Figure 5.

REase delay is disturbed in cells lacking the C regulator as monitored in real-time at the single cell level. (A) Timing of REase expression (red fluorescence) in individual host cells, infected by recombinant M13 phages carrying R-M system with C protein (red points; N = 111) and without C protein (blue points; N = 110). The times are set in reference to appearance of MTase expression (green fluorescence detection), defined as time 0 (x axis). The vertical axis indicates the intensity of rising red fluorescence, which crossed the background red fluorescence baseline at the earliest time. The average time values for REase expression for both +C and ΔC systems are indicated with error bars (standard deviations), which are each about ±15 min. Mean REase expression timing for the ΔC R-M system is ∼15 min earlier than MTase expression, and about 10 min later than MTase expression for the +C R-M system. The two-tailed P value between the ±C groups is <0.0001. (B) Distribution curve of scattered points from panel A, showing the number of cells for each variant (ΔC versus +C) grouped by timing values of appearance of red fluorescence (REase expression) in comparison to appearance of green fluorescence (MTase). The black lines represent the trend lines indicating a roughly normal distribution for both variants. The double-headed arrow indicates the shift in means, of ∼15 min. (C) Representative series of time-lapse images taken independently for cells infected with the two variants of recombinant M13 phages (+C versus ΔC). The 12 frames cover a 60 min time range, with each shot taken at 5 min intervals after M13 infection. Arrows indicate the time of detection of rising fluorescence from no fluorescent background, red fluorescence for REase and green for MTase. In the upper panel for the +C- R-M system, REase expression is detected 10 min after MTase, whereas in the bottom panel for the ΔC R-M system, the REase expression precedes the MTase detection by about 15 min.