Identifying rhodopsin-containing cells using TIRF microscopy
| Author(s) | Keffer, J. L. | |
| Author(s) | Sabanayagam, Chandran Rigor | |
| Author(s) | Lee, M. E. | |
| Author(s) | DeLong, E. F. | |
| Author(s) | Hahn, M. W. | |
| Author(s) | Maresca, Julia Anne | |
| Ordered Author | J.L. Keffer, C.R. Sabanayagam, M.E. Lee, E.F. DeLong, M.W. Hahn and J.A. Maresca | |
| UD Author | Keffer, J. L. | en_US |
| UD Author | Sabanayagam, Chandran Rigor | en_US |
| UD Author | Maresca, Julia Anne | en_US |
| Date Accessioned | 2015-11-17T19:30:35Z | |
| Date Available | 2015-11-17T19:30:35Z | |
| Copyright Date | Copyright © 2015, American Society for Microbiology. | en_US |
| Publication Date | 2015-03-13 | |
| Description | Accepted manuscript posted online. | en_US |
| Abstract | Sunlight 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. | en_US |
| Department | University of Delaware. Department of Civil and Environmental Engineering. | en_US |
| Department | Delaware Biotechnology Institute. | en_US |
| Citation | Keffer, J. L., Sabanayagam, C. R., Lee, M. E., DeLong, E. F., Hahn, M. W., & Maresca, J. A. (2015). Identifying rhodopsin-containing cells using TIRF microscopy. Applied and Environmental Microbiology, AEM-00230. | |
| DOI | doi: 10.1128/AEM.00230-15 | |
| ISSN | 0099-2240 ; e:1098-5336 | en_US |
| URL | http://udspace.udel.edu/handle/19716/17227 | |
| Language | en_US | en_US |
| Publisher | American Society for Microbiology. | en_US |
| dc.rights | Article is made available in accordance with the University of Delaware Faculty Policy on Open Access and the publisher's policy. | |
| dc.source | Applied and Environmental Microbiology | en_US |
| dc.source.uri | http://aem.asm.org/ | en_US |
| Title | Identifying rhodopsin-containing cells using TIRF microscopy | en_US |
| Type | Article | en_US |
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