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The stringent response and cell cycle arrest in Escherichia coli.

Ferullo DJ, Lovett ST - PLoS Genet. (2008)

Bottom Line: In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point.We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation.This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA.

ABSTRACT
The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.

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Release from stringent arrest.Wild type cells in M9 Glucose CAA medium were treated with serine hydroxamate for 1.5 h as previously described. Cells were then washed of serine hydroxamate, incubated in fresh medium, and samples were taken for flow cytometric analysis for DNA content and DAPI staining as previously described. (A) DNA content histograms and (B) corresponding fluorescent DAPI images of cultures incubated with serine hydroxamate for 1.5 h and of cultures at 15, 30, and 60 min after being released from serine hydroxamate. (C) Model for chromosome segregation patterns for 4N cells released from stringent arrest. Cohesion of sister chromosomes help enforce the sequence of segregation of chromosomes from the single nucleoid mass. Pairs of sister chromosomes held in cohesion at their termini segregate first at midcell. After release of cohesion, sisters separate at ¼ positions.
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pgen-1000300-g005: Release from stringent arrest.Wild type cells in M9 Glucose CAA medium were treated with serine hydroxamate for 1.5 h as previously described. Cells were then washed of serine hydroxamate, incubated in fresh medium, and samples were taken for flow cytometric analysis for DNA content and DAPI staining as previously described. (A) DNA content histograms and (B) corresponding fluorescent DAPI images of cultures incubated with serine hydroxamate for 1.5 h and of cultures at 15, 30, and 60 min after being released from serine hydroxamate. (C) Model for chromosome segregation patterns for 4N cells released from stringent arrest. Cohesion of sister chromosomes help enforce the sequence of segregation of chromosomes from the single nucleoid mass. Pairs of sister chromosomes held in cohesion at their termini segregate first at midcell. After release of cohesion, sisters separate at ¼ positions.

Mentions: We wondered how cell cycle patterns would be reset after release from arrest. Would cells resume replication in a pattern similar to that prior to arrest? Or would cells be obligated to segregate each chromosome, divide and initiate replication from 1N progeny? To examine this, after 90 minutes of stringent arrest induced by serine hydroxamate, wild-type cells in minimal M9 Glucose CAA medium were washed and allowed to resume growth. DNA content was followed over time by flow cytometry and nucleoid appearance was monitored by DAPI staining (Figure 5A and 5B). As early as 15 minutes after release, some signs of nucleoid segregation were apparent. Segregation of two nucleoids appeared first at midcell; later segregation at the ¼ positions to form 4 nucleoids was detected. We think this reflects sequential and ordered segregation of chromosomes: sister-chromosome pairs held in cohesion segregate first, followed by separation of sisters (see schematic in Figure 5C). From 15 to 30 minutes, DNA content in the entire population appeared to increase, concomitant with nucleoid condensation and segregation. Therefore, cells appear to assume DNA content equivalent to conditions before the arrest, suggesting that replication patterns are reset quickly upon release.


The stringent response and cell cycle arrest in Escherichia coli.

Ferullo DJ, Lovett ST - PLoS Genet. (2008)

Release from stringent arrest.Wild type cells in M9 Glucose CAA medium were treated with serine hydroxamate for 1.5 h as previously described. Cells were then washed of serine hydroxamate, incubated in fresh medium, and samples were taken for flow cytometric analysis for DNA content and DAPI staining as previously described. (A) DNA content histograms and (B) corresponding fluorescent DAPI images of cultures incubated with serine hydroxamate for 1.5 h and of cultures at 15, 30, and 60 min after being released from serine hydroxamate. (C) Model for chromosome segregation patterns for 4N cells released from stringent arrest. Cohesion of sister chromosomes help enforce the sequence of segregation of chromosomes from the single nucleoid mass. Pairs of sister chromosomes held in cohesion at their termini segregate first at midcell. After release of cohesion, sisters separate at ¼ positions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2586660&req=5

pgen-1000300-g005: Release from stringent arrest.Wild type cells in M9 Glucose CAA medium were treated with serine hydroxamate for 1.5 h as previously described. Cells were then washed of serine hydroxamate, incubated in fresh medium, and samples were taken for flow cytometric analysis for DNA content and DAPI staining as previously described. (A) DNA content histograms and (B) corresponding fluorescent DAPI images of cultures incubated with serine hydroxamate for 1.5 h and of cultures at 15, 30, and 60 min after being released from serine hydroxamate. (C) Model for chromosome segregation patterns for 4N cells released from stringent arrest. Cohesion of sister chromosomes help enforce the sequence of segregation of chromosomes from the single nucleoid mass. Pairs of sister chromosomes held in cohesion at their termini segregate first at midcell. After release of cohesion, sisters separate at ¼ positions.
Mentions: We wondered how cell cycle patterns would be reset after release from arrest. Would cells resume replication in a pattern similar to that prior to arrest? Or would cells be obligated to segregate each chromosome, divide and initiate replication from 1N progeny? To examine this, after 90 minutes of stringent arrest induced by serine hydroxamate, wild-type cells in minimal M9 Glucose CAA medium were washed and allowed to resume growth. DNA content was followed over time by flow cytometry and nucleoid appearance was monitored by DAPI staining (Figure 5A and 5B). As early as 15 minutes after release, some signs of nucleoid segregation were apparent. Segregation of two nucleoids appeared first at midcell; later segregation at the ¼ positions to form 4 nucleoids was detected. We think this reflects sequential and ordered segregation of chromosomes: sister-chromosome pairs held in cohesion segregate first, followed by separation of sisters (see schematic in Figure 5C). From 15 to 30 minutes, DNA content in the entire population appeared to increase, concomitant with nucleoid condensation and segregation. Therefore, cells appear to assume DNA content equivalent to conditions before the arrest, suggesting that replication patterns are reset quickly upon release.

Bottom Line: In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point.We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation.This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA.

ABSTRACT
The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.

Show MeSH
Related in: MedlinePlus