Genome-Wide Functional Screen for Calcium Transients in Escherichia coli Identifies Increased Membrane Potential Adaptation to Persistent DNA Damage

The live-cell screen can identify knockouts with altered calcium flux. (A) Box plot of the calcium AUC from cells treated with DMSO (negative control), CCCP (eliminates transients), or apramycin (increases transients). Each of the box plots has 16 biological replicates. Red lines indicate medians, blue boxes indicate 25/75 limits, black lines indicate 10/90 limits, and red crosses indicate outliers. *, P < 0.001. (B) Calcium AUC from the primary screen. Each gene in the Keio collection is plotted on the y axis, and the log of the calcium AUC is plotted on the y axis. The genes corresponding to the values ranging from 4300 to 5400 on the x axis are not present in the Keio collection. (C) A histogram of the calcium AUC from the entire screen. (D) A random sample of individual cells from wild-type cells, ΔrecG cells (reduced calcium AUC), or ΔbtuR cells (increased calcium AUC). AU, arbitrary units.

Knockouts of outer membrane synthesis and extracellular polysaccharide secretion refine the model for mechanosensation. (A) Box plot of knockouts involved in outer membrane (OM) synthesis and exopolysaccharide (EPS) secretion showed reduced calcium AUC compared to WT cells. Each box plot represents results from 5 biological replicates. (B) Knockouts corresponding to OM synthesis and EPS secretion did not show reduced membrane potential compared to WT cells. Each mark represents the median of the population and corresponds to one biological replicate. Each genotype was measured in biological triplicate. (C) Box plot of calcium AUC of wild-type cells adhered with poly-l-lysine or an agarose pad compared to a Δ₄pol knockout maintained under the same conditions. Each box plot corresponds to 4 biological replicates. *, P < 0.01. (D) Box plot of calcium AUC for E. coli adhered under agarose similarly to the method used for the other measurements or encased in agarose by adding the cells before solidification of the gel. Each box plot corresponds to 5 biological replicates. *, P < 0.01. (E) Proposed model for E. coli mechanosensation. A force is applied by the agarose pad which is opposed by an equal and opposite force from the coverslip. The coverslip force arises from adherence with secreted EPS, which then interacts with lipopolysaccharide (LPS) on the outer membrane of the cell. This force is relayed to a mechanosensor in the inner membrane which remains to be identified. For all box plots, red lines indicate medians, blue boxes indicate 25/75 limits, black lines indicate 10/90 limits, and a red cross indicates an outlier.

Knockouts of genes associated with DNA repair have reduced calcium AUC. (A) Box plot of knockouts involved in DNA repair compared to WT cells. Each box plot corresponds to 5 biological replicates. (B) TUNEL fluorescence measured by a flow cytometer for WT, ΔrecA, and ΔrecG cells. *, P < 0.05. (C) TMRM fluorescence for comparisons of WT cells to rec knockouts. Each marker represents the median of the population for one biological replicate, and 3 biological replicates were measured for each condition. Addition of 50 μM CCCP is used to show fluorescence from zero voltage. (D) Growth curves measured by OD₆₀₀ for comparisons of WT cells to rec knockout cells. Each dark line represents the mean and shaded areas represent standard deviations of results from 3 biological replicates. For all box plots, red lines indicate medians, blue boxes indicate 25/75 limits, and black lines indicate 10/90 limits.