The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the
nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which
ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that
ppGpp blocks new rounds of replication by inhibiting transcription of the essential
initiation factor dnaA, but we found that replication is still inhibited by
ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by
ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of
ppGpp reduces initiation potential at oriC by reducing transcription. We find that maintaining transcription near oriC, either by expressing a
ppGpp-blind
RNA polymerase mutant or by inducing transcription from a
ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by
ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting
topoisomerase I or by deleting the nucleoid-associated
protein gene seqA also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which
ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils into oriCIMPORTANCE To survive bouts of
starvation, cells must inhibit DNA replication. In bacteria,
starvation triggers production of a signaling molecule called
ppGpp (
guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While
ppGpp has been known to block replication initiation in Escherichia coli for decades, the mechanism responsible was unknown. Early work suggested that
ppGpp drives a decrease in levels of the
replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that
ppGpp leads to a change in
DNA topology that prevents initiation.
ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of
ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.