DNA-Membrane Anchor Facilitates Efficient Chromosome Translocation at a Distance inBacillus subtilis

ABSTRACTChromosome segregation in sporulatingBacillus subtilisinvolves the tethering of sister chromosomes at opposite cell poles. RacA is known to mediate chromosome tethering by interacting with both centromere-like elements in the DNA and with DivIVA, a membrane protein which localizes to the cell poles. RacA has a secondary function in which it assists in nucleoid condensation. Here we demonstrate that, in addition to positioning and condensing the chromosome, RacA contributes to efficient transport of DNA by the chromosome segregation motor SpoIIIE. When RacA is deleted, one-quarter of cells fail to capture DNA in the nascent spore, yet 70% of cells fail to form viable spores without RacA. This discrepancy indicates that RacA possesses a role in sporulation beyond DNA capture and condensation. We observed that the mutant cells had reduced chromosome translocation into the forespore across the entire length of the chromosome, requiring nearly twice as much time to move a given DNA locus. Additionally, functional abolition of the RacA-DivIVA interaction reduced translocation to a similar degree as in aracAdeletion strain, demonstrating the importance of the RacA-mediated tether in translocation and chromosome packaging during sporulation. We propose that the DNA-membrane anchor facilitates efficient translocation by SpoIIIE, not through direct protein-protein contacts but by virtue of physical effects on the chromosome that arise from anchoring DNA at a distance.IMPORTANCETo properly segregate their chromosomes, organisms tightly regulate the organization and dynamics of their DNA. Aspects of the process by which DNA is translocated during sporulation are not yet fully understood, such as what factors indirectly influence the activity of the motor protein SpoIIIE. In this work, we have shown that a DNA-membrane tether mediated by RacA contributes to the activity of SpoIIIE. Loss of RacA nearly doubles the time of translocation, despite the physically distinct locations these proteins and their activities occupy within the cell. This is a rare example of an explicit effect that DNA-membrane connections can have on cell physiology and demonstrates that distant changes to the state of the chromosome can influence motor proteins which act upon it.

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Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽

10.1128/mbio.00684-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 61

Author(s):

Matthew J. Bush ◽

Maureen J. Bibb ◽

Govind Chandra ◽

Kim C. Findlay ◽

Mark J. Buttner

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Regulatory Networks ◽

Liquid Culture ◽

Biological Processes ◽

Streptomyces Venezuelae ◽

Content Type ◽

Master Regulators ◽

Aerial Growth ◽

Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

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Young adults’ subjective and objective risk attitude in financial decision making

Review of Behavioral Finance ◽

10.1108/rbf-07-2017-0069 ◽

2018 ◽

Vol 10 (3) ◽

pp. 274-294 ◽

Cited By ~ 5

Author(s):

Andreas Oehler ◽

Matthias Horn ◽

Florian Wedlich

Keyword(s):

Older Adults ◽

Young Adults ◽

Risk Aversion ◽

Real World ◽

Risk Attitude ◽

Similar Degree ◽

Content Type ◽

Financial Decisions ◽

Objective Risk ◽

Subjective Risk

Purpose The purpose of this paper is to derive the determinants of young adults’ subjective and objective risk attitude in theoretical and real-world financial decisions. Furthermore, a comparison of the factors that influence young adults’ and older adults’ risk attitude is provided. Design/methodology/approach The paper relies on an experimental setting and a cross-sectional field study using data of the German central bank’s (Deutsche Bundesbank) PHF-Survey. Findings Young adults’ objective risk aversion is not constant but increases with stake sizes. Furthermore, young adults’ subjective risk attitude is a better predictor for their objective risk attitude than a set of commonly employed socio-demographics and economics like age or income. Moreover, young adults’ subjective risk attitude works as a mediator for the influence of their investable financial wealth on their objective risk attitude. Although young adults’ subjective risk attitude shows a gender effect, the influence of young adults’ gender on their objective risk attitude decreases with higher stake sizes. Compared to older adults, young adults generally show a similar degree of subjective risk aversion. However, due to stronger financial restrictions, young adults show a higher degree of objective risk aversion. Originality/value Although individuals’ financial outcomes depend on the financial behavior established in young adulthood, there is no study that simultaneously analyzes the determinants of young adults’ subjective and objective risk attitude in real-world financial decisions with a focus on young adults as a separate age group. The paper closes this gap in literature and additionally provides a comparison of the subsamples of young adults and older adults. The analysis in this paper reveals that young adults’ lower engagement in financial markets is primarily driven by their tight budget and not by a fundamental different subjective risk attitude.

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Characterization of RelA in Acinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00045-20 ◽

2020 ◽

Vol 202 (12) ◽

Cited By ~ 3

Author(s):

María Pérez-Varela ◽

Aimee R. P. Tierney ◽

Ju-Sim Kim ◽

Andrés Vázquez-Torres ◽

Philip Rather

Keyword(s):

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Multidrug Resistant ◽

Cell Physiology ◽

Content Type ◽

Content Model ◽

Transposon Insertion ◽

Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

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An Archaeal Fluoride-Responsive Riboswitch Provides an Inducible Expression System for Hyperthermophiles

Applied and Environmental Microbiology ◽

10.1128/aem.02306-17 ◽

2018 ◽

Vol 84 (7) ◽

Cited By ~ 15

Author(s):

Michael Clayton Speed ◽

Brett W. Burkhart ◽

Jonathan W. Picking ◽

Thomas J. Santangelo

Keyword(s):

Gene Expression ◽

Transcription Initiation ◽

Expression System ◽

Regulatory Control ◽

Cell Physiology ◽

Content Type ◽

Thermococcus Kodakarensis ◽

Export Pathway ◽

Regulated Expression ◽

Regulated Gene Expression

ABSTRACT Robust genetic systems for the hyperthermophilic Thermococcales have facilitated the overexpression of native genes, enabled the addition of sequences encoding secretion signals, epitope, and affinity tags to coding regions, and aided the introduction of sequences encoding new proteins in these fast-growing fermentative heterotrophs. However, tightly controlled and easily manipulated systems facilitating regulated gene expression are limited for these hosts. Here, we describe an alternative method for regulatory control reliant on a cis -encoded functional riboswitch in the model archaeon Thermococcus kodakarensis . Despite the hyperthermophilic growth temperatures, the proposed structure of the riboswitch conforms to a fluoride-responsive riboswitch encoded in many bacteria and similarly functions to regulate a component-conserved fluoride export pathway. Deleting components of the fluoride export pathway generates T. kodakarensis strains with increased fluoride sensitivity. The mechanism underlying regulated expression suggested that the riboswitch-encoding sequences could be utilized as a tunable expression cassette. When appended to a reporter gene, the riboswitch-mediated control system provides fluoride-dependent tunable regulatory potential, offering an alternative system for regulating gene expression. Riboswitch-regulated expression is thus ubiquitous in extant life and can be exploited to generate regulated expression systems for hyperthermophiles. IMPORTANCE Gene expression is controlled by a myriad of interconnected mechanisms that interpret metabolic states and environmental cues to balance cell physiology. Transcription regulation in Archaea is known to employ both typical repressors-operators and transcription activators to regulate transcription initiation in addition to the regulation afforded by chromatin structure. It was perhaps surprising that the presumed ancient mechanism of riboswitch-mediated regulation is found in Bacteria and Eukarya , but seemingly absent in Archaea . We demonstrate here that a fluoride-responsive riboswitch functions to regulate a detoxification pathway in the hyperthermophilic archaeon Thermococcus kodakarensis . The results obtained define a universal role for riboswitch-mediated regulation, adumbrate the presence of several riboswitch-regulated genes in Thermococcus kodakarensis , demonstrate the utility of RNA-based regulation at high temperatures, and provide a novel riboswitch-regulated expression system to employ in hyperthermophiles.

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Chromosome Dynamics in Bacteria: Triggering Replication at the Opposite Location and Segregation in the Opposite Direction

mBio ◽

10.1128/mbio.01002-19 ◽

2019 ◽

Vol 10 (4) ◽

Author(s):

Ady B. Meléndez ◽

Inoka P. Menikpurage ◽

Paola E. Mera

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Opposite Direction ◽

Caulobacter Crescentus ◽

Chromosome Replication ◽

Opposite Orientation ◽

Circular Chromosome ◽

Content Type ◽

Cell Pole ◽

Protein Gradients

ABSTRACT Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell. IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

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Mentors' approach to practicum mentoring in the Spanish and Thai contexts: a two-cohort comparison using the Mentoring Profile Inventory

International Journal of Mentoring and Coaching in Education ◽

10.1108/ijmce-08-2019-0079 ◽

2020 ◽

Vol 9 (2) ◽

pp. 169-185

Author(s):

Juanjo Mena ◽

Chatree Faikhamta ◽

Anthony Clarke

Keyword(s):

Preservice Teachers ◽

Interpersonal Relationships ◽

Role Model ◽

Cooperating Teachers ◽

Asian Country ◽

Similar Degree ◽

Content Type ◽

Essential Components ◽

And Performance ◽

Validated Instrument

PurposeThe purpose of this study was to better understand the work of mentors or cooperating teachers (CTs) in Spain and Thailand, by comparing how mentors in both contexts conceive of their work in teacher education.Design/methodology/approachA comparative study based on a crossnational research (CNR) approach was used. An internationally validated instrument, the Mentoring Profile Inventory (MPI), was given to 171 Spanish and 170 Thai CTs, and an analysis using t tests and constant comparative methods was conducted.FindingsThe main results indicate that the Spanish cohort reported being significantly more challenged than their Thai counterparts in working with preservice teachers (PSTs). Both groups indicated a similar degree of motivation. The comparative analysis revealed that the Thai CTs emphasized the importance of ethics in teaching and being a role model for PSTs, whereas the Spanish CTs emphasized the need for strong interpersonal relationships as being central to successful mentoring.Research limitations/implicationsUnderstanding mentors' challenges and motivations can form the basis for reflecting upon the essential components of teacher preparation in both contexts. Furthermore, comparative cross-context analyses, as demonstrated in this paper, are vital for the identification of important differences that might otherwise remain invisible or unrecognized from a single-context perspective.Originality/valueThe study is original in its focus in that it offers insights that help better understand attitudes and performance within and across contexts – in this case, a European and an Asian country – using a common metric, the MPI. This study serves as an exemplar for other comparison studies for countries of mentors.

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Micropatterned Macrophage Analysis Reveals Global Cytoskeleton Constraints Induced by Bacillus anthracis Edema Toxin

Infection and Immunity ◽

10.1128/iai.00479-15 ◽

2015 ◽

Vol 83 (8) ◽

pp. 3114-3125 ◽

Cited By ~ 10

Author(s):

Yannick Trescos ◽

Emilie Tessier ◽

Clémence Rougeaux ◽

Pierre L. Goossens ◽

Jean-Nicolas Tournier

Keyword(s):

Bacillus Anthracis ◽

Immune Cells ◽

Adherens Junction ◽

Peritoneal Macrophages ◽

Cell Physiology ◽

Actin Reorganization ◽

Content Type ◽

Cellular Physiology ◽

Edema Toxin ◽

Polar Organization

Bacillus anthracissecretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.

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Pathological effects of angioplasty on vasospastic carotid arteries in a rabbit model

Journal of Neurosurgery ◽

10.3171/jns.1995.83.1.0111 ◽

1995 ◽

Vol 83 (1) ◽

pp. 111-117 ◽

Cited By ~ 22

Author(s):

R. Loch Macdonald ◽

M. Christopher Wallace ◽

Walter J. Montanera ◽

Jennifer A. Glen

Keyword(s):

Carotid Arteries ◽

Rabbit Model ◽

Control Group ◽

Similar Degree ◽

Histopathological Study ◽

Content Type ◽

Tunica Media ◽

Endothelial Proliferation ◽

Empty Control ◽

Arterial Diameters

✓ To define the pathological effects of angioplasty on vasospastic arteries, 36 rabbits underwent angiography and induction of vasospasm by placement of blood-filled (vasospasm groups) or empty (control group) silastic sheaths around the cervical carotid arteries. Two (Day 2) or 7 days (Day 7) later, angiography was repeated and one carotid artery in each animal was dilated by balloon angioplasty. The rabbits were sacrificed 1 day, 7 days, or 3 to 4 weeks after angioplasty. Significant vasospasm developed after placement of silastic sheaths with blood (mean reductions in diameter 39% ± 6% at Day 2 and 48% ± 5% at Day 7). Arterial narrowing was less apparent in the control groups at Day 2 (24% ± 7%). Angioplasty performed on Day 2 significantly increased arterial diameters of vasospastic arteries (50% ± 7%; p < 0.05) but not those of control arteries (10% ± 6%, p > 0.05). Angioplasty performed on Day 7 increased the arterial diameters by a similar degree (47% ± 13%, not significant). Arteries remained dilated after angioplasty, although there was significant vasospasm 7 days after angioplasty when angioplasty was performed on Day 2. Blinded, semiquantitative histopathological study of the arteries showed that 3 to 4 weeks after angioplasty, there was significant endothelial proliferation and a trend for thinning of the tunica media. There were no significant changes in control arteries subjected to angioplasty. Angioplasty was not associated with significant arterial fibrosis as measured by hydroxyproline content (analysis of variance). The increase in endothelial proliferation and decrease in the thickness of the tunica media suggest that, in the rabbit model, angioplasty damages endothelial and smooth-muscle cells. This may be the basis for the observation that vasospastic arteries do not reconstrict after angioplasty.

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Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.00607-16 ◽

2017 ◽

Vol 37 (10) ◽

Cited By ~ 3

Author(s):

Chong Wai Tio ◽

Gregory Omerza ◽

Timothy Phillips ◽

Hua Jane Lou ◽

Benjamin E. Turk ◽

...

Keyword(s):

Protein Kinase ◽

Chromosome Segregation ◽

Kinase Activity ◽

Specific Protein ◽

Mitogen Activated Protein Kinase ◽

Activation Loop ◽

Cyclin Dependent Kinase ◽

Content Type ◽

Dual Specificity ◽

Mitogen Activated Protein

ABSTRACT Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in Saccharomyces cerevisiae that couples spore morphogenesis to the completion of chromosome segregation. Similar to other MAPKs, Smk1 is controlled by phosphorylation of a threonine (T) and a tyrosine (Y) in its activation loop. However, it is not activated by a dual-specificity MAPK kinase. Instead, T207 in Smk1's activation loop is phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (Cak1), and Y209 is autophosphorylated in an intramolecular reaction that requires the meiosis-specific protein Ssp2. In this study, we show that Smk1 is catalytically inert unless it is bound by Ssp2. While Ssp2 binding activates Smk1 by a mechanism that is independent of activation loop phosphorylation, binding also triggers autophosphorylation of Y209 in Smk1, which, along with Cak1-mediated phosphorylation of T207, further activates the kinase. Autophosphorylation of Smk1 on Y209 also appears to modify the specificity of the MAPK by suppressing Y kinase and enhancing S/T kinase activity. We also found that the phosphoconsensus motif preference of Ssp2/Smk1 is more extensive than that of other characterized MAPKs. This study therefore defines a novel mechanism of MAPK activation requiring binding of an activator and also shows that MAPKs can be diversified to recognize unique phosphorylation motifs.

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The Stringent Response Inhibits DNA Replication Initiation inE. coliby Modulating Supercoiling oforiC

mBio ◽

10.1128/mbio.01330-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 16

Author(s):

James A. Kraemer ◽

Allen G. Sanderlin ◽

Michael T. Laub

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Stringent Response ◽

Replication Initiation ◽

Initiation Factor ◽

Cell Physiology ◽

Origin Of Replication ◽

Content Type ◽

Dna Replication Initiation ◽

Negative Supercoils

ABSTRACTThe 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 inEscherichia coliremains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factordnaA, 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 intooriCto help promote replication initiation, so the accumulation of ppGpp reduces initiation potential atoriCby reducing transcription. We find that maintaining transcription nearoriC, 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 geneseqAalso 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 intooriC.IMPORTANCETo 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 inEscherichia colifor 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.

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