scholarly journals Direct visualization of four diffusive LexA states controlling SOS response strength during antibiotic treatment

2020 ◽  
Author(s):  
Leonard Schärfen ◽  
Miloš Tišma ◽  
Andreas Hartmann ◽  
Michael Schlierf
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Sos Response ◽  
Feedback System ◽  
Response Strength ◽  
E Coli ◽  
Detailed Kinetic Model ◽  
Broad Understanding ◽  
Single Molecule Localization Microscopy

AbstractIn bacteria, the key mechanism governing mutation, adaptation and survival upon DNA damage is the SOS response. Through autoproteolytic digestion triggered by single-stranded DNA caused by most antibiotics, the transcriptional repressor LexA controls over 50 SOS genes including DNA repair pathways and drivers of mutagenesis. Efforts to inhibit this response and thereby combat antibiotic resistance rely on a broad understanding of its behavior in vivo, which is still limited. Here, we develop a single-molecule localization microscopy assay to directly visualize LexA mobility in Escherichia coli and monitor the SOS response on the level of transcription factor activity. We identify four diffusive populations and monitor their temporal evolution upon ciprofloxacin-induced continuous DNA damage. With LexA mutants, we assign target bound, non-specifically DNA bound, freely diffusing and cleaved repressors. We develop a strategy to count LexA in fixed cells at different time points after antibiotic stress and combine the time-evolution of LexA sub-populations and the repressor’s overall abundance. Through fitting a detailed kinetic model we obtain in vivo synthesis, cleavage and binding rates and determined that the regulatory feedback system reaches a new equilibrium in ∼100 min. LexA concentrations showed non-constant heterogeneity during SOS response and designate LexA expression, and thereby regulation of downstream SOS proteins, as drivers of evolutionary adaptation. Even under low antibiotic stress, we observed a strong SOS response on the LexA level, suggestion that small amounts of antibiotics can trigger adaptation in E. coli.

Mutagenesis and More: umuDC and the Escherichia coli SOS Response

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1599-1610 ◽  
Author(s):  
Bradley T Smith ◽  
Graham C Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Cellular Response ◽  
Sos Response ◽  
Posttranslational Regulation ◽  
E Coli ◽  
Sos Mutagenesis ◽  
Induced Mutagenesis ◽  
Mechanistic Basis ◽  
Increase Cell Survival

Abstract The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals 702. Zinc Blockade of SOS Response Inhibits Horizontal Transfer of Antibiotic Resistance Genes in Enteric Bacteria

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S253-S253
Author(s):  
John Crane ◽  
Mark Sutton ◽  
Muhammad Cheema ◽  
Michael Olyer
Keyword(s):  
Dna Damage ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Horizontal Transfer ◽  
Antibiotic Resistance Genes ◽  
Sos Response ◽  
Enteric Bacteria ◽  
Extracellular Dna ◽  
In Vitro Assays ◽  
E Coli

Abstract Background The SOS response is a conserved response to DNA damage that is found in Gram negative and Gram-positive bacteria. When DNA damage is sustained and severe, activation of error-prone DNA polymerases can induce a higher mutation rate then normally observed, which is called the mutator phenotype or hypermutation. We previously showed that zinc blocked the hypermutation response induced by quinolone antibiotics and mitomycin C in E. coli and Klebsiella pneumoniae (Bunnell BE, Escobar JF, Bair KL, Sutton MD, Crane JK (2017). Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli. PLoS ONE 12(5): e0178303. https://doi.org/10.1371/journal.pone.0178303.) In addition to causing copying errors in DNA replication, Beaber et al. showed that induction of the SOS response increased the frequency of horizontal gene transfer into Vibrio cholerae, an organism naturally competent at uptake of extracellular DNA. (Beaber JW, Hochhut B, Waldor MK. 2003. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72–74.) Methods. In this study, we tested whether induction of the SOS response could induce transfer of antibiotic resistance from Enterobacter cloacae into E. coli, and whether zinc could inhibit that inter-species transfer of antibiotic resistance. Results. Ciprofloxacin, an inducer of the SOS response, increased the rate of transfer of an extended spectrum β-lactamase (ESBL) gene from Enterobacter into a susceptible E. coli strain. Zinc blocked SOS-induced horizontal transfer of §-lactamase into E. coli. Other divalent metals, such as iron and manganese, failed to inhibit these responses. Conclusion. In vitro assays showed that zinc blocked the ability of RecA to bind to ssDNA, an early step in the SOS response, suggesting the mechanism by which zinc blocks the SOS response. Disclosures All authors: No reported disclosures.

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

scholarly journals Deuterium oxide enhances the SOS response of Escherichia coli induced by bactericidal agents

2020 ◽  
pp. 79-80
Author(s):  
С.В. Смирнова ◽  
Т.Н. Шапиро ◽  
Е.В. Игонина ◽  
С.К. Абилев
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Nalidixic Acid ◽  
Deuterium Oxide ◽  
Gene Promoter ◽  
Sos Response ◽  
Genotoxic Effect ◽  
E Coli ◽  
Lux Biosensor ◽  
First Time

Изучали генотоксическое действие бактерицидных средств диоксидина, фурацилина и налидиксовой кислоты на клетки дейтерированной культуры lux-биосенсора E.coli MG1655 (pColD::lux), люминесцирующего в результате активации промотора гена колицина colD в ответ на повреждение ДНК. Впервые показано, что оксид дейтерия (D2O) в концентрации 9% усиливает SOS-ответ, индуцированный исследуемыми лекарственными препаратами, в 1,6-2,8 раза в клетках E. coli. We studied the genotoxic effect of bactericidal agents: dioxine, furaciline and nalidixic acid on cells of the deuterated culture lux-biosensor E. coli MG1655 (pColD::lux), which luminesces as a result of activation of the colicin gene promoter colD in response to DNA damage. For the first time, it was shown that deuterium oxide (D2O) at a concentration of 9% increases the SOS response by 1.6-2.8 times in E. coli cells induced by the studied drugs.

scholarly journals Genetic analysis of DinG-family helicase YoaA and its interaction with replication clamp-loader protein HolC in E. coli

2021 ◽  
Author(s):  
Vincent A. Sutera ◽  
Thalia H. Sass ◽  
Scott E. Leonard ◽  
Lingling Wu ◽  
David J. Glass ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Tandem Repeats ◽  
Genomic Stability ◽  
Sos Response ◽  
Template Switching ◽  
Clamp Loader ◽  
E Coli ◽  
C Terminus ◽  
Domains Of Life

The XP-D/DinG family of DNA helicases contributes to genomic stability in all three domains of life. We investigate here the role of one of these proteins, YoaA, of Escherichia coli . In E. coli , YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolC’s interaction with HolD and the rest of the replisome; YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind - ), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3’ nascent strand during replication; consistent with this, YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. We provide evidence that YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA. IMPORTANCE Maintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level. The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

scholarly journals FtsW exhibits distinct processive motions driven by either septal cell wall synthesis or FtsZ treadmilling in E. coli

2019 ◽  
Author(s):  
Xinxing Yang ◽  
Ryan McQuillen ◽  
Zhixin Lyu ◽  
Polly Phillips-Mason ◽  
Ana De La Cruz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Single Molecule ◽  
Bacterial Cell ◽  
Spatial Information ◽  
Bacterial Species ◽  
E Coli ◽  
Slow Moving

AbstractDuring bacterial cell division, synthesis of new septal peptidoglycan (sPG) is crucial for successful cytokinesis and cell pole morphogenesis. FtsW, a SEDS (Shape, Elongation, Division and Sporulation) family protein and an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a new monofunctional peptidoglycan glycosyltransferase (PGTase). FtsW and its cognate monofunctional transpeptidase (TPase) class B penicillin binding protein (PBP3 or FtsI in E. coli) may constitute the essential, bifunctional sPG synthase specific for new sPG synthesis. Despite its importance, the septal PGTase activity of FtsW has not been documented in vivo. How its activity is spatiotemporally regulated in vivo has also remained unknown. Here we investigated the septal PGTase activity and dynamics of FtsW in E. coli cells using a combination of single-molecule imaging and genetic manipulations. We show that FtsW exhibits robust activity to incorporate an N-acetylmuramic acid analog at septa in the absence of other known PGTases, confirming FtsW as the essential septum-specific PGTase in vivo. Notably, we identified two populations of processive moving FtsW molecules at septa. A fast-moving population is driven by the treadmilling dynamics of FtsZ and independent of sPG synthesis. A slow-moving population is driven by active sPG synthesis and independent of FtsZ’s treadmilling dynamics. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving, sPG synthesis-dependent population. Our results support a two-track model, in which inactive sPG synthase molecules follow the fast treadmilling “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model explains how the spatial information is integrated into the regulation of sPG synthesis activity and suggests a new mechanistic framework for the spatiotemporal coordination of bacterial cell wall constriction.

scholarly journals SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions

1999 ◽  
Vol 181 (5) ◽  
pp. 1515-1523 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
M. Zafri Humayun
Keyword(s):  
Dna Damage ◽  
Transfection Efficiency ◽  
Sos Response ◽  
Genetic Network ◽  
Dna Lesions ◽  
Site Specific ◽  
E Coli ◽  
Mutational Specificity ◽  
Class 1 ◽  
Ap Sites

ABSTRACT Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N4-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. colistrains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

Ordered Intracellular Reca-Dna Assemblies

2000 ◽  
Vol 6 (S2) ◽  
pp. 860-861
Author(s):  
S. G. Wolf ◽  
S. Levin-Zaidman ◽  
D. Frenkiel-Krispin ◽  
E. Shimoni ◽  
I. Sabanay ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Electron Micrographs ◽  
Reca Protein ◽  
Sos Response ◽  
Structural Features ◽  
Wild Type ◽  
E Coli ◽  
Dna Damaging Agents

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. We find that induction of the SOS system in wild-type E. coli bacteria results in fast and massive intracellular coaggregation of RecA and DNA into lateral assemblies, which comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in-vitro RecA-DNA are consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.Bacterial chromatin is demarcated in electron micrographs of metabolically active cells as amorphous ribosome-free spaces that are irregularly spread over the cytoplasm (Fig. A). Wild-type E. coli cells exposed to DNA-damaging agents that induce the SOS response reveal a strikingly different morphology (Fig. B).

scholarly journals Quinolone Resistance Reversion by Targeting the SOS Response

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
E. Recacha ◽  
J. Machuca ◽  
P. Díaz de Alba ◽  
M. Ramos-Güelfo ◽  
F. Docobo-Pérez ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Agents ◽  
Sos Response ◽  
Resistance Mechanisms ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Health Crisis ◽  
E Coli ◽  
Isogenic Strains

ABSTRACT Suppression of the SOS response has been postulated as a therapeutic strategy for potentiating antimicrobial agents. We aimed to evaluate the impact of its suppression on reversing resistance using a model of isogenic strains of Escherichia coli representing multiple levels of quinolone resistance. E. coli mutants exhibiting a spectrum of SOS activity were constructed from isogenic strains carrying quinolone resistance mechanisms with susceptible and resistant phenotypes. Changes in susceptibility were evaluated by static (MICs) and dynamic (killing curves or flow cytometry) methodologies. A peritoneal sepsis murine model was used to evaluate in vivo impact. Suppression of the SOS response was capable of resensitizing mutant strains with genes encoding three or four different resistance mechanisms (up to 15-fold reductions in MICs). Killing curve assays showed a clear disadvantage for survival (Δlog10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Δlog10 CFU/g of 1.76 log units) in resistant strains with a suppressed SOS response. This effect was evident even after short periods (60 min) of exposure. Suppression of the SOS response reverses antimicrobial resistance across a range of E. coli phenotypes from reduced susceptibility to highly resistant, playing a significant role in increasing the in vivo efficacy. IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this question, we have generated E. coli mutants that exhibited a spectrum of SOS activity, ranging from a natural SOS response to a hypoinducible or constitutively suppressed response. We tested the effects of these mutations on quinolone resistance reversion under therapeutic concentrations in a set of isogenic strains carrying different combinations of chromosome- and plasmid-mediated quinolone resistance mechanisms with susceptible, low-level quinolone resistant, resistant, and highly resistant phenotypes. Our comprehensive analysis opens up a new strategy for reversing drug resistance by targeting the SOS response. IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this question, we have generated E. coli mutants that exhibited a spectrum of SOS activity, ranging from a natural SOS response to a hypoinducible or constitutively suppressed response. We tested the effects of these mutations on quinolone resistance reversion under therapeutic concentrations in a set of isogenic strains carrying different combinations of chromosome- and plasmid-mediated quinolone resistance mechanisms with susceptible, low-level quinolone resistant, resistant, and highly resistant phenotypes. Our comprehensive analysis opens up a new strategy for reversing drug resistance by targeting the SOS response.

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