Functional characterization of flavobacteria rhodopsins reveals a unique class of light-driven chloride pump in bacteria

Abstract

Light-activated, ion-pumping rhodopsins are broadly distributed among many different bacteria and archaea inhabiting the photic zone of aquatic environments. Bacterial proton- or sodium-translocating rhodopsins can convert light energy into a chemiosmotic force that can be converted into cellular biochemical energy, and thus represent a widespread alternative form of photoheterotrophy. Here we report that the genome of the marine flavobacterium Nonlabens marinus S1-08(T) encodes three different types of rhodopsins: Nonlabens marinus rhodopsin 1 (NM-R1), Nonlabens marinus rhodopsin 2 (NM-R2), and Nonlabens marinus rhodopsin 3 (NM-R3). Our functional analysis demonstrated that NM-R1 and NM-R2 are light-driven outward-translocating H(+) and Na(+) pumps, respectively. Functional analyses further revealed that the light-activated NM-R3 rhodopsin pumps Cl(-) ions into the cell, representing the first chloride-pumping rhodopsin uncovered in a marine bacterium. Phylogenetic analysis revealed that NM-R3 belongs to a distinct phylogenetic lineage quite distant from archaeal inward Cl(-)-pumping rhodopsins like halorhodopsin, suggesting that different types of chloride-pumping rhodopsins have evolved independently within marine bacterial lineages. Taken together, our data suggest that similar to haloarchaea, a considerable variety of rhodopsin types with different ion specificities have evolved in marine bacteria, with individual marine strains containing as many as three functionally different rhodopsins.

Keywords: ecology; evolution; photoheterotroph; photoproteins; retinal.

Fig. 1.

Phylogenetic relationships among microbial rhodopsins. (A) Unrooted phylogenetic tree of microbial rhodopsin amino acid sequences. The three rhodopsin genes (NM-R1, NM-R2, and NM-R3) encoded in the S1-08^T genome are indicated by red circles. Orange and yellow indicate the NTQ motif and the NDQ motif rhodopsin clade, respectively. (B) Detailed phylogenetic relationships of NTQ (orange) and NDQ (yellow) motif rhodopsin clades. NM-R2 and NM-R3 are indicated by the red characters. Stars indicate AAP bacteria. Closed and open circles indicate strains containing three opsins and two opsins, respectively. Taxonomic groups and the habitat of origin for each are indicated by the different symbols.

Fig. 2.

Absorption spectra and light-induced pH changes of NM-R1, NM-R2, and NM-R3 proteins. (A) Absorption spectra of purified NM-R1, NM-R2, and NM-R3 proteins. (B) Light-induced pH changes of E. coli cell suspensions expressing NM-R1, NM-R2, or NM-R3 in 100 mM NaCl (blue line). The pH changes after addition of CCCP (red line) or CCCP + TPP⁺ (green line) are indicated as well.

Fig. 3.

Light-induced pH changes of E. coli cell suspensions expressing NM-R2 or NM-R3 in different salt solutions (100 mM). (A) Effect of light on NM-R2 in different salt solutions. (B) Effect of light on NM-R3 in different salt solutions.

Fig. 4.

Light-stimulated growth in N. marinus S1-08^T. S1-08^T was grown in sterile ASW containing 0.14 mM carbon at 22 °C. Error bars denote SD for triplicate cultures.