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Extracellular Electron Uptake in Methanosarcinales Is Independent of Multiheme

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bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Extracellular electron uptake in Methanosarcinales is independent of multiheme 2 c-type cytochromes 3 Mon Oo Yee1 & Amelia-Elena Rotaru1 4 1Nordcee, Department of Biology, University of Southern Denmark, Odense, Denmark 5 6 Correspondence: [email protected] 7 8 Keywords: 9 multiheme c-type cytochromes, direct interspecies electron transfer, Methanosarcina, Methanothrix, 10 Methanosarcina mazei, electromethanogenesis, Rhodoferax ferrireducens, Geobacter hydrogenophilus, 11 Geobacter metallireducens 12 13 14 15 16 17 18 19 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 20 Abstract 21 The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of 22 direct interspecies electron transfer (DIET) in man-made and natural aquatic environments. We previously 23 reported that not all Geobacter are capable of DIET with Methanosarcina. Here we tested 15 new artificial 24 co-culture combinations with methanogens and electrogenic bacteria, including an electrogen outside of the 25 Geobacter clade – Rhodoferax ferrireducens. 26 Consistently, highly effective electrogenic bacteria (G. metallireducens, G. hydrogenophilus and R. 27 ferrireducens) formed successful associations with Methanosarcinales. Highly effective electrogens could 28 not sustain the growth of H2-utilizing methanogens of the genera Methanococcus, Methanobacterium, 29 Methanospirillum, Methanolacinia or Methanoculleus. 30 Methanosarcinales, including strict non-hydrogenotrophic methanogens of the genus Methanothrix (Mtx. 31 harundinacea and Mtx. shoeghenii) and Methanosarcina horonobensis, conserved their ability to interact 32 with electrogens. Methanosarcinales were classified as the only methanogens containing c-type 33 cytochromes, unlike strict hydrogenotrophic methanogens. It was then hypothesized that multiheme c-type 34 cytochromes give Methanosarcinales their ability to retrieve extracellular electrons. However, multiheme c- 35 type cytochromes are neither unique to this group of methanogens nor universal. Only two of the seven 36 Methanosarcinales tested had multiheme c-type cytochromes (MCH). In one of these two species - M. mazei 37 a deletion mutant for its MCH was readily available. Here we tested if the absence of this MHC impacts 38 extracellular electron uptake. Deletion of the MHC in M.mazei did not impact the ability of this methanogens 39 to retrieve extracellular electrons from G. metallireducens or a poised cathode. Since Methanosarcina did 40 not require multiheme c-type cytochromes for direct electron uptake we proposed an alternative strategy for 41 extracellular electron uptake. bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 42 Introduction 43 Direct interspecies electron transfer (DIET) was discovered in an artificial co-culture of an ethanol-oxidizing 44 Geobacter metallireducens with a fumarate-reducing Geobacter sulfurreducens where the possibility of 1,2 45 hydrogen gas (H2) or formate transfer was invalidated through genetic studies . Gene deletions rendering 1,2 46 H2 and formate transfer impossible resulted in active co-cultures , whereas deletion of genes for 47 extracellular electron transfer proteins (EET) such as pili, and multiheme c-type cytochromes made the 48 interspecies interaction impossible 1,3. Remarkably, a deletion mutant lacking an extracellular multiheme c- 49 type cytochrome (OmcS) could be rescued by the addition of extracellular conductive particles, whereas a 50 pili knock-out strain could not be rescued by conductive particles 4. Additionally, previous studies have 51 shown that G. metallireducens is also highly effective as an anode-respiring bacteria (ARB) generating some 52 of the highest current densities of all Geobacter tested 5. During DIET with G. sulfurreducens, G. 53 metallireducens requires pili and certain multiheme c-type cytochromes 3, which were also required during 54 anode respiration 6,7. 55 G. metallireducens is a strict respiratory microorganism unable to ferment its substrates to produce H2 for 8,9 56 interspecies H2-transfer . Consequently, G. metallireducens could not provide reducing equivalents for 57 strict hydrogenotrophic methanogens (Methanospirilum hungatei and Methanobacterium formicicum)10,11. 58 However, G. metallireducens did interact syntrophically with Methanosarcinales (Methanosarcina. barkeri 59 800, Methanosarcina horonobensis, Methanothrix harundinacea)10–12 of which the last two are unable to 13,14 60 consume H2 . When incubated with Methanosarcinales, G. metallireducens upregulated EET-proteins, 61 and if some of these EET proteins were deleted, co-cultures became inviable demonstrating the direct 62 electron transfer nature of the interaction 10,12. 63 For co-culture incubations, G. metallireducens has been typically provided with an ethanol as electron donor, 64 but without a soluble electron acceptor. In the absence of a soluble electron acceptor, G. metallireducens 65 releases electrons extracellularly (reaction 1) and uses the methanogen as its extracellular terminal electron 66 acceptor (reaction 2 & 3). If electrons released by G. metallireducens cannot reach a terminal electron bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 67 acceptor, ethanol oxidation would stop, because the electron transport chain would become ineffective, and 68 NADH produced during ethanol oxidation could not get re-oxidized. 69 Reaction 1. Ethanol oxidation with electrons release by G. metallireducens: + - 70 2CH3CH2OH + 2H2O → CH3COOH + 2CO2 + [8H + 8e ] 71 Reaction 2. Electron uptake coupled with CO2 reductive methanogenesis by Methanosarcinales: + - 72 CO2 + [8H + 8e ] → CH4 + 2H2O 73 Reaction 3. Acetoclastic methanogenesis: CH3COOH → CH4 + CO2 74 Reaction 4. Total DIET reaction by a syntrophic association between Geobacter and Methanosarcinales: 75 2CH3CH2OH → 3CH4 + CO2 76 There are indications for direct interspecies electron transfer occurs in methane producing environments such 77 as anaerobic digesters 15, rice paddy soils 16, and aquatic sediments 17,18 , as well as in methane consuming 78 environments such as hydrothermal vents 19,20. In these environments, DIET is typically inferred either 79 because conductive materials stimulate the syntrophic metabolism but also by the co-presence of DNA 80 and/or RNA of phylotypes related to DIET-microorganisms. DIET-pairing of Geobacter with methanogens 81 was only described in two Geobacter species (G. metallireducens, G. hydrogenophilus) and three 82 Methanosarcinales (Ms. barkeri 800, Ms. horonobensis, Mtx. harundinacea) 5,10–12. On the other hand, a 83 series of six Geobacter species were unable to interact syntrophically with Ms. barkeri 800. All these 84 Geobacter were modest anode respiring bacteria and did not produce high current densities at the anode 5. 85 No species outside of the Geobacter-clade have been shown to do DIET with methanogens. 86 In this study, we expand the list of syntrophic-DIET pairs and investigated whether other electrogenic 87 bacteria but Geobacter could interact syntrophically with methanogens. We determined whether DIET was 88 spread among other methanogens or was a specific trait of Methanosarcinales because of their high c-type 89 cytochrome content 21. Multiheme c-type cytochromes (MHC) were previously implicated in EET in bacteria bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 90 22 and an MHC of Methanosarcina acetivorans was required for AQDS respiration 23. Here we asked 91 whereas the OMCs of a Methanosarcina is required for DIET and electron uptake from electrodes. 92 Materials and methods 93 Cultures were purchased from the German culture collection (DSMZ) and grown on the media advised by 94 the collection until pre-adaption to co-cultivation media. The following strains of methanogens were tested: 95 Methanococcus maripaludis JJ (DSM 2067), Methanococcus voltae PS (DSM 1537), Methanoculleus 96 marisnigri JR1 (DSM 1498), Methanolacinia petrolearia (DSM 11571), Methanosarcina mazei Gö1 (DSM 97 3647), Methanospirillum hungatei JF1 (DSM 864), Methanosaeta harundinacea 8Ac (DSM 17206), three 98 strains of Methanosarcina
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  • Characterization of Methanosarcina Barkeri MST and 227, Methanosarcina Mazei S-6T, and Methanosarcina Vacuolata Z-76IT GLORIA M

    Characterization of Methanosarcina Barkeri MST and 227, Methanosarcina Mazei S-6T, and Methanosarcina Vacuolata Z-76IT GLORIA M

    INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 267-274 Vol. 41, No. 2 0020-7713/91/020267-08$02.OO/O Copyright 0 1991, International Union of Microbiological Societies Characterization of Methanosarcina barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-76IT GLORIA M. MAESTROJUAN' AND DAVID R. BOONE172* Departments of Environmental Science and Engineering' and Chemical and Biological Science,2 Oregon Graduate Institute, 19600 N.W. von Neumann Drive, Beaverton, Oregon 97006-1999 Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H, plus methanol, growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCI.