A blue-shifted light-driven proton pump for neural silencing.

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λmax = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the ß-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.

Conversion of mono-polar to peritrichous flagellation in Vibrio alginolyticus.

Precise regulation of the number and positioning of flagella are critical in order for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no ΔflhF cells have flagella, while a very small fraction of ΔflhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of ΔflhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.

Spectroscopic studies of a sensory rhodopsin I homologue from the archaeon Haloarcula vallismortis.

Sensory rhodopsin I (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new information about structural changes and Cl(-) binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (HvSRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in HvSRI (unlike in SrSRI), and HvSRI is extremely stable in buffers without Cl(-). Taking advantage of the high stability, we characterized the photochemical properties of HvSRI under acidic and basic conditions and observed the effects of Cl(-) on the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving our understanding of the molecular mechanism of the dual photosensing by SRI.

Characterization of two sets of subpolar flagella in Bradyrhizobium japonicum.

Bradyrhizobium japonicum is one of the soil bacteria that form nodules on soybean roots. The cell has two sets of flagellar systems, one thick flagellum and a few thin flagella, uniquely growing at subpolar positions. The thick flagellum appears to be semicoiled in morphology, and the thin flagella were in a tight-curly form as observed by dark-field microscopy. Flagellin genes were identified from the amino acid sequence of each flagellin. Flagellar genes for the thick flagellum are scattered into several clusters on the genome, while those genes for the thin flagellum are compactly organized in one cluster. Both types of flagella are powered by proton-driven motors. The swimming propulsion is supplied mainly by the thick flagellum. B. japonicum flagellar systems resemble the polar-lateral flagellar systems of Vibrio species but differ in several aspects.

The flagellar-specific transcription factor, sigma28, is the Type III secretion chaperone for the flagellar-specific anti-sigma28 factor FlgM.

The sigma(28) protein is a member of the bacterial sigma(70)-family of transcription factors that directs RNA polymerase to flagellar late (class 3) promoters. The sigma(28) protein is regulated in response to flagellar assembly by the anti-sigma(28) factor FlgM. FlgM inhibits sigma(28)-dependent transcription of genes whose products are needed late in assembly until the flagellar basal motor structure, the hook-basal body (HBB), is constructed. A second function for the sigma(28) transcription factor has been discovered: sigma(28) facilitates the secretion of FlgM through the HBB, acting as the FlgM Type III secretion chaperone. Transcription-specific mutants in sigma(28) were isolated that remained competent for FlgM-facilitated secretion separating the transcription and secretion-facilitation activities of sigma (28). Conversely, we also describe the isolation of mutants in sigma(28) that are specific for FlgM-facilitated secretion. The data demonstrate that sigma(28) is the Type III secretion chaperone for its own anti-sigma factor FlgM. Thus, a novel role for a sigma(70)-family transcription factor is described.

Roles of the intramolecular disulfide bridge in MotX and MotY, the specific proteins for sodium-driven motors in Vibrio spp.

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.