Catastrophic chromosome fragmentation probes the nucleoid structure and dynamics in Escherichia coli

Abstract

Abstract Escherichia coli cells treated with a combination of cyanide (CN) and hydrogen peroxide (HP) succumb to catastrophic chromosome fragmentation (CCF), detectable in pulsed-field gels as >100 double-strand breaks per genome equivalent. Here we show that CN + HP-induced double-strand breaks are independent of replication and occur uniformly over the chromosome,—therefore we used CCF to probe the nucleoid structure by measuring DNA release from precipitated nucleoids. CCF releases surprisingly little chromosomal DNA from the nucleoid suggesting that: (i) the nucleoid is a single DNA-protein complex with only limited stretches of protein-free DNA and (ii) CN + HP-induced breaks happen within these unsecured DNA stretches, rather than at DNA attachments to the central scaffold. Mutants lacking individual nucleoid-associated proteins (NAPs) release more DNA during CCF, consistent with NAPs anchoring chromosome to the central scaffold (Dps also reduces the number of double-strand breaks directly). Finally, significantly more broken DNA is released once ATP production is restored, with about two-thirds of this ATP-dependent DNA release being due to transcription, suggesting that transcription complexes act as pulleys to move DNA loops. In addition to NAPs, recombinational repair of double-strand breaks also inhibits DNA release by CCF, contributing to a dynamic and complex nucleoid structure.