Figure 1.
Optimization of EdU-click in gut bacterial isolates. Gut bacterial isolates underwent the EdU-click procedure as described in the main text. (a) Overview of the EdU-click and FACS-Seq procedure. In Step 1, bacterial cells are incubated in vitro with EdU. In Step 2, the alkyne group of the EdU molecule, located within the bacterial DNA, is exposed to a fluorescent azide and undergoes a copper-catalyzed ‘click’ reaction. In Step 3, bacteria which have incorporated EdU become fluorescent, are detected, and physically separated from unlabeled bacteria. In Step 4, bacterial DNA from both labeled and unlabeled bacteria undergo 16S amplicon sequencing and taxonomic analysis. (b) Top: Representative flow cytograms of Enterococcus faecalis grown either with or without EdU. Isolates grown without EdU were used to setup the gating strategy and determine background noise or unspecific labeling. Quadrant 3 (Q3): unlabeled, or EdU-negative (EdU−), population. Quadrant 2 (Q2): EdU+ population. Bottom: epifluorescence microscopy images of the same isolate in the flow cytograms above. Scale bar: 10 microns. (c) Top: Representative flow cytograms of Bacteroides caccae grown either with or without EdU. Bottom: epifluorescence microscopy images of the same isolate in the flow cytograms above. Scale bar: 10 microns. (d) Quantification of the proportion of EdU+ cells for each bacterial isolate. The proportion of EdU+ cells for each isolate was calculated as the average number of cells present in Q2, minus background noise, divided by the sum of cells in Q2 and Q3. (e) Growth rates for each of the nine bacterial isolates were calculated as the steepest slope of their exponential growth curve (Max V) and were correlated with percent of cells labeled with EdU (Pearson correlation). B. infantis = Bifidobacterium longum subspecies infantis; B. caccae = Bacteroides caccae; B. fragilis = Bacteroides fragilis; P. intermedia = Prevotella intermedia; C. beijerinckii = Clostridium beijerinckii; E. faecalis = Enterococcus faecalis; R. bromii = Ruminococcus bromii; E. coli = Escherichia coli; A. muciniphila = Akkermansia muciniphila.