RNA-seq Analyzes Transcriptomes of dnaB-Ts and dnaB-Ts ?ahpC at Both Permissive and Non-permissive Temperature

To generate a bona fide model to study post-stress baterial programmed cell death (PCD), we used a temperature-sensitive E. coli mutant (dnaB-Ts) since temperature stress can be rapidly reversed while other stress, such as antibotic treatment, is difficult to be rapidly and completely stopped and removed for post-stress PCD analysis. When cells were shifted from permissive (30 °C) to non-permissive temperature (42 °C), the dnaB-Ts ?ahpC double mutant maintained full surival while survival of the dnaB-Ts single mutant dropped 3 orders of magnitude; similarly, the double mutant showed much higher survival during treatments with quinolones and ß-lactams at permissive temperature. The purpose of the RNA-seq analyses is to uncover molecular mechanisms underlying the increased survival of the dnaB-Ts ?ahpC double mutant by comparing its transcriptional profiles with that of the dnaB-Ts single mutant at both permissive and non-permissive temperature. The results showed that multiple antioxidative systems and stress response pathways were highly upregulated due to a deficiency of ahpC when combined with a dnaB-Ts mutation. The high expression of these genes are consistent with the lower levels of toxic reactive oxygen species (ROS) in the dnaB-Ts ?ahpC mutant than in the dnaB-Ts single mutant. Thus, the RNA-seq data supported that ROS are an important lethal factor in bacterial suicide pathways when bacterial cells are exposed to harsh stress. Overall design: Two independent early log-phase cells of a dnaB-Ts mutant or a dnaB-Ts ?ahpC mutant, incubated at 30 °C or at 42 °C for 60 min, were collected and mixed together for total RNA extraction and subsequenct RNA-seq sequencing.