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Proteorhodopsin Photosystem Gene Expression Enables Photophosphorylation in a Heterologous Host

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Proteorhodopsin Photosystem Gene Expression Enables Photophosphorylation in a Heterologous Host Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host A. Martinez*, A. S. Bradley†, J. R. Waldbauer‡, R. E. Summons†, and E. F. DeLong*§ *Department of Civil and Environmental Engineering, Division of Biological Engineering, and †Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; and ‡Joint Program in Chemical Oceanography, Woods Hole Oceanographic Institution and Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by W. Ford Doolittle, Dalhousie University, Halifax, NS, Canada, and approved February 9, 2007 (received for review December 22, 2006) Proteorhodopsins (PRs) are retinal-containing proteins that cata- To further characterize PR photosystem structure and func- lyze light-activated proton efflux across the cell membrane. These tion, we directly screened large-insert DNA libraries derived photoproteins are known to be globally distributed in the ocean’s from marine picoplankton for visibly detectable PR-expressing photic zone, and they are found in a diverse array of Bacteria and phenotypes. In this report, we describe completely intact PR- Archaea. Recently, light-enhanced growth rates and yields have based photosystems that can be functionally expressed in E. coli, been reported in at least one PR-containing marine bacterium, but without addition of exogenous photopigment (e.g., retinal or its the physiological basis of light-activated growth stimulation has precursors). Analyses of insertional mutants verified the func- not yet been determined. To describe more fully PR photosystem tional annotation of each gene product in the photosystem genetics and biochemistry, we functionally surveyed a marine biosynthetic pathway. We also show that light-activated, PR- picoplankton large-insert genomic library for recombinant clones catalyzed proton translocation, by the chemiosmotic potential it expressing PR photosystems in vivo. Our screening approach ex- generates, activates photophosphorylation in E. coli. ploited transient increases in vector copy number that significantly Results enhanced the sensitivity of phenotypic detection. Two genetically distinct recombinants, initially identified by their orange pigmen- Screening a Fosmid Library for in Vivo PR Photosystem Expression. tation, expressed a small cluster of genes encoding a complete When E. coli expresses a PR apoprotein from an inducible PR-based photosystem. Genetic and biochemical analyses of trans- promoter on a high-copy number plasmid, the cells acquire a red or orange pigmentation in the presence of exogenous all-trans poson mutants verified the function of gene products in the retinal (1, 12). Retinal addition is required because E. coli lacks photopigment and opsin biosynthetic pathways. Heterologous the ability to biosynthesize retinal or its precursor, ␤-carotene. expression of six genes, five encoding photopigment biosynthetic Based on these observations, we screened for PR-containing proteins and one encoding a PR, generated a fully functional PR clones on retinal-containing LB agar plating medium, which we photosystem that enabled photophosphorylation in recombinant expected would display an orange to red phenotype under these Escherichia coli cells exposed to light. Our results demonstrate that conditions. To enhance assay sensitivity, we used the copy- a single genetic event can result in the acquisition of phototrophic control system present in our fosmid vector that allowed a capabilities in an otherwise chemoorganotrophic microorganism, controlled transition from one copy per cell to multiple (up to and they explain in part the ubiquity of PR photosystems among 100) vector copies upon addition of the inducer L-arabinose (13). diverse microbial taxa. A fosmid library prepared from ocean surface water pico- plankton containing 12,280 clones (Ϸ440 Mb of cloned DNA) photoheterotrophy ͉ rhodopsin ͉ lateral gene transfer ͉ marine ͉ (14) was screened by using the above approach. Three orange metagenomics colonies were identified as potential PR-expressing clones on the LB-retinal-L-arabinose agar plates. All three showed no pigmen- roteorhodopsins (PRs) are retinal-binding membrane pro- tation in the absence of the high-copy number inducer. Unex- Pteins belonging to the rhodopsin family. Prokaryotic mem- pectedly, these clones also displayed an orange phenotype in the bers of this family include photosensors (sensory rhodopsins), absence of L-retinal when induced to high copy number. The transmembrane proton pumps (bacteriorhodopsins, xanthoro- sequence of one clone, HF10࿝19P19, revealed the presence of a hodopsin, and PRs), and transmembrane chloride pumps (halor- PR gene near the fosmid vector junction (see below). Because hodopsins). Originally discovered in Archaea, rhodopsins were the clones exhibited orange pigmentation in the absence of later identified in Gammaproteobacteria of the SAR86 group exogenous retinal, we expected that they must also be expressing ࿝ during a cultivation-independent genomic survey. Dubbed pro- retinal biosynthetic genes. Two clones, HF10 25F10 and teorhodopsin, this photoprotein functions as a light-activated proton pump when expressed in Escherichia coli in the presence Author contributions: A.M. and E.F.D. designed research; A.M., A.S.B., and J.R.W. per- of exogenously added retinal (1). Since then, numerous molec- formed research; A.M., A.S.B., J.R.W., R.E.S., and E.F.D. analyzed data; and A.M. and E.F.D. ular surveys have demonstrated that PR genes are ubiquitous in wrote the paper. bacteria inhabiting the ocean’s photic zone (2–9). An estimated The authors declare no conflict of interest. 13% of bacteria in marine picoplankton populations, as well as This article is a PNAS Direct Submission. a significant fraction of planktonic Euryarchaeota, contain a PR Abbreviations: CCCP, carbonylcyanide m-chlorophenylhydrazone; DCCD, N,NЈ-dicyclo- gene (4, 8). In a number of marine bacteria, retinal biosynthetic hexylcarbodiimide; FPP, farnesyl diphosphate; GGPP, geranylgeranyl pyrophosphate; IPP, genes and PR are genetically linked, and their lateral transfer isopentenyl diphosphate; PR, proteorhodopsin. and retention appear to be relatively common events, indicating Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. EF100190 (HF10࿝19P19) and EF100191 (HF10࿝25F10)]. that the photosystem confers a significant fitness advantage (3, §To whom correspondence should be addressed at: Department of Civil and Environmental 4, 7, 10, 11). A recent report of light-stimulated growth in a Engineering, 48-427, Massachusetts Institute of Technology, 15 Vassar Street, Cambridge, PR-containing marine flavobacterium supports this hypothesis MA 02139. E-mail: [email protected]. (11). Despite all of these observations however, the various This article contains supporting information online at www.pnas.org/cgi/content/full/ specific functions and physiological roles of diverse marine 0611470104/DC1. microbial PRs remain to be fully described. © 2007 by The National Academy of Sciences of the USA 5590–5595 ͉ PNAS ͉ March 27, 2007 ͉ vol. 104 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611470104 Downloaded by guest on September 30, 2021 A idi blh crtY crtB crtI crtE PR HF10_19P19 HF10_25F10 B HF10_19P19 idi blh crtY crtB crtI crtE PR C D IPP δ-isomerase (idi) IPP CH 2 O P P CH O P P DMAPP 2 Lycopene FPP synthase IPP CrtY - IPP nm λ=473nm 200 250 300 350 400 450 500 550 600 650 700 750 800 (ispA) λ (nm) Lycopene CH O P P - 2 FPP polyisoprenoids CrtY ubiquinones HF10_19P19 GGPP synthase IPP CrtI - Blh - crtE 25.00 30.00 35.00 40.00 45.00 50.00 Rt (min) CH 2 O P P GGPP Phytoene synthase GGPP crtB β-carotene Blh- n 200 250 300 350 400 450 500 550 600 650 700 750 800 Phytoene λ =454nm λ (nm) β-carotene Blh- Phytoene dehydrogenase HF10_19P19 crtI CrtY- CrtI- 20.00 25.00 30.00 35.00 40.00 45.00 Lycopene Rt (min) 1 9 .2 MICROBIOLOGY Lycopene cyclase crtY Retinal β-Carotene HF10_19P19 n λ=386nm 200 250 300 350 400 450 500 550 600 650 700 750 800 λ (nm) O Retinal 2 15,15’-β-carotene dioxygenase HF10_19P19 blh CrtY- CrtI- Blh- COH Retinal 10.0 0000 115.005 00 220.00 000 2525.0000 3030.00 00 3535.00 tR ( m )ni 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. HF10࿝19P19, were analyzed further for PR photosystem gene PR-containing BAC clones from Alphaproteobacteria from the expression and function. Mediterranean and Red Seas (8). This similarity was evident across the entire cloned insert, although some large-scale rear- Genomic Analyses of Candidate PR Photosystem-Expressing Clones. rangements were apparent. The HF10࿝19P19 PR-inferred pro- The full DNA sequence of the two putative PR photosystem- tein sequence was most similar to a homologue from another containing fosmids was obtained by sequencing a collection of environmental BAC, MedeBAC66A03
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