Using Total Internal Reflection Fluorescence Microscopy To Visualize Rhodopsin-Containing Cells

Abstract

e 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, ␤-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.

Marine proteorhodopsins rival photosynthesis in solar energy capture

“…The highest PR concentrations were observed in the low nutrient regions of the Eastern Mediterranean Sea, with maximum values ranging from 14.4 to 61.2 pM (stations [2][3][4][5][6][7][8][9][10][11][12]. This trend contrasts with the geographical distribution of Chl-a, which peaked in the most coastal influenced regions (68.7-695.6 pM; stations [13][14][15][16][17][18][19][20][21][22][23][24]. Therefore, the longitudinal distribution of PR photoheterotrophy showed highest values in areas where oxygenic photosynthesis was the lowest.…”

“…However, PR quantification that relies on genes or transcripts does not necessarily reflect PR abundance, due to post-transcriptional regulation and other factors that result in a disconnect between gene transcription and protein abundance. Since the discovery of PRs, only a handful of estimates of PR abundance have been reported [13][14][15].…”

Biochemical Analysis of Microbial Rhodopsins

“…Note that volumes here are optimized for a pellet from ß500 ml Actinobacterial culture or ß50 ml E. coli culture. For E. coli expressing rhodopsin from the pMCL200 vector and the retinal biosynthetic pathway cloned into the pBAD-TOPO vector (Keffer et al, 2015b), grow cells overnight at 37°C with shaking at ß200 rpm, in medium supplemented with arabinose (0.02% to 0.2%), chloramphenicol (34 μg/m), and ampicillin (100 μg/ml). For R. lacicola, grow for several days at 28°C in 3 g/liter NSY medium (Hahn et al, 2003) in the light.…”

“…autofluorescence. However, because TIRF microscopy only excites molecules within 100 to 200 nm of the coverglass-sample interface, background fluorescence is low enough to allow visualization of rhodopsin fluorescence (Keffer et al, 2015b). This technique allows rhodopsin-containing cells in natural bacterial assemblages to be differentiated from other cells based on the wavelengths of light used to excite the fluorophores.…”

“…In the absence of cultivable organisms encoding these genes, they can be characterized biochemically in heterologous expression systems such as E. coli. However, E. coli cannot synthesize retinal or any of its precursors, so the retinal must be provided exogenously in the medium (Beja et al, 2001;Martinez et al, 2007), or the entire retinal biosynthesis pathway must be supplied in trans (Keffer et al, 2015a(Keffer et al, , 2015b.…”