Tunable phenotypic variability through an autoregulatory alternative sigma factor circuit

A

It is unclear how genetic circuits (centre) tune phenotypic variability in a bacterial population in response to genetic perturbation, environmental stress and history (left) to modulate the fraction of cells that activate a given pathway (right).

B

Schematic of the σ^V circuit. In its inactive form, σ^V is bound to its anti‐sigma factor RsiV. If there is lysozyme present in the environment, RsiV binds to lysozyme and undergoes a conformational change. Only then the proteases SipS/ SipT and RasP can cleave RsiV to release σ^V. Once σ^V is released from RsiV, it can bind to RNA polymerase to redirect transcription to the σ^V regulon. σ^V can initiate transcription of its own operon which includes sigV (σ^V), rsiV (R), oatA (O, one of the main lysozyme resistance genes) and yrhK (Y, unknown function).

C

Time‐lapse microscopy of cells containing a P _sigV ‐YFP promoter reporter reveals heterogeneous activation of σ^V in response to lysozyme stress. The stress (red line) was added between the 200 and 300 min time points (at 240 min). The red arrow highlights a cell with a delayed activation of σ^V. Scale bar: 5 μm.

D

Time traces of mean YFP fluorescence per cell. Each trace represents a single‐cell lineage's response to 1 µg/ml lysozyme. (109 traces from two independent experiments). Lysozyme added at the black dashed line.

E

Figure D replotted to allow examination of variable activation times.

F

The observed heterogeneity is reduced with increasing stress levels. Each line represents the cumulative fraction of cells (N = ˜ 50) with P _sigV ‐YFP values that are higher than the half maximum of their final values (representing cells that have activated).

G

The fold change in mean YFP fluorescence increases with increasing stress levels, shown for two biological repeats (adjacent boxplots). Each day's fold change distribution consisted of > 40 manually corrected single‐cell traces. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25^th and 75^th percentiles, respectively. The lower and higher whiskers of boxplot are extended to the first quartile minus 1.5 * interquartile range and the third quartile plus 1.5 * interquartile range, respectively. The black dashed line is the mean fold change.

Figure 1. σV is activated heterogeneously in response to lysozyme stress.

Figure 1. σ^V is activated heterogeneously in response to lysozyme stress.