Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Johnson, Ethan T.; Baron, Daniel; Naranjo, Belén; Bond, Daniel R.; Schmidt‐Dannert, Claudia; Gralnick, Jeffrey A.

doi:10.1128/aem.02425-09

Cited by 84 publications

(74 citation statements)

References 44 publications

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“…Subsequent studies on aquatic PR-containing bacteria have confirmed this finding (146). In a heterologous expression system, S. oneidensis MR-1 produced 4.0 ϫ 10 4 PR molecules per cell while growing anaerobically in minimal medium (15). In another heterologous expression system, Martinez et al (127) found that starved E. coli harboring recombinant PR retrieved 2.2 ϫ 10 5 molecules of ATP from 5 min of illumination, which was compared to 9 ϫ 10 5 molecules of ATP from heterotrophic utilization of succinate under similar conditions.…”

Section: Toward Quantification Of Marine Photoheterotrophymentioning

confidence: 50%

“…Johnson et al investigated the benefit of PR proton pumping for physiological functions in another heterotrophic Gammaproteobacteria species, Shewanella oneidensis MR-1, in part also to explore potential practical aspects of metabolic engineering (15). The PR gene from a SAR86 clade bacterium expressed from a broad-host-range vector in S. oneidensis resulted in an estimated 40,000 PR molecules per cell, and light exposure of starving cells caused similar levels of energized cells as amendment of the medium with lactate as an organic matter source under anaerobic conditions.…”

Section: Clues To the Mechanisms Of Proteorhodopsin Photophysiology Fmentioning

confidence: 99%

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Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology

Pinhassi

DeLong

Béjà

et al. 2016

Microbiol Mol Biol Rev

175

172

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SUMMARYThe recognition of a new family of rhodopsins in marine planktonic bacteria, proton-pumping proteorhodopsin, expanded the known phylogenetic range, environmental distribution, and sequence diversity of retinylidene photoproteins. At the time of this discovery, microbial ion-pumping rhodopsins were known solely in haloarchaea inhabiting extreme hypersaline environments. Shortly thereafter, proteorhodopsins and other light-activated energy-generating rhodopsins were recognized to be widespread among marine bacteria. The ubiquity of marine rhodopsin photosystems now challenges prior understanding of the nature and contributions of “heterotrophic” bacteria to biogeochemical carbon cycling and energy fluxes. Subsequent investigations have focused on the biophysics and biochemistry of these novel microbial rhodopsins, their distribution across the tree of life, evolutionary trajectories, and functional expression in nature. Later discoveries included the identification of proteorhodopsin genes in all three domains of life, the spectral tuning of rhodopsin variants to wavelengths prevailing in the sea, variable light-activated ion-pumping specificities among bacterial rhodopsin variants, and the widespread lateral gene transfer of biosynthetic genes for bacterial rhodopsins and their associated photopigments. Heterologous expression experiments with marine rhodopsin genes (and associated retinal chromophore genes) provided early evidence that light energy harvested by rhodopsins could be harnessed to provide biochemical energy. Importantly, some studies with native marine bacteria show that rhodopsin-containing bacteria use light to enhance growth or promote survival during starvation. We infer from the distribution of rhodopsin genes in diverse genomic contexts that different marine bacteria probably use rhodopsins to support light-dependent fitness strategies somewhere between these two extremes.

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Section: Toward Quantification Of Marine Photoheterotrophymentioning

confidence: 50%

Section: Clues To the Mechanisms Of Proteorhodopsin Photophysiology Fmentioning

confidence: 99%

Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology

Pinhassi

DeLong

Béjà

et al. 2016

Microbiol Mol Biol Rev

175

172

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“…PRs have also been heterologously expressed in non-photosynthetic hosts (5,(21)(22)(23)(24)(25). For example, the introduction of a PR in Shewanella oneidensis increased the pmf, which resulted in increases in electrical current generation, lactate uptake, and survival under starvation conditions (23,24).…”

Section: Introductionmentioning

confidence: 99%

“…For example, the introduction of a PR in Shewanella oneidensis increased the pmf, which resulted in increases in electrical current generation, lactate uptake, and survival under starvation conditions (23,24). In Escherichia coli, the pmf generated by a PR resulted in ATP synthesis (22), was able to drive the flagellar motor (21), and could be used to significantly increase the production of hydrogen by a co-introduced hydrogenase (25,26).…”

Section: Introductionmentioning

confidence: 99%

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Chen

Steen

Dekker

et al. 2016

Metabolic Engineering

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Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10 5 molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize alltrans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a nonpumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

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“…Characterization studies of proton motive force (PMF) in MR-1 have not definitively determined whether the source of anaerobic proton pumping or translocation is electron transport, ATP synthase, or metabolite transport (13,19). Myers et al demonstrated that anaerobic MR-1 cells starved of electron acceptor generate PMF in response to fumarate addition (19).…”

mentioning

confidence: 99%

Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of Shewanella oneidensis Strain MR-1

et al. 2010

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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.Shewanella oneidensis strain MR-1 is a nonfermentative, facultative anaerobe which respires various substrates, including oxygen, soluble metals, insoluble iron and manganese oxide minerals, electrodes, and organic compounds (8,12,18,22). Other bacteria with the ability to respire electrodes and oxide minerals, such as Geobacter and Geothrix, oxidize acetate to carbon dioxide (4, 7, 9), consistent with these organisms generating ATP primarily from oxidative phosphorylation rather than substrate-level phosphorylation. Yet, an examination of metabolic end products and a variety of central metabolism and flux analyses of MR-1 show that acetate is the major product under anaerobic conditions (18,27,29,31). The general anaerobic metabolism model for MR-1, as depicted in Fig.

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Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Cited by 84 publications

References 44 publications

Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology

Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of Shewanella oneidensis Strain MR-1

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