Presence of a Haloarchaeal Halorhodopsin-Like Cl⁻ Pump in Marine Bacteria

Abstract

Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl−-pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl− pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl−-transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl−-pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.

Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl− concentrations

“…These results indicate that Psu ACR1 binds Cl − in the initial state and that the bound Cl − increases the acid dissociation constant (p K a ) of the protonated retinal Schiff base. The same behavior was also observed for HRs 26 , 27 . We then estimated the Cl − binding affinity of Psu ACR1 (the dissociation constant, K d ) from the Cl − dependent shift of the λ max .…”

“…With regard to the Cl − binding affinity, the K d was estimated to be 5.5 ± 1.6 mM from the Hill equation (Fig. 1C ), which was in the same order as HsHR (2.6 mM) 34 , NpHR (5.0 mM) 35 , bacterial Rubricoccus marinus HR (RmHR; 7.6 mM) 26 and MrHR (2.0 mM) 30 . This result indicates that the natively expressed Psu ACR1 in P. sulcata binds Cl − in the initial state under physiological conditions (the Cl − concentration in the marine environment is a few hundreds of millimolar).…”

“…In the P 1 – P 4 states, physically defined photo-intermediates such as K, L, and M were populated at equilibrium. Details for the analysis are described in our previous reports 25 , 26 , 29 .…”

“…Focusing on the channel functions of ACRs in which several photo-intermediates are involved, anion concentration dependency, which is a useful parameter to characterize anion transport function, is still unclear. In anion pumping rhodopsins, such as archaeal and cyanobacterial halorhodopsins (HRs) and marine bacterial Cl − pumping rhodopsins (ClRs), their anion transport mechanisms, including their anion binding ability, photocycle kinetics, sequence and timing of anion uptake and release, and residues important for anion transport, have been revealed based on the anion concentration dependency in their spectroscopic properties 18 – 26 . Therefore, in this study we used static and time-resolved absorption spectroscopy to characterize the anion channel function of ACR with varying anion concentrations, especially for Cl − .…”

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage

“…PCC 7509 17,18 . Most of the genes in the CyHR clade are possessed only by cyanobacteria; however, they have also been found in a few marine bacteria 19 . Together, this fragmented evidence implies that rhodopsin-mediated photosystems may have evolved in the cyanobacterial lineage together with chlorophyll-based photosynthesis systems to allow for more efficient utilization of solar energy, although the distribution of rhodopsin genes in the cyanobacterial lineage remains to be clarified 20 .…”