From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction-modification gene complex

Abstract

Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants' deaths. With type II restriction-modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and sigma(E)-regulon was noticeable. With time, more SOS genes, stress-response genes (including sigma(S)-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of sigma(E)-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several sigma(E)-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes.

Figure 1.

Postsegregational cell killing triggered by loss of PaeR7I RM gene complex. (A) Experimental design. Cell death was induced by blocking the replication of a plasmid with temperature-sensitive replication machinery and PaeR7I RM gene complex. (B) The plasmid-carrying viable cell counts. (C) The viable cell counts. (D) OD₆₆₀. The values are relative to those at the time of the temperature shift (0 h). The culture was diluted 100-fold when its OD₆₆₀ reached 0.5 (2 h after the temperature shift).

Figure 2.

Functional classification of genes demonstrating differential expression with respect to r⁺/r⁻. Genes with a significantly higher expression level in r⁺m⁺ than r⁻m⁺ strains (left, reddish color), and a lower expression level in r⁺m⁺ than r⁻m⁺ strains (right, greenish color) were categorized by COG codes (http://www.ncbi.nlm.nih.gov/COG/new/) (24,25), with an additional J’ category. The colors show the significance of category overrepresentation based on the chi-squared test.

Figure 3.

Hierarchical clustering of genes demonstrating differential expression with respect to r⁺/r⁻. (Left) The 1328 genes with a statistically significant difference in expression at, at least, one of the four time points were hierarchically clustered according to their temporal expression. The red (higher) and green (lower) colors of the bars represent the transcript ratio of r⁺/r⁻. (Right) Examples of the genes from each cluster are shown with their functional categories by COG as in Figure 2.

Figure 4.

Protein release associated with the RM-mediated cell death. Protein fractions outside of the cells were obtained 6 h after the shift and analyzed by SDS–PAGE. The bacterial culture was centrifuged at 17 000g for 5 min, the supernatant was collected. The protein fraction of MW >10 kDa obtained from 0.2 ml of the supernatant was applied to 10% SDS–PAGE.

Figure 5.

Effects of over-expression of σ^E regulon genes. (A) OD₆₆₀. (B) Viable cell counts. (C) Cell morphology. (D) Proteins outside of the cells were analyzed 3.1 h after IPTG addition for expression of rpoE, htrG and yfeK. The bacterial culture was centrifuged at 17 000g for 5 min, the supernatant was collected. The protein fraction of MW >10 kDa obtained from 0.2 ml of the supernatant was applied to 10% SDS–PAGE.

Figure 6.

Summary of the transcriptome changes during the death process following the loss of RM gene complex. Induction (red) and repression (green) are shown. Transcriptome features reported for ofloxacin-induced (yellow) and ampicillin-induced (purple) deaths are indicated.