Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication

Ferullo, Daniel J.; Cooper, Deani L.; Moore, Hayley R.; Lovett, Susan T.

doi:10.1016/j.ymeth.2009.02.010

Cited by 106 publications

(99 citation statements)

References 37 publications

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“…This change in DNA content meant that the ratio of REL8593A to REL607 cells in the control mixtures had to be corrected to reflect the actual number of copies of the evolved and ancestral alleles in these samples. To account for this difference, we stained stationary-phase cultures of the ancestral Ara -clone, REL606, and the 20,000-generation clone, REL8593A, with a PicoGreen fluorescent DNA stain (Ferullo et al 2009) and used flow cytometry to quantify the average genomic DNA content per cell in DM100 media ( Figure S1). We used six replicate measurements to calculate a correction factor for the allele frequencies in the REL8593A/REL607 mixtures.…”

Section: Allele Frequency Measurementsmentioning

confidence: 99%

Adaptation, Clonal Interference, and Frequency-Dependent Interactions in a Long-Term Evolution Experiment withEscherichia coli

2015

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Twelve replicate populations of Escherichia coli have been evolving in the laboratory for .25 years and 60,000 generations. We analyzed bacteria from whole-population samples frozen every 500 generations through 20,000 generations for one wellstudied population, called Ara21. By tracking 42 known mutations in these samples, we reconstructed the history of this population's genotypic evolution over this period. The evolutionary dynamics of Ara21 show strong evidence of selective sweeps as well as clonal interference between competing lineages bearing different beneficial mutations. In some cases, sets of several mutations approached fixation simultaneously, often conveying no information about their order of origination; we present several possible explanations for the existence of these mutational cohorts. Against a backdrop of rapid selective sweeps both earlier and later, two genetically diverged clades coexisted for .6000 generations before one went extinct. In that time, many additional mutations arose in the clade that eventually prevailed. We show that the clades evolved a frequency-dependent interaction, which prevented the immediate competitive exclusion of either clade, but which collapsed as beneficial mutations accumulated in the clade that prevailed. Clonal interference and frequency dependence can occur even in the simplest microbial populations. Furthermore, frequency dependence may generate dynamics that extend the period of coexistence that would otherwise be sustained by clonal interference alone.KEYWORDS experimental evolution; clonal interference; frequency-dependent selection; beneficial mutations; asexual populations; mutational cohorts T HE long-term evolution experiment (LTEE) spans .25 years and 60,000 generations of bacterial evolution. In this experiment, 12 replicate populations of Escherichia coli have been propagated in a simple environment, and samples of each population have been frozen at 500-generation intervals. This experiment originally focused on whether and to what extent the populations would diverge in their mean fitness and other phenotypic properties as they adapted to identical environments (Lenski et al. 1991;Lenski and Travisano 1994). Over time, this experiment has become a model for exploring many other aspects of evolution, including the emergence of new functions (Blount et al. 2008), the evolution of mutation rates (Sniegowski et al. 1997), the maintenance of genetic diversity (Elena and Lenski 1997;Rozen and Lenski 2000;Le Gac et al. 2012), and the structure of the fitness landscape (Khan et al. 2011;Woods et al. 2011;Wiser et al. 2013). The ability to examine these and other issues has grown tremendously as data that were difficult or impossible to obtain when the LTEE began have yielded to new technologies, particularly genome sequencing Lenski 2009, 2013;Blount et al. 2012;Wielgoss et al. 2013).The LTEE has also inspired theoretical work, especially on the dynamics of adaptation in large asexual populations (Gerrish and Lenski 1998;Hegreness et al....

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Section: Allele Frequency Measurementsmentioning

confidence: 99%

Adaptation, Clonal Interference, and Frequency-Dependent Interactions in a Long-Term Evolution Experiment withEscherichia coli

2015

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“…However, an effective method for synchronizing populations of Escherichia coli involves growing cells in conditions that promote the highly conserved stringent response, which induces a G1 arrest (27). The stringent response of S. meliloti is stimulated by both carbon and nitrogen starvation and is dependent on ppGpp synthesis by the enzyme Rel Sm (28,29).…”

Section: Synchronization Of S Meliloti Cell Populations Via Nutrientmentioning

confidence: 99%

Global analysis of cell cycle gene expression of the legume symbiontSinorhizobium meliloti

Nisco

Abo

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

118

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In α-proteobacteria, strict regulation of cell cycle progression is necessary for the specific cellular differentiation required for adaptation to diverse environmental niches. The symbiotic lifestyle of Sinorhizobium meliloti requires a drastic cellular differentiation that includes genome amplification. To achieve polyploidy, the S. meliloti cell cycle program must be altered to uncouple DNA replication from cell division. In the α-proteobacterium Caulobacter crescentus, cell cycle-regulated transcription plays an important role in the control of cell cycle progression but this has not been demonstrated in other α-proteobacteria. Here we describe a robust method for synchronizing cell growth that enabled global analysis of S. meliloti cell cycle-regulated gene expression. This analysis identified 462 genes with cell cycle-regulated transcripts, including several key cell cycle regulators, and genes involved in motility, attachment, and cell division. Only 28% of the 462 S. meliloti cell cycle-regulated genes were also transcriptionally cell cycle-regulated in C. crescentus. Furthermore, CtrA-and DnaA-binding motif analysis revealed little overlap between the cell cycle-dependent regulons of CtrA and DnaA in S. meliloti and C. crescentus. The predicted S. meliloti cell cycle regulon of CtrA, but not that of DnaA, was strongly conserved in more closely related α-proteobacteria with similar ecological niches as S. meliloti, suggesting that the CtrA cell cycle regulatory network may control functions of central importance to the specific lifestyles of α-proteobacteria.cell cycle regulation | symbiosis | alpha-proteobacteria

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“…Flow cytometry was used to measure the direct DNA content per cell and was performed using the PicoGreen staining method previously described (Ferullo et al 2009). ''Run-out'' conditions included the addition of 300 mg/ml rifampicin and 3.2 mg/ml cephalexin to cultures grown to an OD 600 of 0.2.…”

Section: Methodsmentioning

confidence: 99%

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

Molt

Sutera

Moore³

et al. 2009

Genetics

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The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgETTn5-EZ seqAD, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaAD 96-120 , ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaAD 96-120 mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC.

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Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication

Cited by 106 publications

References 37 publications

Adaptation, Clonal Interference, and Frequency-Dependent Interactions in a Long-Term Evolution Experiment withEscherichia coli

Adaptation, Clonal Interference, and Frequency-Dependent Interactions in a Long-Term Evolution Experiment withEscherichia coli

Global analysis of cell cycle gene expression of the legume symbiontSinorhizobium meliloti

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

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