in strain PCA promoted current generation, while flagellum deletion in strain KN400 impaired current production. However, the expression of conductive pili and outer surface c-type cytochromes was not affected. Further microscopic analyses demonstrated that flagella promoted the formation of thicker biofilms and served as biofilm matrix scaffolds to accommodate more extracellular cytochromes with an orderly arrangement, which increased the electron diffusion rate within the biofilm. Our findings reveal an unprecedented structural role for flagella in stabilizing electroactive biofilms and highlight the importance of cytochromes in electron transfer across biofilms, which will deepen our understanding of biofilm conductivity.

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

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 ArchaeaIMPORTANCE 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.

Iron Corrosion via Direct Metal-Microbe Electron Transfer.

The concept that anaerobic microorganisms can directly accept electrons from Fe(0) has been controversial because direct metal-microbe electron transfer has previously only been indirectly inferred. Fe(0) oxidation was studied with Geobacter sulfurreducens strain ACL, an autotrophic strain that was previously shown to grow with electrons derived from a graphite cathode as the sole electron donor. Strain ACL grew with Fe(0) as the sole electron donor and fumarate as the electron acceptor. However, it appeared that at least a portion of the electron transfer was via H2 produced nonenzymatically from the oxidation of Fe(0) to Fe(II). H2, which accumulated in abiotic controls, was consumed during the growth of strain ACL, the cells were predominately planktonic, and genes for the uptake hydrogenase were highly expressed. Strain ACLHF was constructed to prevent growth on H2 or formate by deleting the genes for the uptake of hydrogenase and formate dehydrogenases from strain ACL. Strain ACLHF also grew with Fe(0) as the sole electron donor, but H2 accumulated in the culture, and cells heavily colonized Fe(0) surfaces with no visible planktonic growth. Transcriptomics suggested that the outer surface c-type cytochromes OmcS and OmcZ were important during growth of strain ACLHF on Fe(0). Strain ACLHF did not grow on Fe(0) if the gene for either of these cytochromes was deleted. The specific attachment of strain ACLHF to Fe(0), coupled with requirements for known extracellular electrical contacts, suggest that direct metal-microbe electron transfer is the most likely option for Fe(0) serving as an electron donor.IMPORTANCE The anaerobic corrosion of iron structures is expensive to repair and can be a safety and environmental concern. It has been known for over 100 years that the presence of anaerobic respiratory microorganisms can accelerate iron corrosion. Multiple studies have suggested that there are sulfate reducers, methanogens, and acetogens that can directly accept electrons from Fe(0) to support sulfate or carbon dioxide reduction. However, all of the strains studied can also use H2 as an electron donor for growth, which is known to be abiotically produced from Fe(0). Furthermore, no proteins definitely shown to function as extracellular electrical contacts with Fe(0) were identified. The studies described here demonstrate that direct electron transfer from Fe(0) can support anaerobic respiration. They also map out a simple genetic approach to the study of iron corrosion mechanisms in other microorganisms. A better understanding of how microorganisms promote iron corrosion is expected to lead to the development of strategies that can help reduce adverse impacts from this process.

Potential of direct interspecies electron transfer in synergetic enhancement of methanogenesis and sulfate removal in an up-flow anaerobic sludge blanket reactor with magnetite

Anaerobic digestion (AD) has been widely applied in the treatment of industrial wastewater containing oxidized sulfur compounds. However, the production of hydrogen sulfide usually limits the syntrophic metabolism proceeded by interspecies hydrogen transfer (IHT), due to its corrosive and toxic properties. The current study was in an attempt to establish direct interspecies electron transfer (DIET) to resist the toxic inhibition from hydrogen sulfide and keep syntrophic metabolism stable. The results showed that, in the presence of magnetite, the methane production was improved about 3–10 folds at each ratio of COD/SO42−, while the enhancement of methanogenesis had almost no negative effect on sulfate reduction. With magnetite, the sludge conductance increased about 3 folds, but the concentration of c-type cytochromes decreased, suggesting that the potential DIET via both electrically conductive pili and outer surface c-type cytochromes was established. Microbial community revealed that, Veillonella species, the Fe(III)-reducing genus capable of reducing sulfate to hydrogen sulfide, were specially enriched with magnetite. Together with the relatively higher abundance of Methanothrix and Methanosarcina species, the novel DIET between Fe(III)/sulfate-reducing genus and methanogens was inferred to be responsible for the synergetic enhancement of methanogenesis and sulfate removal.

Syntrophy Goes Electric: Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) has biogeochemical significance, and practical applications that rely on DIET or DIET-based aspects of microbial physiology are growing. Mechanisms for DIET have primarily been studied in defined cocultures in which Geobacter species are one of the DIET partners. Electrically conductive pili (e-pili) can be an important electrical conduit for DIET. However, there may be instances in which electrical contacts are made between electron transport proteins associated with the outer membranes of the partners. Alternatively, DIET partners can plug into conductive carbon materials, such as granular activated carbon, carbon cloth, and biochar, for long-range electron exchange without the need for e-pili. Magnetite promotes DIET, possibly by acting as a substitute for outer-surface c-type cytochromes. DIET is the primary mode of interspecies electron exchange in some anaerobic digesters converting wastes to methane. Promoting DIET with conductive materials shows promise for stabilizing and accelerating methane production in digesters, permitting higher organic loading rates. Various lines of evidence suggest that DIET is important in terrestrial wetlands, which are an important source of atmospheric methane. DIET may also have a role in anaerobic methane oxidation coupled to sulfate reduction, an important control on methane releases. The finding that DIET can serve as the source of electrons for anaerobic photosynthesis further broadens its potential environmental significance. Microorganisms capable of DIET are good catalysts for several bioelectrochemical technologies and e-pili are a promising renewable source of electronic materials. The study of DIET is in its early stages, and additional investigation is required to better understand the diversity of microorganisms that are capable of DIET, the importance of DIET to carbon and electron flow in anaerobic environments, and the biochemistry and physiology of DIET.

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

Syntrophic growth via quinone-mediated interspecies electron transfer.

The mechanisms by which microbial species exchange electrons are of interest because interspecies electron transfer can expand the metabolic capabilities of microbial communities. Previous studies with the humic substance analog anthraquinone-2,6-disulfonate (AQDS) suggested that quinone-mediated interspecies electron transfer (QUIET) is feasible, but it was not determined if sufficient energy is available from QUIET to support the growth of both species. Furthermore, there have been no previous studies on the mechanisms for the oxidation of anthrahydroquinone-2,6-disulfonate (AHQDS). A co-culture of Geobacter metallireducens and G. sulfurreducens metabolized ethanol with the reduction of fumarate much faster in the presence of AQDS, and there was an increase in cell protein. G. sulfurreducens was more abundant, consistent with G. sulfurreducens obtaining electrons from acetate that G. metallireducens produced from ethanol, as well as from AHQDS. Co-cultures initiated with a citrate synthase-deficient strain of G. sulfurreducens that was unable to use acetate as an electron donor also metabolized ethanol with the reduction of fumarate and cell growth, but acetate accumulated over time. G. sulfurreducens and G. metallireducens were equally abundant in these co-cultures reflecting the inability of the citrate synthase-deficient strain of G. sulfurreducens to metabolize acetate. Evaluation of the mechanisms by which G. sulfurreducens accepts electrons from AHQDS demonstrated that a strain deficient in outer-surface c-type cytochromes that are required for AQDS reduction was as effective at QUIET as the wild-type strain. Deletion of additional genes previously implicated in extracellular electron transfer also had no impact on QUIET. These results demonstrate that QUIET can yield sufficient energy to support the growth of both syntrophic partners, but that the mechanisms by which electrons are derived from extracellular hydroquinones require further investigation.

A severe reduction in the cytochrome C content of Geobacter sulfurreducens eliminates its capacity for extracellular electron transfer.

The ability of Geobacter species to transfer electrons outside the cell enables them to play an important role in a number of biogeochemical and bioenergy processes. Gene deletion studies have implicated periplasmic and outer-surface c-type cytochromes in this extracellular electron transfer. However, even when as many as five c-type cytochrome genes have been deleted, some capacity for extracellular electron transfer remains. In order to evaluate the role of c-type cytochromes in extracellular electron transfer, Geobacter sulfurreducens was grown in a low-iron medium that included the iron chelator (2,2'-bipyridine) to further sequester iron. Haem-staining revealed that the cytochrome content of cells grown in this manner was 15-fold lower than in cells exposed to a standard iron-containing medium. The low cytochrome abundance was confirmed by in situ nanoparticle-enhanced Raman spectroscopy (NERS). The cytochrome-depleted cells reduced fumarate to succinate as well as the cytochrome-replete cells do, but were unable to reduce Fe(III) citrate or to exchange electrons with a graphite electrode. These results demonstrate that c-type cytochromes are essential for extracellular electron transfer by G. sulfurreducens. The strategy for growing cytochrome-depleted G. sulfurreducens will also greatly aid future physiological studies of Geobacter species and other microorganisms capable of extracellular electron transfer.

Two Isoforms of Geobacter sulfurreducens PilA Have Distinct Roles in Pilus Biogenesis, Cytochrome Localization, Extracellular Electron Transfer, and Biofilm Formation

Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides.

Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells

Geobacter sulfurreducens produces current densities in microbial fuel cells that are among the highest known for pure cultures. The possibility of adapting this organism to produce even higher current densities was evaluated. A system in which a graphite anode was poised at −400 mV (versus Ag/AgCl) was inoculated with the wild-type strain of G. sulfurreducens, strain DL-1. An isolate, designated strain KN400, was recovered from the biofilm after 5 months of growth on the electrode. KN400 was much more effective in current production than strain DL-1. This was apparent with anodes poised at −400 mV, as well as in systems run in true fuel cell mode. KN400 had current (7.6 A/m 2) and power (3.9 W/m 2) densities that respectively were substantially higher than those of DL1 (1.4 A/m 2 and 0.5 W/m 2). On a per cell basis KN400 was more effective in current production than DL1, requiring thinner biofilms to make equivalent current. The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance (0.015 versus 0.130 Ω/m 2) and mass transfer limitation in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. Surprisingly, KN400 had much less outer-surface c-type cytochromes than DL1. KN400 also had a greater propensity to form biofilms on glass or graphite than DL1, even when growing with the soluble electron acceptor, fumarate. These results demonstrate that it is possible to enhance the ability of microorganisms to electrochemically interact with electrodes with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide insights into the mechanisms for microbe–electrode interactions.