Mechanism of Escherichia coli Lethality Caused by Overexpression of flhDC, the Flagellar Master Regulator Genes, as Revealed by Transcriptome Analysis

The flhDC operon of Escherichia coli encodes a transcription factor that initiates flagella synthesis, elevates flagella construction and enhances cell motility, which all are energetically costly and highly regulated processes. In this study, we found that overexpression of flhDC genes from a strong regulatable pN15E6 plasmid could inhibit the growth of E. coli host cells and even eventually cause death. We used transcriptome analysis to investigate the mechanism of flhDC overexpression lethal to host bacteria. The results showed that a total of 568 differentially expressed genes (DEGs), including 378 up-regulated genes and 190 down-regulated genes were detected when the flhDC genes were over-expressed. Functional enrichment analysis results showed that the DEGs are related to a series of crucial biomolecular processes, including flagella synthesis, oxidative phosphorylation and pentose phosphate pathways, etc. We then examined, using RT-qPCR, the expression of key genes of the oxidative phosphorylation pathway at different time points after induction. Results showed that their expression increased in the early stage and decreased afterward, which was suggested to be the result of feedback on the overproduction of ROS, a strong side effect product of the elevated oxidative phosphorylation process. To further verify the level of ROS output, flhDC over-expressed bacteria cells were stained with DCHF-DA and a fluorescence signal was detected using flow cytometry. Results showed that the level of ROS output was higher in cells with over-expressed flhDC than in normal controls. Besides, we found upregulation of other genes (recN and zwf) that respond to ROS damage. This leads to the conclusion that the bacterial death led by the overexpression of flhDC genes is caused by damage from ROS overproduction, which leaked from the oxidative phosphorylation pathway.