Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase

Mand, Thomas D.; Kulkarni, Gargi; Metcalf, William W.

doi:10.1128/jb.00342-18

Cited by 34 publications

(41 citation statements)

References 39 publications

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“…On the other hand, in reactors inoculated with M. barkeri , the detected H 2 stabilized at 0.065 ± 0.02 mM, similar to concentrations observed for co-cultures of M. barkeri with (0.077 ± 0.03 mM) or without conductive particles (0.076 ± 0.06 mM) and in pure culture (0.068 mM). This is supported by previous research, which demonstrated H 2 -cycling (H 2 -production and H 2 -uptake) in M. barkeri (Kulkarni et al, 2009, 2018; Mand et al, 2018). The cellular evolved H 2 is well above the H 2 -uptake threshold for M. barkeri (296 nM −376 nM) (Kral et al, 1998; Lovley, 1985) possibly because in these cultures there is an alternative, competitive electron donor.…”

Section: Resultssupporting

confidence: 90%

Extracellular Electron Uptake by TwoMethanosarcinaSpecies

Snoeyenbos-West

Thamdrup

Ottosen

et al. 2018

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9Direct electron uptake by prokaryotes is a recently described mechanism with a potential application for 10 energy and CO 2 storage into value added chemicals. Members of Methanosarcinales, an 11 environmentally and biotechnologically relevant group of methanogens, were previously shown to 12 retrieve electrons from an extracellular electrogenic partner performing Direct Interspecies Electron 13 45 (Holmes et al., 2018b; Shrestha et al., 2013). Geobacter's requirement for outer-surface proteins during 46 DIET was confirmed earlier with gene-deletion studies (Rotaru et al., 2014a(Rotaru et al., , 2014b. Thus, if 47 Geobacter lacked the ability to produce e.g. pili it was incapable of DIET. 48Although we understand how Geobacter releases electrons outside their cells during DIET, the way 49 Methanosarcinales retrieve DIET-electrons is poorly understood. A glimpse at this mechanism was 50 provided in a recent comparative transcriptomic study (Holmes et al., 2018b). In this study, the 51 transcriptomes of DIET co-cultures (G. metallireducens -Methanosarcina barkeri) were compared to 52 those of co-cultures performing interspecies H 2 -transfer (Pelobacter carbinolicus -M. barkeri). During 53 DIET, M. barkeri had higher expression of membrane-bound redox-active proteins like cupredoxins, 54 thioredoxins, pyrroloquinoline, and quinone-, cytochrome-or Fe-S containing proteins (Holmes et al., 55 2018b). Still, the exact mechanism of electron uptake by Methanosarcina has not been validated and 56 warrants further investigation. 57 Moreover, Methanosarcina can also retrieve electrons from electrically conductive particles charged by 58 Geobacter oxidizing organics (Shrestha and Rotaru, 2014). In effect, DIET is accelerated by electrically 59 conductive particles/minerals perhaps because they replace conductive and redox active surface proteins 60 diminishing cellular energy expenditure required to overexpress such surface constituents (Liu et al., 61 2012). For instance, co-cultures of G. metallireducens and M. barkeri were stimulated at the addition of 62 conductive particles, such as GAC (Liu et al., 2012), carbon cloth (Chen et al., 2014a), biochar (Chen et 63 al., 2014b), or magnetite (Wang et al., 2018). On the other hand, the addition of non-conductive 64 materials did not stimulate DIET (Chen et al., 2014a; Rotaru et al., 2018a). In addition, conductive 65 particles appear to play a significant role in interspecies interactions from natural and artificial 66 ecosystems such as sediments, soils, rice paddies or anaerobic digesters (Holmes et al., 2017a; Rotaru et 67 al., 2018b; Ye et al., 2018; Zhang et al., 2018). In these cases, the addition of conductive particles 68 enriched for DIET-related Methanosarcinales (Dang et al., 2016; Holmes et al., 2017b; Rotaru et al., 69 2018a; Zheng et al., 2015). However, exceptions were observed since occasionally conductive particles 70 enriched for H 2 -utilizing methanogens of the genus Methanospirillum (Lee et al., 2016) or 71Methanobacterium (Lin et al., 2017; Zhua...

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Section: Resultssupporting

confidence: 90%

Extracellular Electron Uptake by TwoMethanosarcinaSpecies

Snoeyenbos-West

Thamdrup

Ottosen

et al. 2018

Preprint

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“…Bacterial Culturing Conditions. The strains of Methanosarcina barkeri used have 325 been described previously (24,27). In brief, a background strain containing the Δhpt::ΦC31int-attP promoter fusion was utilized for wild-type experiments as this strain was utilized for genetic manipulations (28,29).…”

Section: Methodsmentioning

confidence: 99%

“…In brief, a background strain containing the Δhpt::ΦC31int-attP promoter fusion was utilized for wild-type experiments as this strain was utilized for genetic manipulations (28,29). A hydrogenase deletion mutant with the Ech, Vhu, and Fhr hydrogenases removed was utilized for delta-hydrogenase experiments (24). Each strain was provided by William Metcalf at the University of 330 Illinois at Urbana-Champaign.…”

Section: Methodsmentioning

confidence: 99%

“…electrodes poised below -400 mV. 195 To confirm the impact of a non-hydrogenase mediated mechanism of electron uptake that can be utilized for methane formation, electrochemical experiments were performed using a hydrogenase deletion mutant of M. barkeri (24). This mutant lacks the genes for all three types of hydrogenase present in M. barkeri: the ferredoxin-dependent energy converting hydrogenase (Ech), the cytoplasmic F420-dependent hydrogenase (Frh), and 200 the periplasmic methanophenazine-dependent hydrogenase (Vht).…”

Section: Current Generation Observed In M Barkeri Hydrogenase Deletimentioning

confidence: 99%

“…Furthermore, this mutant does not grow with hydrogen as an electron donor or via acetate disproportionation. However, this strain is capable of growth via methanol disproportionation (24). Cathodic currents (i.e.…”

Section: Current Generation Observed In M Barkeri Hydrogenase Deletimentioning

confidence: 99%

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Methane-linked mechanisms of electron uptake from cathodes byMethanosarcina barkeri

Rowe

Gardel

et al. 2018

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20The Methanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes by Methanosarcina barkeri, which is an important model organism that is genetically 25 tractable and utilizes a wide range of substrates for methanogenesis. Here we confirm the ability of M. barkeri to perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g. 30 hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g. hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production in M. barkeri. Our electrochemical 35 measurements of wild-type and mutant strains point to a novel and extracellularenzyme-free mode of electron uptake able to take up electrons at potentials lower than -498 mV vs. SHE (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest that M. barkeri can perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent) of 40 electrode interactions on cathodes including a mechanism pointing to a direct interaction, which has significant applied and ecological implications. ImportanceMethanogenic Archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating 45 gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens has been well studied with respect to soluble substrates, mechanistic understanding of their interaction with solid phase redox active compounds is limited. This work provides insight into solid phase redox interactions in Methanosarcina using electrochemical methods. We highlight a previously undescribed 50 mode of electron uptake from cathodes, that is potentially informative of direct interspecies electron transfer interactions in the Methanosarcinales.

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