Thalia H. Sass and Susan T. Lovett

PNAS; June 27, 2024; 121 (27) e2407832121, https://doi.org/10.1073/pnas.2407832121

Significance

The DNA damage response in bacteria allows them to persist in unfavorable environments, including in human hosts. Genes induced by the DNA damage response promote the acquisition and spread of antibiotic resistance and virulence factors. This study identifies a major determinant of expression following replication stress in Escherichia coli, the SspA protein, and shows that its action is redirected upon DNA damage. It also identifies groups of genes induced or repressed by sublethal exposure to the replication inhibitor, azidothymidine.

Abstract

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli, which later came to be called the “SOS response.” We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor “stringent starvation protein” SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance.

See https://www.pnas.org/doi/10.1073/pnas.2407832121

Fig.1: LexA-dependent and independent genes. (Upper) AZT induction values (log2 fold change) in wt vs. lexA3 (self-cleavage defective, SOS noninducible) strains. Dashed yellow line indicates identical dependence in both backgrounds. Expression of genes to the right of the red dashed line are elevated by AZT in wt strains; those to the left are repressed by AZT. (Lower) Genetic effects on expression across the e14 cryptic prophage. Coverage is the normalized frequency of read counts at each genomic position × 4641652 (genome size of MG1655, GenBank reference U000913) after AZT treatment; colors indicate genotype of strains: gray, wt; green, lexA3; gold, sspAΔ, light blue; lexA3 sspAΔ double mutant.