Quick replication fork stop by overproduction of &lt;i&gt;Escherichia coli&lt;/i&gt; DinB produces non-proliferative cells with an aberrant chromosome

Ikeda, Mio; Shinozaki, Yutaka; Uchida, Kaori; Ohshika, Yasuha; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro

doi:10.1266/ggs.87.221

Cited by 4 publications

(2 citation statements)

References 54 publications

(79 reference statements)

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“…Moreover, pGB- dinB was not deleterious for growth in HolD + LexA + or LexAdef backgrounds, confirming previous results showing that DinB expressed from a pSC101 replicon is not deleterious for growth, even in the absence of the LexA repressor [29] . In these conditions, DinB is expressed at 8- and 30-times the wild-type chromosomal level, respectively [29] , and replication in wild-type cells is only sensitive to the higher levels of DinB over-expression [30] , [31] . However, pGB- dinB could not be introduced into Δ holD argE :: ssb or JJC2394 cells on MM ( Figure 4 , Table S2 ); on LB, Δ holD argE :: ssb [pGB- dinB ] clones were obtained at 37°C and 42°C but could not be propagated ( Figure S5 ).…”

Section: Resultsmentioning

confidence: 99%

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

Duigou

Silvain²,

Viguera

et al. 2014

PLoS Genet

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The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.

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Section: Resultsmentioning

confidence: 99%

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

Duigou

Silvain²,

Viguera

et al. 2014

PLoS Genet

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“…The peak of cells with 2 chromosomes represents bacteria that had not initiated replication when antibiotics were added, and a delay in initiation would increase the proportion of such cells in a population 27 . Such a delay is observed in most of the mutants whilst problems with completing ongoing replication are apparent from poorly resolved peaks [28][29][30] .…”

Section: Mutant Strains Display Diverse Replication Patterns Dna Conmentioning

confidence: 99%

Disruption of glycogen metabolism alters cell size inEscherichia coli

Walt¹,

Bürgy²,

Engelbrecht³

et al. 2020

Preprint

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The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation -glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) -were disrupted, we examined if endogenous polyglucan degradation affects cell size. It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ∆malP mutant cells did, however, accumulate increased DnaA amounts at late growth stages indicating a potential effect on DNA replication. Replication run-out experiments demonstrated that this was indeed the case, and that DNA replication was also affected in the other mutants. Bacteria with a disruption in glgX accumulated glycogen and protein inclusion bodies that coincided with each other at inter-nucleoid and polar regions.Robust adaptive survival strategies are employed by bacteria to promote fitness. Most notably, when nutrients are abundant, bacterial cells demonstrate increases in length, width and growth rate 1 which permit parental cells to promote vigour of future generations by giving rise to larger daughter cells 2 . Enhanced rates of macromolecular biosynthesis accompany nutrientdependent size increases to ensure that larger cells are born with more DNA, RNA and protein 3-6 . This positive scaling relationship between cell size and external nutrient-imposed growth rate has historically been termed Schaechter's nutrient growth law 1 . Escherichia coli is known to passively correct deviant changes to cell size by adding a constant volume of cellular material (cell unit) between division events, regardless of size at birth 7,8 which has led to the proposal of what is known as the adder model 9 .Under steady-state conditions, the cell unit postulated in the adder model is dictated by two mechanisms 10 . The first is an initiation adder that ensures DNA origin firing is triggered only when the cell has grown and accumulated protein factors, such as DnaA, to a threshold required to initiate DNA replication. Synchronous origin firing under conditions of slow growth results in the birth of two daughters, each containing one parental chromosome; however, in environments that promote rapid growth, cell division can occur at a rate faster than the time it takes to duplicate the genome. Bacteria overcome this problem by initiating overlapping replication cycles, so daughter cells are born with two, four or even eight replication origins that ensure genomic integrity is maintained when the cell is faced with rapid division rates 11 . Several studies have reported that the volume of the cell unit proposed in the adder model is prop...

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Disruption of Glycogen Metabolism Alters Cell Size in Escherichia Coli

Walt

Bürgy

Engelbrecht

et al. 2021

Preprint

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Background: The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation - glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) - were disrupted, we examined if degradation of this energy storage compound affects cell size. Results: It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ∆malP mutant cells did, however, show abnormal nuclear staining patterns and accumulate increased DnaA amounts at late growth stages, indicating a potential effect on DNA replication. Conclusion: This study reveals a link between glycogen metabolism and cell size in E. coli. A disrupted malP allele is associated with cell size heterogeneity and elongation at all stages of growth, without detectable changes to mass doubling time, FtsZ protein levels or division ring frequency.

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Quick replication fork stop by overproduction of <i>Escherichia coli</i> DinB produces non-proliferative cells with an aberrant chromosome

Cited by 4 publications

References 54 publications

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

Disruption of glycogen metabolism alters cell size inEscherichia coli

Disruption of Glycogen Metabolism Alters Cell Size in Escherichia Coli

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