50, p = 0.0385) AZD1152 supplier and CVs (F [1,2] = 46.24, p = 0.0209). A similar negative relationship was also Everolimus apparent for
the MLTs. However, because of the case of the LB medium, in which the higher growth rate actually resulted in a slightly longer MLT, the observed negative relationship was not significant (F [1,2] = 6.44, p = 0.1265). Interestingly, neither the SDs (F [1,2] = 16.11, p = 0.0568) nor the CVs (F [1,2] = 6.04, p = 0.133) was significantly associated with the MLTs. Effects of KCN Addition The energy poison potassium cyanide, KCN, has long been used in phage research to trigger premature lysis [43]. Typically, after KCN addition, culture turbidity declines precipitously [44], indicating that individual lysis events are relatively synchronous. The KCN-induced premature lysis is thought to be mediated through a collapsed proton motive force (PMF) resulting from a inhibition of the bacterial respiratory chain. As has been shown with λ S holin, a 40% drop in the PMF triggers lysis [45]. Without
a constant supply of ATP, the production of holin protein would also be terminated. If KCN is added soon after thermal induction of the lysogen culture, few holin proteins would have been made before the termination of holin production. Consequently, it should take a longer time for the holin proteins Rapamycin in vivo in the membrane to transition from a diffused state to aggregated rafts. Therefore, after the cessation of holin production by KCN addition, it may take a longer time, on average, before any lysis events are observed. On the other hand, if KCN is added late, a larger proportion of the thermally-induced lysogenic cells should have accumulated enough holin proteins in the cell membrane such that they could be triggered to form holin holes quickly. That is, the addition of KCN should prompt the rapid formation of holin holes, thus resulting in an almost immediate and synchronous lysis of most of the cells in the population. Based on the aforementioned scenarios, we expected that
(1) the time delay between the time of KCN addition (t KCN) and the eventual mean lysis time (t L) (i.e., t L – t KCN) would be negatively correlated with the timing of KCN addition, and (2) t KCN would be negatively correlated with lysis time stochasticity. Figure CHIR-99021 clinical trial 4A shows a significant negative relationship between t L – t KCN and t KCN. As KCN was added later in time (i. e., closer to the normal lysis time of 65.1 min), the time delay between addition of KCN and the MLT was reduced (a quadratic fit, F [2,4] = 12.87, p = 0.0181, adjusted R 2 = 0.798). In fact, when added 55 min after induction (i.e., 10 min before the normal MLT), the time delay was only 2.6 min, almost instantaneous when compared to the 2 min sampling rate of the sipper-equipped spectrophotometer method of lysis time determination [46].