Figure 4 Effects of t KCN (timing of KCN addition). (A) On time delay t L – t KCN. The solid curve shows the quadratic fit of y = 54.52 – 1.09x + 0.02(x – 36.57)2. Error bars indicate the associated SDs. As an example, when
t KCN = 45 min, the observed t L is 50.11 min, thus the time delay is t L – t KCN = 5.11 min. (B) On lysis time SD (closed circles) and CV (closed triangles). Solid curve shows the quadratic fit of SD against t KCN (y = 13.24 – 0.28x + 0.01(x – 36.57)2). The effects of t KCN on lysis time SDs and CVs are shown in Figure 4B. Again, we witnessed the expected pattern of a significant negative relationship between t KCN and the SDs (a quadratic fit, F [2,4] = 9.91, p = 0.0123, adjusted R 2 = 0.748) and between t KCN and the CVs (a quadratic fit, F [2,4] = 16.03, ABT888 p = 0.0282, adjusted R 2 = 0.834). These results showed that the later in time KCN was added, the less variation there was in individual lysis times. In fact, the lowest SD (1.45 min) and lowest CV (2.53%) were observed when KCN was added 55 min after induction. This was a significant THZ1 cost two-fold reduction
in the SD when compared normal lysis conditions (see Table 1 for strain IN56 with the SD = 3.24 min; Student’s t = 15.45, p < 0.0001, using the standard deviation for the SD in Box 7.1 of [56]). This observation indicated that individual triggering for hole formation during the normal progression of cell lysis was relatively asynchronous when compared to the artificial method of acute triggering by KCN addition.
Similar to the effect of growth rate, a linear regression of the SDs (F [1,5] = 0.60, p = 0.4726) or CVs (F [1,5] = 0.328, p = 0.5917) against the MLTs did not yield significant result. Another Endonuclease interesting aspect of the relationship between t KCN and the lysis time SDs is that the SDs drop precipitously when KCN is added about 35 min after induction. This observation TGF-beta inhibitor suggests that, approximately 35 min after thermal induction, the majority of the lysogenic cells have accumulated enough holin proteins in the cell membrane to form holes immediately if triggered. Discussion The current model of holin hole formation hypothesizes that λ phage lysis timing is mainly determined by when a critical concentration of holin proteins is reached in the cell membrane [40] (Figure 1, dark arrows). According to this model, any factor that influences the holin protein production should also affect the timing of lysis. Furthermore, the realized rate of holin production in each cell should also be subjected to stochastic influences impacting the various upstream biochemical reactions, such as gene transcription and translation, that lead to holin production. As has been shown by others, the lower the average rates of the biochemical reactions, the more prominent the cell-to-cell variation is [51, 52].