Escherichia coli Genes and Pathways Involved in Surviving Extreme Exposure to Ionizing Radiation

Abstract

To further an improved understanding of the mechanisms used by bacterial cells to survive extreme exposure to ionizing radiation (IR), we broadly screened nonessential Escherichia coli genes for those involved in IR resistance by using transposon-directed insertion sequencing (TraDIS). Forty-six genes were identified, most of which become essential upon heavy IR exposure. Most of these were subjected to direct validation. The results reinforced the notion that survival after high doses of ionizing radiation does not depend on a single mechanism or process, but instead is multifaceted. Many identified genes affect either DNA repair or the cellular response to oxidative damage. However, contributions by genes involved in cell wall structure/function, cell division, and intermediary metabolism were also evident. About half of the identified genes have not previously been associated with IR resistance or recovery from IR exposure, including eight genes of unknown function.

Plasmidic Expression ofnemAandyafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

“…2A). The yafC gene encodes a putative transcriptional regulator of unknown function (33). A single base mutation was found in the C terminus of the yafC coding region (D275G), designated yafC* (Fig.…”

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

“…The underrepresentation of mutants within the IR-challenged samples would suggest that the corresponding genes were involved in mitigating the stress of IR treatment, which is established to cause DNA damage (23,24). Despite decades of research on E. coli DNA repair (24), the authors identified approximately 20 genes not previously known to be important for survival following IR exposure that were missed using classical genetic approaches (18). Similarly, a recent report used Tn-seq to identify genes not previously implicated in the well-studied process of sporulation in Bacillus subtilis (16).…”

“…1B). There have been multiple methods reported to determine which of the mutants have statistically significant deviations from the wild-type population (5,6,9,(16)(17)(18). The most straightforward method is to apply a Mann-Whitney U test, which makes no assumptions about the underlying distribution of the data (16).…”

“…Many Tn-seq-type experiments have been published for a variety of bacteria (1)(2)(3)(4)(5). As an example, a recent investigation utilized a Tn-seq-like approach to isolate Escherichia coli mutants that are sensitive to ionizing radiation (IR) (18). The underrepresentation of mutants within the IR-challenged samples would suggest that the corresponding genes were involved in mitigating the stress of IR treatment, which is established to cause DNA damage (23,24).…”

“…The next method to identify over-or underrepresented mutants in a transposon insertion mutant library is to calculate a ratio of mutant abundance for each gene (5,6,9,(16)(17)(18)). An advantage of calculating a ratio of mutant abundance is that the population does not have to be growing exponentially during the experiment, which greatly expands the range of experimental designs amenable to analysis.…”