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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

Construction of plasmid pJC1 and strain verification. (A) Map of plasmid pJC1 containing the hdrABC* operon under the control of the Ptet promoter. The asterisks indicate the locations of point mutations in the hdrABC operon. (B) Agarose gel showing PCR identification of each strain.
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Figure 2: Construction of plasmid pJC1 and strain verification. (A) Map of plasmid pJC1 containing the hdrABC* operon under the control of the Ptet promoter. The asterisks indicate the locations of point mutations in the hdrABC operon. (B) Agarose gel showing PCR identification of each strain.

Mentions: To determine the effect of increased HdrABC enzyme activity on cell physiology, we created a plasmid to introduce a second hdrABC* operon on the M. acetivorans chromosome (Fig. 2A). The open reading frames corresponding to the methylotrophic hdrABC operon, MA3126-MA3128, were amplified from the M. acetivorans C2A chromosome and cloned into the pJK027A plasmid, resulting in plasmid pJC1. The hdrABC operon in pJC1 was designed to include five mutations to distinguish the inserted genes from the wild-type genes by removing NdeI restriction sites (see Table S1 in the supplemental material). Four of the mutations are silent mutations that do not change the translated protein sequence. One mutation necessitated a change to the translated protein primary sequence so that the pJC1 plasmid expressed HdrBM249V mutant protein. The HdrB M249V mutation is in a predicted disordered region within the second cysteine-rich motif at residues 166 to 257 (InterProScan) (27).


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

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

Construction of plasmid pJC1 and strain verification. (A) Map of plasmid pJC1 containing the hdrABC* operon under the control of the Ptet promoter. The asterisks indicate the locations of point mutations in the hdrABC operon. (B) Agarose gel showing PCR identification of each strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Construction of plasmid pJC1 and strain verification. (A) Map of plasmid pJC1 containing the hdrABC* operon under the control of the Ptet promoter. The asterisks indicate the locations of point mutations in the hdrABC operon. (B) Agarose gel showing PCR identification of each strain.
Mentions: To determine the effect of increased HdrABC enzyme activity on cell physiology, we created a plasmid to introduce a second hdrABC* operon on the M. acetivorans chromosome (Fig. 2A). The open reading frames corresponding to the methylotrophic hdrABC operon, MA3126-MA3128, were amplified from the M. acetivorans C2A chromosome and cloned into the pJK027A plasmid, resulting in plasmid pJC1. The hdrABC operon in pJC1 was designed to include five mutations to distinguish the inserted genes from the wild-type genes by removing NdeI restriction sites (see Table S1 in the supplemental material). Four of the mutations are silent mutations that do not change the translated protein sequence. One mutation necessitated a change to the translated protein primary sequence so that the pJC1 plasmid expressed HdrBM249V mutant protein. The HdrB M249V mutation is in a predicted disordered region within the second cysteine-rich motif at residues 166 to 257 (InterProScan) (27).

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