Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host

Abstract

Proteorhodopsins (PRs) are retinal-containing proteins that catalyze light-activated proton efflux across the cell membrane. These photoproteins are known to be globally distributed in the ocean's photic zone, and they are found in a diverse array of Bacteria and Archaea. Recently, light-enhanced growth rates and yields have been reported in at least one PR-containing marine bacterium, but the physiological basis of light-activated growth stimulation has not yet been determined. To describe more fully PR photosystem genetics and biochemistry, we functionally surveyed a marine picoplankton large-insert genomic library for recombinant clones expressing PR photosystems in vivo. Our screening approach exploited transient increases in vector copy number that significantly enhanced the sensitivity of phenotypic detection. Two genetically distinct recombinants, initially identified by their orange pigmentation, expressed a small cluster of genes encoding a complete PR-based photosystem. Genetic and biochemical analyses of transposon mutants verified the function of gene products in the photopigment and opsin biosynthetic pathways. Heterologous expression of six genes, five encoding photopigment biosynthetic proteins and one encoding a PR, generated a fully functional PR photosystem that enabled photophosphorylation in recombinant Escherichia coli cells exposed to light. Our results demonstrate that a single genetic event can result in the acquisition of phototrophic capabilities in an otherwise chemoorganotrophic microorganism, and they explain in part the ubiquity of PR photosystems among diverse microbial taxa.photoheterotrophy ͉ rhodopsin ͉ lateral gene transfer ͉ marine ͉ metagenomics

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…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).…”

“…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). Very recently, functional expression of a proteorhodopsin in E. coli was shown to cause a minute increase the growth rate of this organism when growing fermentatively (27).…”

Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria

“…Rhodopsin proteins are documented to contribute to similar pigmentation in other rhodopsin-expressing organisms (Bolhuis et al, 2006;Gomez-Consarnau et al, 2007). Alternatively, such coloration is often caused by the presence of carotenoid pigments (Trutko et al, 2005), which increases the probability that these organisms carry rhodopsin genes, because the retinal chromophore of these photoproteins is a byproduct of carotenoid biosynthesis (Martinez et al, 2007).…”

Adaptation and spectral tuning in divergent marine proteorhodopsins from the eastern Mediterranean and the Sargasso Seas

“…Proteorhodopsins (PRs) (Béjà et al, 2000(Béjà et al, , 2001 are bacterial retinal-binding membrane pigments that belong to the microbial rhodopsin superfamily (Spudich et al, 2000) and are predicted to have an important role in supplying light energy for microbial metabolism in marine ecosystems (Béjà et al, 2000(Béjà et al, , 2001Sabehi et al, 2005;Martinez et al, 2007;Walter et al, 2007). PRs have been observed in different ocean regions (Béjà et al, 2000(Béjà et al, , 2001de la Torre et al, 2003;Man et al, 2003;Sabehi et al, 2003Sabehi et al, , 2004Sabehi et al, , 2005Venter et al, 2004;Frigaard et al, 2006) and are found in diverse taxonomic backgrounds, including the ubiquitous marine gammaproteobacterial SAR86 (Béjà et al, 2000;Sabehi et al, 2004Sabehi et al, , 2005 and alphaproteobacterial SAR11 (Giovannoni et al, 2005a, b;Sabehi et al, 2005) groups, as well as in marine Bacteroidetes (Venter et al, 2004;Gó mez-Consarnau et al, 2007), planktonic Archaea (Frigaard et al, 2006) and other microbial taxa (de la Torre et al, 2003;Sabehi et al, 2003Sabehi et al, , 2005Venter et al, 2004;McCarren and DeLong, 2007).…”