The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>

ABSTRACT 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, in...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	James A. Kraemer, Allen G. Sanderlin, Michael T. Laub
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	DNA topology DnaA ppGpp replication initiation stringent response Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/2e4890f49a714dfa9faa376a7b10132c
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:2e4890f49a714dfa9faa376a7b10132c
record_format	dspace
spelling	oai:doaj.org-article:2e4890f49a714dfa9faa376a7b10132c2021-11-15T16:22:11ZThe Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>10.1128/mBio.01330-192150-7511https://doaj.org/article/2e4890f49a714dfa9faa376a7b10132c2019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01330-19https://doaj.org/toc/2150-7511ABSTRACT 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 oriC. IMPORTANCE 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.James A. KraemerAllen G. SanderlinMichael T. LaubAmerican Society for MicrobiologyarticleDNA topologyDnaAppGppreplication initiationstringent responseMicrobiologyQR1-502ENmBio, Vol 10, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA topology DnaA ppGpp replication initiation stringent response Microbiology QR1-502
spellingShingle	DNA topology DnaA ppGpp replication initiation stringent response Microbiology QR1-502 James A. Kraemer Allen G. Sanderlin Michael T. Laub The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>
description	ABSTRACT 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 oriC. IMPORTANCE 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.
format	article
author	James A. Kraemer Allen G. Sanderlin Michael T. Laub
author_facet	James A. Kraemer Allen G. Sanderlin Michael T. Laub
author_sort	James A. Kraemer
title	The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>
title_short	The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>
title_full	The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>
title_fullStr	The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>
title_full_unstemmed	The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>
title_sort	stringent response inhibits dna replication initiation in <named-content content-type="genus-species">e. coli</named-content> by modulating supercoiling of <italic toggle="yes">oric</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/2e4890f49a714dfa9faa376a7b10132c
work_keys_str_mv	AT jamesakraemer thestringentresponseinhibitsdnareplicationinitiationinnamedcontentcontenttypegenusspeciesecolinamedcontentbymodulatingsupercoilingofitalictoggleyesoricitalic AT allengsanderlin thestringentresponseinhibitsdnareplicationinitiationinnamedcontentcontenttypegenusspeciesecolinamedcontentbymodulatingsupercoilingofitalictoggleyesoricitalic AT michaeltlaub thestringentresponseinhibitsdnareplicationinitiationinnamedcontentcontenttypegenusspeciesecolinamedcontentbymodulatingsupercoilingofitalictoggleyesoricitalic AT jamesakraemer stringentresponseinhibitsdnareplicationinitiationinnamedcontentcontenttypegenusspeciesecolinamedcontentbymodulatingsupercoilingofitalictoggleyesoricitalic AT allengsanderlin stringentresponseinhibitsdnareplicationinitiationinnamedcontentcontenttypegenusspeciesecolinamedcontentbymodulatingsupercoilingofitalictoggleyesoricitalic AT michaeltlaub stringentresponseinhibitsdnareplicationinitiationinnamedcontentcontenttypegenusspeciesecolinamedcontentbymodulatingsupercoilingofitalictoggleyesoricitalic
_version_	1718426893654425600

The Stringent Response Inhibits DNA Replication Initiation in <named-content content-type="genus-species">E. coli</named-content> by Modulating Supercoiling of <italic toggle="yes">oriC</italic>

Ejemplares similares