A light-driven sodium ion pump in marine bacteria

ARTICLE Received 16 Jan 2013 | Accepted 1 Mar 2013 | Published 9 Apr 2013 DOI: 10.1038/ncomms2689 A light-driven sodium ion pump in marine bacteria Keiichi Inoue1,2, Hikaru Ono1, Rei Abe-Yoshizumi1, Susumu Yoshizawa3, Hiroyasu Ito1, Kazuhiro Kogure3 & Hideki Kandori1 Light-driven proton-pumping rhodopsins are widely distributed in many microorganisms. They convert sunlight energy into proton gradients that serve as energy source of the cell. Here we report a new functional class of a microbial rhodopsin, a light-driven sodium ion pump. We discover that the marine flavobacterium Krokinobacter eikastus possesses two rhodopsins, the first, KR1, being a prototypical proton pump, while the second, KR2, pumps sodium ions outward. Rhodopsin KR2 can also pump lithium ions, but converts to a proton pump when presented with potassium chloride or salts of larger cations. These data indicate that KR2 is a compatible sodium ion–proton pump, and spectroscopic analysis showed it binds sodium ions in its extracellular domain. These findings suggest that light-driven sodium pumps may be as important in situ as their proton-pumping counterparts. 1 Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan. 2 PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan. 3 Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan. Correspondence and requests for materials should be addressed to H.K. (email: ). NATURE COMMUNICATIONS | 4:1678 | DOI: 10.1038/ncomms2689 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2689 M icroorganisms utilize ion-transporting microbial rhodopsins for light-energy conversion and light-signal transduction1,2. Light-driven outward proton and inward chloride pumps are used to create membrane potential for ATP synthesis3–5, while a light-gated ion channel depolarizes cells for phototaxis of Chlamydomonas6–8. These rhodopsins are important tools for optogenetics, where channelrhodopsin (ChR) excites neurons by light, and ion pumps are used for neural silencing9–11. The light-driven proton pump bacteriorhodopsin (BR) and chloride pump halorhodopsin (HR) were first discovered in 1971 (ref. 12) and 1977 (ref. 13), respectively, from Halophilic archaea. Since 2000, metagenomic analysis revealed that microbial rhodopsins are widely distributed among marine prokaryotes, most of which were classified as light-driven proton pumps (proteorhodopsin; PR)2,14–16. Although there were no data on the proton pump activity of PR in native marine bacteria, light-induced pH drops of native cell suspensions have been successfully recorded for flavobacteria17 and g-proteobacteria18. These facts suggest that the contribution of PR phototrophy is significant in marine environment, where light-driven proton pumps convert the energy of sunlight to proton gradients as the energy source of the cell. Similarly, sodium ion gradients are also widely used to drive uptake of nutrients19,20, but there have been no reports of a light-driven sodium ion pump. Possibility of HR as the outward sodium ion pump was originally discussed13, but it is now established that HR is an inward chloride pump21. Absence of sodium ion-pumping rhodopsins may be reasonable from the chemical point of view. All microbial rhodopsins possess an all-trans retinal as the chromophore, which binds to a lysine residue through a protonated retinal Schiff base (RSB) linkage (Supplementary Fig. S1)3,4,22. Therefore, light energy is captured by the positively charged retinal chromophore. In case of BR and PR, transient drop of the RSB pKa by retinal photoisomerization causes the proton transfer to the extracellular side, and reprotonation from the cytoplasmic side leads to the outward proton pump3,4,22. In case of HR, chloride binding stabilizes the protonated RSB, and retinal photoisomerization alters the interaction of the ion pair, leading to the inward chloride pump23,24. In contrast, cations other than proton are probably unstable near the protonated RSB because of an electrostatic repulsion. Despite the above explanation has been generally accepted, we report the discovery of a light-driven sodium ion pump in this paper. The marine flavobacterium K. eikastus possesses two rhodopsins (KR1 and 2). While KR1 is a typical proton pump, KR2 pumps sodium ion outward. The sodium ion-pumping rhodopsin (NaR) can also pump lithium ion, but it becomes a proton pump in potassium chloride or salts of larger cations. KR2, a compatible sodium ion–proton pump, was converted to uni-functional sodium ion and proton pumps by mutations, from which important amino acids were revealed for sodium ion and proton pumps. The molecular mechanism of the light-driven sodium ion pump will be discussed. Results K. eikastus KR2 is a light-driven sodium ion pump. We first measured light-driven pump activity in native cell suspensions of K. eikastus25, a marine Flavobacterium. The pump activity was monitored by a pH electrode at various growth phases. Although no pumping activity was observed before stationary phase (10 h in Fig. 1a), the cells showed pH drop upon illumination at 96 h, which was abolished by the protonophore carbonylcyanidem-chlorophenylhydrazone (CCCP). This reproduced our previous 2 observation17, implying that K. eikastus contains a PR-like outward proton pump. However, at 48 h, we observed lightinduced pH increase, which was not affected by CCCP (Fig. 1a). This result (pumping activities at all growth phases; Supplementary Fig. S2) suggests that there are multiple ion-pumping rhodopsins that function at different stages of the growth of K. eikastus. Genomic analysis shows two microbial rhodopsins in K. eikastus (KR1 and KR2), where KR1 belongs to PR-clade, and KR2 is located in an isolated clade of unknown function (Fig. 1b and Supplementary Fig. S3; detailed phylogenic tree). It is suggested that KR1 and KR2 are the major gene products at 96 and 48 h, respectively, in Fig. 1a. To study the function of the rhodopsins, C-terminal his-tagged KR1 or KR2 were overexpressed in Escherichia coli (C41 (DE3) strain). KR1 and KR2 show very similar absorption spectra (Fig. 2a), and the retinal configurations of both proteins are 490% all-trans form in both dark and light conditions (Supplementary Fig. S4). Therefore, the all-trans isomer is the functional form as in the other microbial rhodopsins1. We then measured light-induced pH changes of E. coli cell suspensions containing KR1 or KR2. In case of KR1, we observed pH drop upon illumination, which was prevented by the protonophore CCCP (Fig. 2b). This indicates that KR1 is a light-driven proton pump, which is consistent with the phylogenic analysis (Fig. 1b). In contrast, we observed light-induced pH increase for KR2, which is accelerated with CCCP, but completely (...truncated)