著者

Yu Nakajima Keiichi Kojima Yuichiro Kashiyama Satoko Doi Ryosuke Nakai Yuki Sudo Kazuhiro Kogure Susumu Yoshizawa

出版者

Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles

雑誌

Microbes and Environments (ISSN:13426311)

巻号頁・発行日

vol.35, no.4, pp.ME20085, 2020 (Released:2020-12-05)

参考文献数

36

被引用文献数

14

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Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether Aurantimicrobium minutum KNCT, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNCT possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.

著者

Keiichi Kojima Hiroshi C. Watanabe Satoko Doi Natsuki Miyoshi Misaki Kato Hiroshi Ishikita Yuki Sudo

出版者

The Biophysical Society of Japan

雑誌

Biophysics and Physicobiology (ISSN:21894779)

巻号頁・発行日

vol.15, pp.179-188, 2018 (Released:2018-09-07)

参考文献数

42

被引用文献数

7 9

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Anion channelrhodopsin-2 (ACR2), a light-gated channel recently identified from the cryptophyte alga Guillardia theta, exhibits anion channel activity with exclusive selectivity. In addition to its novel function, ACR2 has become a focus of interest as a powerful tool for optogenetics. Here we combined experimental and computational approaches to investigate the roles of conserved carboxylates on the anion transport activity of ACR2 in Escherichia coli membrane. First, we replaced six conserved carboxylates with a neutral residue (i.e. E9Q, E56Q, E64Q, E159Q, E219Q and D230N), and measured anion transport activity using E. coli expression system. E159Q and D230N exhibited significantly lower anion transport activity compared with wild-type ACR2 (1/12~1/3.4), which suggests that E159 and D230 play important roles in the anion transport. Second, to explain its molecular aspects, we constructed a homology model of ACR2 based on the crystal structure of a cation channelrhodopsin (ChR). The model structure showed a cavity formed by four transmembrane helices (TM1, TM2, TM3 and TM7) similar to ChRs, as a putative anion conducting pathway. Although E159 is not located in the putative pathway, the model structure showed hydrogen bonds between E159 and R129 with a water molecule. D230 is located in the pathway near the protonated Schiff base (PSB) of the chromophore retinal, which suggests that there is an interaction between D230 and the PSB. Thus, we demonstrated the functional importance and the hypothetical roles of two conserved carboxylates, E159 and D230, in the anion transport activity of ACR2 in E. coli membrane.