Browsing by Author "Maresca, Julia Anne"
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Item Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola(American Society for Microbiology., 2015-06-08) Keffer, J. L.; Hahn, M. W.; Maresca, Julia Anne; J.L. Keffer, M.W. Hahn, J.A. Maresca; Keffer, J. L.; Maresca, Julia AnneRhodopsin-encoding microorganisms are common in many environments. However, knowing that rhodopsin genes are present provides little insight into how the host cells utilize light. The genome of the freshwater actinobacterium, Rhodoluna lacicola encodes a rhodopsin of the uncharacterized actinorhodopsin family. We hypothesized that actinorhodopsin was a light-activated proton pump, and confirmed this by heterologously expressing R. lacicola actinorhodopsin in retinal-producing Escherichia coli. However, cultures of R. lacicola did not pump protons, even though actinorhodopsin mRNA and protein were both detected. Proton pumping in R. lacicola was induced by providing exogenous retinal, suggesting that the cells lacked the retinal cofactor. We used HPLC and oxidation of accessory pigments to confirm that R. lacicola does not synthesize retinal. These results suggest that in some organisms, the actinorhodopsin gene is constitutively expressed, but rhodopsin-based light capture may require cofactors obtained from the environment.Item Identifying rhodopsin-containing cells using TIRF microscopy(American Society for Microbiology., 2015-03-13) Keffer, J. L.; Sabanayagam, Chandran Rigor; Lee, M. E.; DeLong, E. F.; Hahn, M. W.; Maresca, Julia Anne; J.L. Keffer, C.R. Sabanayagam, M.E. Lee, E.F. DeLong, M.W. Hahn and J.A. Maresca; Keffer, J. L.; Sabanayagam, Chandran Rigor; Maresca, Julia AnneSunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low—comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls—these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, B-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.