TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor.

In many rhodopsins, the retinal Schiff base pKa remains very high, ensuring Schiff base protonation captures visible light. Nevertheless, recently we found that TAT rhodopsin contains protonated and unprotonated forms at physiological pH. The protonated form displays a unique photochemical behavior in which the primary K intermediate returns to the original state within 10-5 s, and the lack of photocycle completion poses questions about the functional role of TAT rhodopsin. Here we studied the molecular properties of the protonated and unprotonated forms of the Schiff base in TAT rhodopsin. We confirmed no photointermediate formation at >10-5 s for the protonated form of TAT rhodopsin in microenvironments such as detergents, nanodiscs, and liposomes. In contrast, the unprotonated form features a very long photocycle with a time constant of 15 s. A low-temperature study revealed that the primary reaction of the unprotonated form is all-trans to 13-cis photoisomerization, which is usual, but with a proton transfer reaction occurring at 77 K, which is unusual. The active intermediate contains the unprotonated Schiff base as well as the resting state. Electrophysiological measurements excluded ion-transport activity for TAT rhodopsin, while transient outward proton movement only at an alkaline extracellular pH indicates that TAT rhodopsin senses the extracellular pH. On the basis of the findings presented here, we propose that TAT rhodopsin is an ultraviolet (UV)-dependent environmental pH sensor in marine bacteria. At acidic pH, absorbed visible light energy is quickly dissipated into heat without any function. In contrast, when the environmental pH becomes high, absorption of UV/blue light yields formation of the long-lived intermediates, possibly driving the signal transduction cascade in marine bacteria.

Saccharibacteria harness light energy using type-1 rhodopsins that may rely on retinal sourced from microbial hosts.

Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the candidate phyla radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. These Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from ß-carotene in microorganisms co-occurring with Saccharibacteria, and this genetic potential was dominated by members of the Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.

Genomic and Metabolomic Analyses of a Piezosensitive Mutant of Saccharomyces cerevisiae and Application for Generation of Piezosensitive Niigata-Sake Yeast Strains.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Unique Photochemistry Observed in a New Microbial Rhodopsin.

Light energy is first captured in animal and microbial rhodopsins by ultrafast photoisomerization, whose relaxation accompanies protein structural changes for each function. Here, we report a microbial rhodopsin, marine bacterial TAT rhodopsin, that displays no formation of photointermediates at >10-5 s. Low-temperature ultraviolet-visible and Fourier transform infrared spectroscopy revealed that TAT rhodopsin features all-trans to 13-cis photoisomerization like other microbial rhodopsins, but a planar 13-cis chromophore in the primary K intermediate seems to favor thermal back isomerization to the original state without photocycle completion. The molecular mechanism of the early photoreaction in TAT rhodopsin will be discussed.

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy.

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.