Substrate-level Phosphorylation Is The Primary Source Of ... - PubMed

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Abstract

It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that Shewanella oneidensis strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking ackA (SO2915) and pta (SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking ackA and pta were also shown to grow slowly using N-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on N-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although S. oneidensis requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an ackA deletion in Shewanella sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of Shewanella.

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Figures

FIG. 1.

FIG. 1.

Simplified model of S. oneidensis …

FIG. 1.

Simplified model of S. oneidensis central metabolism. Entner-Doudoroff glycolysis yields two molecules of…

FIG. 1. Simplified model of S. oneidensis central metabolism. Entner-Doudoroff glycolysis yields two molecules of pyruvate. Under aerobic conditions, pyruvate facilitates the reduction of NAD+ to NADH before being completely oxidized to carbon dioxide in the TCA cycle. Anaerobically, pyruvate oxidation to acetyl-CoA yields formate before the pyruvate is converted to acetate. Formate is subsequently oxidized to carbon dioxide. Reactions catalyzed by acetate kinase and phosphate acetyltransferase are denoted AckA and Pta, respectively. QH2 is reduced quinone. The model is based on several references (5, 24, 29, 31, 36).
FIG. 2.

FIG. 2.

AckA and Pta are required…

FIG. 2.

AckA and Pta are required for anaerobic growth of S. oneidensis using lactate.…

FIG. 2. AckA and Pta are required for anaerobic growth of S. oneidensis using lactate. (A) Cultures of wild-type MR-1 (•) and the ΔackA (▴), Δpta (▾), and ΔackA Δpta (⧫) strains were grown anaerobically in minimal medium using 20 mM lactate and 40 mM fumarate. Data presented are averages from quadruplicate independent cultures; error bars represent the standard errors of the mean (SEM). (B) Complementation of the ackA and pta mutants grown anaerobically with 20 mM lactate and 40 mM fumarate. Strains containing the empty vector (pBBR1MCS-2) are MR-1 (•), the ΔackA mutant (▴), and the Δpta mutant (▾). Strains containing complementation vectors for the ackA (▵) and pta (▿) mutations in the respective deletion backgrounds. Data presented are averages from triplicate independent cultures; error bars represent the SEM. OD600, OD at 600 nm.
FIG. 3.

FIG. 3.

Mutant strains lacking ackA and/or …

FIG. 3.

Mutant strains lacking ackA and/or pta are strongly defective when grown anaerobically with…

FIG. 3. Mutant strains lacking ackA and/or pta are strongly defective when grown anaerobically with NAG. Cultures of wild-type MR-1 (•) and the ΔackA (▴), Δpta (▾), and ΔackA Δpta (⧫) strains were grown anaerobically in minimal medium using 10 mM NAG and 40 mM fumarate. Doubling times of 25 ± 4 h, 130 ± 20 h, 116 ± 5 h, and 127 ± 13 h were observed for the wild-type ΔackA, Δpta, and ΔackA Δpta strains, respectively. Cultures reached maximum optical densities of 0.48, 0.13, 0.25, and 0.25 for the wild-type, ΔackA, Δpta, and ΔackA Δpta strains, respectively. Data presented are averages from triplicate independent cultures, and error bars represent the SEM.
FIG. 4.

FIG. 4.

ATP synthase is not essential…

FIG. 4.

ATP synthase is not essential for anaerobic growth of MR-1. (A) Cultures of…

FIG. 4. ATP synthase is not essential for anaerobic growth of MR-1. (A) Cultures of wild-type MR-1 (•) and the Δatp mutant (▪) were grown aerobically in minimal medium with 40 mM NAG. (B) Cultures of MR-1 with the empty vector (pBBR1MCS-2) (•), the Δatp mutant with the empty vector (▪), and the Δatp mutant with a complementation vector (patp) (□) grown aerobically with 40 mM NAG. (C) Anaerobic growth of MR-1 (•) and the Δatp mutant (▪) with 10 mM NAG and 40 mM fumarate. (D) Anaerobic growth of the same strains described for panel C with 20 mM lactate and 40 mM fumarate. Data presented are averages from triplicate independent cultures, with error bars representing the SEM.
FIG. 5.

FIG. 5.

Proteorhodopsin enhances growth of strains…

FIG. 5.

Proteorhodopsin enhances growth of strains lacking ATP synthase. Cultures of the wild-type MR-1…

FIG. 5. Proteorhodopsin enhances growth of strains lacking ATP synthase. Cultures of the wild-type MR-1 (circles) and Δatp (squares) strains of S. oneidensis expressing PR were grown anaerobically in minimal medium with retinal and kanamycin, using 20 mM lactate and 40 mM fumarate. Results from cultures grown with and without light are represented by solid and dashed lines, respectively. Data presented are averages from an experiment performed in duplicate, and error bars are omitted for clarity (SEM, <5% deviation).
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References

    1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296. - PMC - PubMed
    1. Beja, O., L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong. 2000. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289:1902-1906. - PubMed
    1. Beliaev, A. S., D. M. Klingeman, J. A. Klappenbach, L. Wu, M. F. Romine, J. M. Tiedje, K. H. Nealson, J. K. Fredrickson, and J. Zhou. 2005. Global transcriptome analysis of Shewanella oneidensis MR-1 exposed to different terminal electron acceptors. J. Bacteriol. 187:7138-7145. - PMC - PubMed
    1. Bond, D. R., and D. R. Lovley. 2005. Evidence for involvement of an electron shuttle in electricity generation by Geothrix fermentans. Appl. Environ. Microbiol. 71:2186-2189. - PMC - PubMed
    1. Chai, Y., R. Kolter, and R. Losick. 2009. A widely conserved gene cluster required for lactate utilization in Bacillus subtilis and its involvement in biofilm formation. J. Bacteriol. 191:2423-2430. - PMC - PubMed
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