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Functional responses of methanogenic archaea to syntrophic growth.

Walker CB, Redding-Johanson AM, Baidoo EE, Rajeev L, He Z, Hendrickson EL, Joachimiak MP, Stolyar S, Arkin AP, Leigh JA, Zhou J, Keasling JD, Mukhopadhyay A, Stahl DA - ISME J (2012)

Bottom Line: These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis.A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)).The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, University of Washington, Seattle, WA, USA.

ABSTRACT
Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism.

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Related in: MedlinePlus

Conceptual schematic of M. maripaludis and D. vulgaris syntrophic interaction, highlighting the central energy-generating and -consuming the pathways of the methanogen. Relative changes (Table 2) in transcript abundance during syntrophic growth are indicated by red (increase) and green (decrease) coloration. Blue coloration indicates no statistically significant change as specified in the Materials and methods section. Hydrogen-limited monocultures served as the control growth condition. Oxidation of formate to CO2 and H2 coupled with coenzyme F420-reduction (not depicted) is catalyzed by two alternative formate dehydrogenases, Fdh1 and Fdh2. One of two membrane-bound energy-conserving hydrogenases (Eha and Ehb) couple the chemiosmotic energy of ion gradients to H2 oxidation and ferredoxin reduction. Of these, Ehb generates the low potential electron carrier used for anabolism, whereas Eha is hypothesized to function primarily in the energy-generating methanogenesis pathway, generating low potential-reducing equivalents for the reduction of CO2 to formylmethanofuran (Major et al., 2010). Two different formylmethanofuran dehydrogenases catalyze this first step in methanogenesis, tungsten (Fwd) and molybdenum (Fmd) forms. Transfer of the formyl group from methanofuran to methanopterin by Ftr and subsequent elimination of H2O by Mch yields methenyl-H4-methanopterin. Two different enzymes can then reduce methenyl-H4-MPT to methylene-H4MPT, one (Mtd) using H2 as reductant and the other (Hmd) using reduced coenzyme F420. M. maripaludis has an Hmd paralog of unknown function (Mmp1716, HmdII) that may also function in this step (Hendrickson et al., 2004). Reduction of methylene-H4MPT by another F420-dependent reductase (Mer) yields methyl-H4MPT. The reduced coenzyme F420 required for the formation of methyl-H4MPT by these two steps is generated by one of two alternative F420-reducting hydrogenase (Fru and Frc). The final steps to methane production are catalyzed by a methyl transferase (Mtr) and a reductase (Mcr) coupled to two forms of a F420-nonreducing hydrogenase (Vhu and Vhc). The mixed disulfide (CoM-CoB) produced by reduction of methyl coenzyme M is then reduced by one of two forms of the heterodisulfide reductase determined by the composition of the HdrA subunit (HdrAU or HdrAV). Fdh/ Hdr/Vhu/ Fwd are reported to have protein–protein interactions (Costa et al., 2010). (Note: The interaction with Fwd could not be depicted here without compromising the clarity of the figure. Vhc is not part of this interaction). Other reactions include the transport of alanine (AlsT), and subsequent coversion to pyruvate via an alanine racemase (Alr) and dehydrogenase (Ald). Additional M. maripaludis proteins shown: pyruvate oxidoreductase, acetyl-CoA decarbonylase/synthase. The D. vulgaris metabolic pathway is based upon results as described in Walker et al., 2009 and is updated to include an unspecified sodium/alanine transporter (Na+/ala sym). Other D. vulgaris proteins shown: lactate permease (lac per), lactate deydrogenase (ldh), membrane-bound Coo hydrogenase (Coo), high-molecular weight cytochrome (Hmc), periplasmic hydrogenases (Hyd and Hyn), cytochrome c3 (Cyt c3) and oxidized and reduced ferredoxin (Fd).
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fig1: Conceptual schematic of M. maripaludis and D. vulgaris syntrophic interaction, highlighting the central energy-generating and -consuming the pathways of the methanogen. Relative changes (Table 2) in transcript abundance during syntrophic growth are indicated by red (increase) and green (decrease) coloration. Blue coloration indicates no statistically significant change as specified in the Materials and methods section. Hydrogen-limited monocultures served as the control growth condition. Oxidation of formate to CO2 and H2 coupled with coenzyme F420-reduction (not depicted) is catalyzed by two alternative formate dehydrogenases, Fdh1 and Fdh2. One of two membrane-bound energy-conserving hydrogenases (Eha and Ehb) couple the chemiosmotic energy of ion gradients to H2 oxidation and ferredoxin reduction. Of these, Ehb generates the low potential electron carrier used for anabolism, whereas Eha is hypothesized to function primarily in the energy-generating methanogenesis pathway, generating low potential-reducing equivalents for the reduction of CO2 to formylmethanofuran (Major et al., 2010). Two different formylmethanofuran dehydrogenases catalyze this first step in methanogenesis, tungsten (Fwd) and molybdenum (Fmd) forms. Transfer of the formyl group from methanofuran to methanopterin by Ftr and subsequent elimination of H2O by Mch yields methenyl-H4-methanopterin. Two different enzymes can then reduce methenyl-H4-MPT to methylene-H4MPT, one (Mtd) using H2 as reductant and the other (Hmd) using reduced coenzyme F420. M. maripaludis has an Hmd paralog of unknown function (Mmp1716, HmdII) that may also function in this step (Hendrickson et al., 2004). Reduction of methylene-H4MPT by another F420-dependent reductase (Mer) yields methyl-H4MPT. The reduced coenzyme F420 required for the formation of methyl-H4MPT by these two steps is generated by one of two alternative F420-reducting hydrogenase (Fru and Frc). The final steps to methane production are catalyzed by a methyl transferase (Mtr) and a reductase (Mcr) coupled to two forms of a F420-nonreducing hydrogenase (Vhu and Vhc). The mixed disulfide (CoM-CoB) produced by reduction of methyl coenzyme M is then reduced by one of two forms of the heterodisulfide reductase determined by the composition of the HdrA subunit (HdrAU or HdrAV). Fdh/ Hdr/Vhu/ Fwd are reported to have protein–protein interactions (Costa et al., 2010). (Note: The interaction with Fwd could not be depicted here without compromising the clarity of the figure. Vhc is not part of this interaction). Other reactions include the transport of alanine (AlsT), and subsequent coversion to pyruvate via an alanine racemase (Alr) and dehydrogenase (Ald). Additional M. maripaludis proteins shown: pyruvate oxidoreductase, acetyl-CoA decarbonylase/synthase. The D. vulgaris metabolic pathway is based upon results as described in Walker et al., 2009 and is updated to include an unspecified sodium/alanine transporter (Na+/ala sym). Other D. vulgaris proteins shown: lactate permease (lac per), lactate deydrogenase (ldh), membrane-bound Coo hydrogenase (Coo), high-molecular weight cytochrome (Hmc), periplasmic hydrogenases (Hyd and Hyn), cytochrome c3 (Cyt c3) and oxidized and reduced ferredoxin (Fd).

Mentions: The most general change associated with syntrophic growth was a decrease in transcripts for energy-consuming biosynthetic functions (for example, pyruvate oxidoreductase (Por), acetyl-CoA decarbonylase/synthase and energy-conserving hydrogenase B (Ehb), see Figure 1 for enzymatic reaction depictions) and an increase in transcripts in the energy-generating methanogenesis pathway (see Table 2 and Supplementary Tables S2 and S4; Supplementary Figure S1) plots transcriptional changes according to function as defined by clusters of orthologous groups). Figure 1 illustrates the differential expression of transcripts observed in coculture compared with hydrogen-limited M. maripaludis monocultures. However, compared with growth in monoculture under hydrogen limitation, transcript levels for isofunctional genes often diverged, with transcripts of one variant significantly increasing, whereas the other was little changed or significantly decreased in their relative abundances. A common feature was an apparent increase in transcripts for functions using H2 directly as reductant, versus those using the reduced deazaflavin (coenzyme F420). In several cases, the proteins were confidently identified (Table 3 and Figure 2) and the corresponding changes generally corroborated the observations at the transcript level. The greater importance of direct reduction by H2 was supported by improved syntrophic growth of a deletion mutant in an F420-dependent dehydrogenase of M. maripaludis (described in more detail below). Metabolite, transcript and proteomic analyses also pointed to a unique, although undefined, role for alanine utilization within this syntrophic coupling.


Functional responses of methanogenic archaea to syntrophic growth.

Walker CB, Redding-Johanson AM, Baidoo EE, Rajeev L, He Z, Hendrickson EL, Joachimiak MP, Stolyar S, Arkin AP, Leigh JA, Zhou J, Keasling JD, Mukhopadhyay A, Stahl DA - ISME J (2012)

Conceptual schematic of M. maripaludis and D. vulgaris syntrophic interaction, highlighting the central energy-generating and -consuming the pathways of the methanogen. Relative changes (Table 2) in transcript abundance during syntrophic growth are indicated by red (increase) and green (decrease) coloration. Blue coloration indicates no statistically significant change as specified in the Materials and methods section. Hydrogen-limited monocultures served as the control growth condition. Oxidation of formate to CO2 and H2 coupled with coenzyme F420-reduction (not depicted) is catalyzed by two alternative formate dehydrogenases, Fdh1 and Fdh2. One of two membrane-bound energy-conserving hydrogenases (Eha and Ehb) couple the chemiosmotic energy of ion gradients to H2 oxidation and ferredoxin reduction. Of these, Ehb generates the low potential electron carrier used for anabolism, whereas Eha is hypothesized to function primarily in the energy-generating methanogenesis pathway, generating low potential-reducing equivalents for the reduction of CO2 to formylmethanofuran (Major et al., 2010). Two different formylmethanofuran dehydrogenases catalyze this first step in methanogenesis, tungsten (Fwd) and molybdenum (Fmd) forms. Transfer of the formyl group from methanofuran to methanopterin by Ftr and subsequent elimination of H2O by Mch yields methenyl-H4-methanopterin. Two different enzymes can then reduce methenyl-H4-MPT to methylene-H4MPT, one (Mtd) using H2 as reductant and the other (Hmd) using reduced coenzyme F420. M. maripaludis has an Hmd paralog of unknown function (Mmp1716, HmdII) that may also function in this step (Hendrickson et al., 2004). Reduction of methylene-H4MPT by another F420-dependent reductase (Mer) yields methyl-H4MPT. The reduced coenzyme F420 required for the formation of methyl-H4MPT by these two steps is generated by one of two alternative F420-reducting hydrogenase (Fru and Frc). The final steps to methane production are catalyzed by a methyl transferase (Mtr) and a reductase (Mcr) coupled to two forms of a F420-nonreducing hydrogenase (Vhu and Vhc). The mixed disulfide (CoM-CoB) produced by reduction of methyl coenzyme M is then reduced by one of two forms of the heterodisulfide reductase determined by the composition of the HdrA subunit (HdrAU or HdrAV). Fdh/ Hdr/Vhu/ Fwd are reported to have protein–protein interactions (Costa et al., 2010). (Note: The interaction with Fwd could not be depicted here without compromising the clarity of the figure. Vhc is not part of this interaction). Other reactions include the transport of alanine (AlsT), and subsequent coversion to pyruvate via an alanine racemase (Alr) and dehydrogenase (Ald). Additional M. maripaludis proteins shown: pyruvate oxidoreductase, acetyl-CoA decarbonylase/synthase. The D. vulgaris metabolic pathway is based upon results as described in Walker et al., 2009 and is updated to include an unspecified sodium/alanine transporter (Na+/ala sym). Other D. vulgaris proteins shown: lactate permease (lac per), lactate deydrogenase (ldh), membrane-bound Coo hydrogenase (Coo), high-molecular weight cytochrome (Hmc), periplasmic hydrogenases (Hyd and Hyn), cytochrome c3 (Cyt c3) and oxidized and reduced ferredoxin (Fd).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475374&req=5

fig1: Conceptual schematic of M. maripaludis and D. vulgaris syntrophic interaction, highlighting the central energy-generating and -consuming the pathways of the methanogen. Relative changes (Table 2) in transcript abundance during syntrophic growth are indicated by red (increase) and green (decrease) coloration. Blue coloration indicates no statistically significant change as specified in the Materials and methods section. Hydrogen-limited monocultures served as the control growth condition. Oxidation of formate to CO2 and H2 coupled with coenzyme F420-reduction (not depicted) is catalyzed by two alternative formate dehydrogenases, Fdh1 and Fdh2. One of two membrane-bound energy-conserving hydrogenases (Eha and Ehb) couple the chemiosmotic energy of ion gradients to H2 oxidation and ferredoxin reduction. Of these, Ehb generates the low potential electron carrier used for anabolism, whereas Eha is hypothesized to function primarily in the energy-generating methanogenesis pathway, generating low potential-reducing equivalents for the reduction of CO2 to formylmethanofuran (Major et al., 2010). Two different formylmethanofuran dehydrogenases catalyze this first step in methanogenesis, tungsten (Fwd) and molybdenum (Fmd) forms. Transfer of the formyl group from methanofuran to methanopterin by Ftr and subsequent elimination of H2O by Mch yields methenyl-H4-methanopterin. Two different enzymes can then reduce methenyl-H4-MPT to methylene-H4MPT, one (Mtd) using H2 as reductant and the other (Hmd) using reduced coenzyme F420. M. maripaludis has an Hmd paralog of unknown function (Mmp1716, HmdII) that may also function in this step (Hendrickson et al., 2004). Reduction of methylene-H4MPT by another F420-dependent reductase (Mer) yields methyl-H4MPT. The reduced coenzyme F420 required for the formation of methyl-H4MPT by these two steps is generated by one of two alternative F420-reducting hydrogenase (Fru and Frc). The final steps to methane production are catalyzed by a methyl transferase (Mtr) and a reductase (Mcr) coupled to two forms of a F420-nonreducing hydrogenase (Vhu and Vhc). The mixed disulfide (CoM-CoB) produced by reduction of methyl coenzyme M is then reduced by one of two forms of the heterodisulfide reductase determined by the composition of the HdrA subunit (HdrAU or HdrAV). Fdh/ Hdr/Vhu/ Fwd are reported to have protein–protein interactions (Costa et al., 2010). (Note: The interaction with Fwd could not be depicted here without compromising the clarity of the figure. Vhc is not part of this interaction). Other reactions include the transport of alanine (AlsT), and subsequent coversion to pyruvate via an alanine racemase (Alr) and dehydrogenase (Ald). Additional M. maripaludis proteins shown: pyruvate oxidoreductase, acetyl-CoA decarbonylase/synthase. The D. vulgaris metabolic pathway is based upon results as described in Walker et al., 2009 and is updated to include an unspecified sodium/alanine transporter (Na+/ala sym). Other D. vulgaris proteins shown: lactate permease (lac per), lactate deydrogenase (ldh), membrane-bound Coo hydrogenase (Coo), high-molecular weight cytochrome (Hmc), periplasmic hydrogenases (Hyd and Hyn), cytochrome c3 (Cyt c3) and oxidized and reduced ferredoxin (Fd).
Mentions: The most general change associated with syntrophic growth was a decrease in transcripts for energy-consuming biosynthetic functions (for example, pyruvate oxidoreductase (Por), acetyl-CoA decarbonylase/synthase and energy-conserving hydrogenase B (Ehb), see Figure 1 for enzymatic reaction depictions) and an increase in transcripts in the energy-generating methanogenesis pathway (see Table 2 and Supplementary Tables S2 and S4; Supplementary Figure S1) plots transcriptional changes according to function as defined by clusters of orthologous groups). Figure 1 illustrates the differential expression of transcripts observed in coculture compared with hydrogen-limited M. maripaludis monocultures. However, compared with growth in monoculture under hydrogen limitation, transcript levels for isofunctional genes often diverged, with transcripts of one variant significantly increasing, whereas the other was little changed or significantly decreased in their relative abundances. A common feature was an apparent increase in transcripts for functions using H2 directly as reductant, versus those using the reduced deazaflavin (coenzyme F420). In several cases, the proteins were confidently identified (Table 3 and Figure 2) and the corresponding changes generally corroborated the observations at the transcript level. The greater importance of direct reduction by H2 was supported by improved syntrophic growth of a deletion mutant in an F420-dependent dehydrogenase of M. maripaludis (described in more detail below). Metabolite, transcript and proteomic analyses also pointed to a unique, although undefined, role for alanine utilization within this syntrophic coupling.

Bottom Line: These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis.A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)).The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, University of Washington, Seattle, WA, USA.

ABSTRACT
Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism.

Show MeSH
Related in: MedlinePlus