Figures and data in Optogenetic control of gut bacterial metabolism to promote longevity

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4 figures, 1 table and 2 additional files

Figures

Figure 1 with 4 supplements

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Optogenetic control of *C.elegans* gut bacterial gene expression.

(a) Strain LH01, which harbors two plasmids: pSR43.6, which encodes the biosynthesis of the PCB chromophore, and pLH401, which encodes the CcaSR TCS with GFP as output and constitutive mCherry production. DNA element legend: origins of replication (circles), promoters (bent arrows), ribosome binding sites (semi-circles), open reading frames (block arrows), transcriptional terminators (vertical lines with flat tops). Protein legend: Dark green protein (CcaS), light green protein (CcaR), black circle (phosphoryl group). Signal legend: filled gray arrows (biochemical or biophysical process), open gray arrows (signal transduction). (b) Microscopy and cytometry workflows. All begin with worms fed bacteria grown in the precondition light condition. Microscopy samples were then paralyzed and imaged while exposed to the experimental light condition. Cytometry samples were washed and paralyzed, exposed to the experimental light condition, and finally homogenized before cytometry. (c) Fluorescence microscopy images 0 and 8 h after green light exposure in the step ON experiment. Scale bar: 10 μm. (d) Response dynamics in the step ON (green) and step OFF (red) microscopy experiments. Black: ΔPCB strain (step ON experiment). Individual- (light lines) and multi-worm average (dark lines) data are shown. n = 7, 4, 6 worms for green, red, black data sets (measured over 2, 3, 1 days, respectively). Error bars: SEM. (**e–g**) Flow cytometry histograms for response dynamics experiments. MEFL: molecules of equivalent fluorescein.

Figure 1—figure supplement 1

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In vitro characterization of GFP reporter strains used in this study.

All optogenetic strains carry our previously published CcaSR v2.0 system, which is encoded on plasmids (a) pSR43.6, (b) pSR58.6, (c) pLH401 (used for microscopy) and (d) pLH405 (used for cytometry) genetic device schematics. We replaced *sfgfp* with gfpmut3* in pLH405 as we hypothesized that the latter may be less stable and thus result in faster response dynamics. However, we observed no difference in dynamics. (e) Batch culture light responses of CcaSR v2.0 and all GFP reporter strains used in this study. GFP fluorescence was measured by flow cytometry. The dynamic range (ratio of GFP fluorescence in green versus red light) is shown above each data set. We note that CcaSR v2.0 exhibits 77-fold dynamic range in the reference strain BW29655 (Δ*envZ*, Δ*ompR*), which is similar to our previous measurement of this strain at 120-fold (Schmidl et al., 2014). The calculated fold-change is sensitive to fluctuations in the measured *E. coli* autofluorescence and red-light expression level, which likely explains the slight discrepancy. CcaSR v2.0 dynamic range increases slightly to 84.2-fold in *∆rcsA* (JW1935-1), which is used throughout this work, due to higher sfGFP expression in green light. The mCherry cassette in pLH401 decreases dynamic range to 38.3-fold due to higher leaky sfGFP expression in red light. In worms, the fold-change in response to green light decreases further to 5.52 ± 2.4 fold (Figure 1d). The use of gfpmut3* in pLH405 further decreases dynamic range to 13.5 ± 0.0 fold for reasons that are not clear, while in worms the fold-change is 8.63 ± 3.6 fold (Figure 1e). Data represent the mean of three independent, autofluorescence-subtracted, biological replicates acquired on three separate days. Error bars: standard deviation.

Figure 1—figure supplement 2

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Flow cytometry gating.

(a) Strain LH05 (Supplementary file 1b), which expresses only mCherry, was fed to worms, isolated, and analyzed by flow cytometry to quantify *E. coli* autofluorescence through the FL1 (GFP) channel. To eliminate events corresponding to cytometer noise and autofluorescence of homogenized worm samples, three gates were then applied: (1) a density gate for the most homogeneous 75% of samples in forward scatter (FSC) vs side-scatter (SSC) (the area within the bold black line in the plots in Column 1), (2) a threshold gate on the FL1 (GFP) channel (marked by a red dashed line; Methods), and (3) a threshold gate for events exhibiting high FL3 (mCherry, red dashed line; Methods). (b) Applying these gates to samples from the step ON experiment (Figure 1e) reveals robust isolation of bacteria with a FSC/SSC profile consistent with our previous in vitro experiments^24,47 and an increase in expression of GFPmut3* in response to green light. Ungated data are shown for comparison to gated data in all plots.

Figure 1—figure supplement 3

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Gene expression dynamics from flow cytometry experiments.

(a) LH03 and (b) LH04 (ΔPCB) step ON results. (c) LH03 and (d) LH04 (ΔPCB) step OFF results. Each individual trajectory (faint lines) corresponds to a single biological replicate. A biological replicate comprises the homogenized contents of five worms collected on a single day. Each biological replicate on a given plot was run on a different day. Population medians taken across all trajectories are shown in bold (Methods). Data are composed of six and eight trajectories in the step ON and step OFF experiments, respectively. Error bars: SEM.

Figure 1—figure supplement 4

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Bacteria on the exterior of worms do not contribute to the measured light response in flow cytometry experiments.

Worms were suspended in clear tubes containing M9Ce+Lev media and levamisole in the flow cytometry experiments. To demonstrate that bacterial cells outside the worm gut (e.g. on the exterior of the worm) do not respond to light in these conditions, samples of pre-conditioned bacteria from the NGM plates were suspended in the M9Ce+Lev or *E. coli* M9 media supplemented with casamino acids and glucose (M9Ec). These samples were then exposed to either green or red light for 8 hr, then measured via flow cytometry after the fluorophore maturation protocol (Methods). As expected, the bacterial populations are only responsive to light when grown in M9Ec and when the PCB operon is intact (strain JW1935-1/pLH405/pSR43.6, aka LH03). We conclude that the responses shown in data in Figure 1e–f and Figure 1—figure supplement 3 are not due to bacteria outside the worm, nor to bacteria that escape the worm over the course of the experiment, as such cells do not respond to light in the experimental buffer (M9Ce). Data represent 7, 6, 3, and 5 replicates (left); and 8, 3, 8, and 3 replicates (right) over 8 and 10 days, respectively; error bars: SEM. Individual data points for each condition are overlaid as white markers.

Figure 2

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Optogenetic control of colanic acid biosynthesis.

(a) Strain MVK29, harboring two plasmids: pSR43.6, which encodes the biosynthesis of the PCB chromophore, and pMK201.2, which includes *rcsA* under the optogenetic control of CcaSR. RcsA activates transcription of the CA biosynthetic operon, ultimately producing CA. (b) CA secretion levels for MVK29 and control strains exposed to red and green light. JW1935-1 is the *E. coli rcsA* background strain used in this study. N.D.: below assay limit of detection. (c) Green light intensity versus CA secretion level for MVK29. Data points represent three biological replicates collected on a single day. Dashed line: limit of detection. Error bars indicate standard deviation of the three biological replicates.

Figure 3 with 1 supplement

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Light-regulated CA secretion modulates *C.elegans* mitochondrial dynamics.

(a) Schematic of experiment for activating CA biosynthesis in situ using unparalyzed light-exposure conditions. (b) Representative images of the mitochondrial network of anterior intestinal cells visualized by either mitochondrially-localized GFP (mito-GFP) or RFP (mito-RFP) immediately distal to the pharynx are scored as fragmented, intermediate, or tubular. Scale bars: 5 μm. (c) Mitochondrial morphology profiles of intestinal cells in unparalyzed worms fed MVK29 while exposed to red or green light for 6 hr. (d) Schematic of experiment for activating CA biosynthesis in situ using paralyzed light-exposure conditions (e) Mitochondrial morphology profiles of intestinal cells in worms fed the indicated strain, and then paralyzed for 6 hr while exposed to red or green light. (f) Representative images of mitochondrial morphology in muscular cells, and the quantification of these images using worms fed the indicated strain, and then paralyzed for 6 hr while exposed to red or green light. The Chi-Squared Test of Homogeneity was used to calculate p-values between conditions in c, e, and f. Combined results are shown from multiple independent replicates and see Figure 3—figure supplement 1 for data of each replicate.

Figure 3—figure supplement 1

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Optogenetic induction of CA in situ regulates mitochondrial dynamics in the gut of *C.elegans.*

(a) Mitochondrial morphology profiles of intestinal cells in unparalyzed worms fed the indicated strain while exposed to red or green light for 6 hr. The combined result is presented in Figure 3c. (**b,c**) Mitochondrial morphology profiles of intestinal cells in worms fed the indicated strain, and then paralyzed for 6 hr while exposed to red or green light. The combined result is presented in Figure 3e. (d) Mitochondrial morphology profiles of muscle cells in worms fed the indicated strain, and then paralyzed for 6 hr while exposed to red or green light. The combined result is presented in Figure 3f. **p<0.01, ***p<0.001 by the Chi-Squared Test of Homogeneity.

Figure 4 with 1 supplement

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Optogenetically-regulated CA biosynthesis extends worm lifespan.

(a) Green light induces CA overproduction in MVK29 to a level comparable to the *∆lon* mutant, and neither the *∆lon* or the *∆rcsA* mutant changes its CA levels in response to the green light exposure. Green-H indicates a high green light intensity yielding high levels of CA production. **p<0.01 by student’s t-test. Experiments were conducted at 26°C. (b) When exposed to green light, worms grown on MVK29 live longer than those exposed to red light, and the magnitude of lifespan extension correlates with green light intensity (p<0.0001 green vs. red, log-rank test). Green-L indicates a low level of green light intensity resulting in intermediate levels of CA production. Experiments were conducted at 26°C. (**c–d**) The lifespans of worms grown on the *∆lon* (c) or the *∆rcsA* (d) controls are not affected by light condition (p>0.1 green vs. red, log-rank test). Experiments were conducted at 26°C.

Figure 4—figure supplement 1

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Light intensity calibration.

Calibration of light intensities used to compare CA secretion levels from MVK29 to those from Δ*lon* and Δ*rcsA* in Figure 4. **p<0.01 by student’s t-test.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Recombinant DNA reagent	pLH401	This paper	GenBank: MN617156	Constitutive ccaR expression, P_cpcG2_-172:sfgfp, constitutive mcherry expression
Recombinant DNA reagent	pLH405	This paper	GenBank: MN617157	P_cpcG2_-172:gfpmut3, constitutive mcherry* expression
Recombinant DNA reagent	pLH407	This paper	GenBank: MN617158	Constitutive mcherry expression
Recombinant DNA reagent	pLH412	This paper	GenBank: MN617159	Constitutive ccaS(H534A), ho1, and pcyA expression
Recombinant DNA reagent	pLH413	This paper	GenBank: MN617160	Constitutive ccaR(D51N) expression, P_cpcG2_-172:rcsA
Recombinant DNA reagent	pSR43.6	Schmidl et al., 2014	GenBank: MN617163	Constitutive expression of ccaS, ho1, and pcyA
Recombinant DNA reagent	pSR49.2	Schmidl et al., 2014	GenBank: MN617164	Constitutive expression of ccaS
Recombinant DNA reagent	pMVK201.2	This paper	GenBank: MN617161	Constitutive expression of ccaR, P_cpcG2_-172:rcsA
Recombinant DNA reagent	pMVK228	This paper	GenBank: MN617162	Constitutive expression of ccaS
Stain, strain background (Escherichia coli)	BW25113	Baba et al., 2006	CGSC#: 7626	Keio parent strain
Stain, strain background (Escherichia coli)	ΔrcsA	Baba et al., 2006	CGSC: JW1935-1	Low CA production mutant from the Keio collection
Stain, strain background (E. coli)	Δlon	Baba et al., 2006	CGSC: JW0429-1	High CA production mutant from the Keio collection
Genetic reagent (C. elegans)	zu391	Hermann et al., 2005	CGC ID: JJ1271	glo-1(zu391) X
Genetic reagent (C. elegans)	zcIs17	Han et al., 2017	CGC ID: SJ4143	zcIs17[P_ges-1::mito-GFP]
Genetic reagent (C. elegans)	zcIs14	Han et al., 2017	CGC ID: SJ4103	zcIs14[P_myo-3::mito-GFP]
Genetic reagent (C. elegans)	MW2241	This paper		raxIs145[P_ges-1::mito-RFP]
Genetic reagent (C. elegans)	e2117	Han et al., 2017	CGC ID: CB4121	sqt-3(e2117) V