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.

Fig. 1.

Genetic and phenotypic analysis of PR photosystem transposon mutants. (A) Schematic representation of the PR gene clusters identified in this work. Predicted transcription terminators in the clusters are indicated. (B) Color phenotype of intact cells of transposon-insertion mutants grown in liquid cultures with arabinose. (C) Retinal biosynthesis pathway. Names of genes encoding pathway enzymes are indicated. The genes that are present in E. coli are in parentheses. (D) HPLC profiles of wild-type and transposon mutant extracts. Detection wavelengths are indicated. Absorption spectrum of relevant peaks, including standards used for identification, are shown on the top for each panel.

Fig. 2.

Proton-pumping assays. pH measurements are expressed as pH change with respect to the pH at time 0 for each sample. Gray boxes indicate dark periods.

Fig. 3.

Light-activated, PR-enabled photophosphorylation in E. coli. (A) Diagram of the proposed mechanism of PR-dependent ATP synthesis. The effects of the inhibitors used are indicated. (B) Proton-pumping assays with HF10_19P19 cells. pH measurements are expressed as the pH change respect to the pH at time 0 for each sample. CCCP, 25 μM; DCCD, 1 mM. Gray boxes indicate dark periods. (C) ATP assays with HF10_19P19 cells. Results are expressed as the difference between the ATP level in the light and the ATP level in the dark, ΔATP, for each treatment.