Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis: Proteorhodopsin expression increases growth rate and fitness of a ∆PSI strain of Synechocystis sp. PCC6803.

To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction.

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803.

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10(5) molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

Optogenetics and the future of neuroscience.

Over the last 10 years, optogenetics has become widespread in neuroscience for the study of how specific cell types contribute to brain functions and brain disorder states. The full impact of optogenetics will emerge only when other toolsets mature, including neural connectivity and cell phenotyping tools and neural recording and imaging tools. The latter tools are rapidly improving, in part because optogenetics has helped galvanize broad interest in neurotechnology development.

Optogenetics: 10 years of microbial opsins in neuroscience.

Over the past 10 years, the development and convergence of microbial opsin engineering, modular genetic methods for cell-type targeting and optical strategies for guiding light through tissue have enabled versatile optical control of defined cells in living systems, defining modern optogenetics. Despite widespread recognition of the importance of spatiotemporally precise causal control over cellular signaling, for nearly the first half (2005-2009) of this 10-year period, as optogenetics was being created, there were difficulties in implementation, few publications and limited biological findings. In contrast, the ensuing years have witnessed a substantial acceleration in the application domain, with the publication of thousands of discoveries and insights into the function of nervous systems and beyond. This Historical Commentary reflects on the scientific landscape of this decade-long transition.

Photo-induced regulation of the chromatic adaptive gene expression by Anabaena sensory rhodopsin.

Rhodopsin molecules are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices, which bind the chromophore retinal (vitamin A aldehyde). They are roughly divided into two groups according to their basic functions: (i) ion transporters such as proton pumps, chloride pumps, and cation channels; and (ii) photo-sensors such as sensory rhodopsin from microbes and visual pigments from animals. Anabaena sensory rhodopsin (ASR), found in 2003 in the cyanobacterium Anabaena PCC7120, is categorized as a microbial sensory rhodopsin. To investigate the function of ASR in vivo, ASR and the promoter sequence of the pigment protein phycocyanin were co-introduced into Escherichia coli cells with the reporter gene crp. The result clearly showed that ASR functions as a repressor of the CRP protein expression and that this is fully inhibited by the light activation of ASR, suggesting that ASR would directly regulate the transcription of crp. The repression is also clearly inhibited by the truncation of the C-terminal region of ASR, or mutations on the C-terminal Arg residues, indicating the functional importance of the C-terminal region. Thus, our results demonstrate a novel function of rhodopsin molecules and raise the possibility that the membrane-spanning protein ASR could work as a transcriptional factor. In the future, the ASR activity could be utilized as a tool for arbitrary protein expression in vivo regulated by visible light.