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Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production.

Catlett JL, Ortiz AM, Buan NR - Appl. Environ. Microbiol. (2015)

Bottom Line: In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates.When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates.These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.

No MeSH data available.


Related in: MedlinePlus

Phenotypes of parent and hdrABC mutant strains. (A) Growth curve of methanol-adapted strains. Growth data were collected from triplicate biological replicates. (B and C) The rates of methane production by cell suspensions were measured from cells grown on methanol (B) and acetate (C). Methane production was measured in triplicate biological and triplicate technical replicates. *, P < 0.0001. (D) Heterodisulfide reductase activity in methanol-grown cell extracts. Assays were performed in triplicate. The error bars indicate the standard deviations and may not be visible behind the symbols.
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Figure 3: Phenotypes of parent and hdrABC mutant strains. (A) Growth curve of methanol-adapted strains. Growth data were collected from triplicate biological replicates. (B and C) The rates of methane production by cell suspensions were measured from cells grown on methanol (B) and acetate (C). Methane production was measured in triplicate biological and triplicate technical replicates. *, P < 0.0001. (D) Heterodisulfide reductase activity in methanol-grown cell extracts. Assays were performed in triplicate. The error bars indicate the standard deviations and may not be visible behind the symbols.

Mentions: Hdr enzyme activity in cell extracts of each strain was measured to check that the HdrB M249V point mutations did not abolish enzyme activity (Fig. 3D). Assaying Hdr enzyme activity in cell extracts is complicated by the fact that M. acetivorans expresses multiple hdr genes during growth on methanol. The hdrA1C1B1 (MA3126-MA3128) operon is methanol specific but nonessential, while genes encoding the membrane-bound HdrED (MA0687-MA0688) are essential. M. acetivorans also expresses hdrD2 (MA0526), hdrA2, polyferredoxin genes (MA2867-MA2868), and hdrC2B2 (MA4236-MA4237), which are constitutively expressed. We assayed CoM-S-S-CoB-dependent methyl viologen oxidation in clarified cell extracts to minimize HdrED protein levels, which could mask HdrABC activity. Methyl viologen was used as the electron donor because the specific electron donor for M. acetivorans HdrABC is unknown.


Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production.

Catlett JL, Ortiz AM, Buan NR - Appl. Environ. Microbiol. (2015)

Phenotypes of parent and hdrABC mutant strains. (A) Growth curve of methanol-adapted strains. Growth data were collected from triplicate biological replicates. (B and C) The rates of methane production by cell suspensions were measured from cells grown on methanol (B) and acetate (C). Methane production was measured in triplicate biological and triplicate technical replicates. *, P < 0.0001. (D) Heterodisulfide reductase activity in methanol-grown cell extracts. Assays were performed in triplicate. The error bars indicate the standard deviations and may not be visible behind the symbols.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561719&req=5

Figure 3: Phenotypes of parent and hdrABC mutant strains. (A) Growth curve of methanol-adapted strains. Growth data were collected from triplicate biological replicates. (B and C) The rates of methane production by cell suspensions were measured from cells grown on methanol (B) and acetate (C). Methane production was measured in triplicate biological and triplicate technical replicates. *, P < 0.0001. (D) Heterodisulfide reductase activity in methanol-grown cell extracts. Assays were performed in triplicate. The error bars indicate the standard deviations and may not be visible behind the symbols.
Mentions: Hdr enzyme activity in cell extracts of each strain was measured to check that the HdrB M249V point mutations did not abolish enzyme activity (Fig. 3D). Assaying Hdr enzyme activity in cell extracts is complicated by the fact that M. acetivorans expresses multiple hdr genes during growth on methanol. The hdrA1C1B1 (MA3126-MA3128) operon is methanol specific but nonessential, while genes encoding the membrane-bound HdrED (MA0687-MA0688) are essential. M. acetivorans also expresses hdrD2 (MA0526), hdrA2, polyferredoxin genes (MA2867-MA2868), and hdrC2B2 (MA4236-MA4237), which are constitutively expressed. We assayed CoM-S-S-CoB-dependent methyl viologen oxidation in clarified cell extracts to minimize HdrED protein levels, which could mask HdrABC activity. Methyl viologen was used as the electron donor because the specific electron donor for M. acetivorans HdrABC is unknown.

Bottom Line: In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates.When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates.These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.

No MeSH data available.


Related in: MedlinePlus