scholarly journals Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-Deficient Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 7027-7037 ◽  
Author(s):  
Jennifer L. Robbins-Manke ◽  
Zoran Z. Zdraveski ◽  
Martin Marinus ◽  
John M. Essigmann
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Mismatch Repair ◽  
Gene Expression Regulation ◽  
Replication Initiation ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Adenine Methyltransferase

ABSTRACT DNA adenine methylation by DNA adenine methyltransferase (Dam) in Escherichia coli plays an important role in processes such as DNA replication initiation, gene expression regulation, and mismatch repair. In addition, E. coli strains deficient in Dam are hypersensitive to DNA-damaging agents. We used genome microarrays to compare the transcriptional profiles of E. coli strains deficient in Dam and mismatch repair (dam, dam mutS, and mutS mutants). Our results show that >200 genes are expressed at a higher level in the dam strain, while an additional mutation in mutS suppresses the induction of many of the same genes. We also show by microarray and semiquantitative real-time reverse transcription-PCR that both dam and dam mutS strains show derepression of LexA-regulated SOS genes as well as the up-regulation of other non-SOS genes involved in DNA repair. To correlate the level of SOS induction and the up-regulation of genes involved in recombinational repair with the level of DNA damage, we used neutral single-cell electrophoresis to determine the number of double-strand breaks per cell in each of the strains. We find that dam mutant E. coli strains have a significantly higher level of double-strand breaks than the other strains. We also observe a broad range in the number of double-strand breaks in dam mutant cells, with a minority of cells showing as many as 10 or more double-strand breaks. We propose that the up-regulation of recombinational repair in dam mutants allows for the efficient repair of double-strand breaks whose formation is dependent on functional mismatch repair.

scholarly journals ATP-Dependent Persister Formation in Escherichia coli

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Yue Shan ◽  
Autumn Brown Gandt ◽  
Sarah E. Rowe ◽  
Julia P. Deisinger ◽  
Brian P. Conlon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Tolerance ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Chronic Infections ◽  
Stochastic Variation ◽  
Strand Breaks ◽  
E Coli ◽  
Atp Levels ◽  
Energy Dependent

ABSTRACT Persisters are dormant variants that form a subpopulation of cells tolerant to antibiotics. Persisters are largely responsible for the recalcitrance of chronic infections to therapy. In Escherichia coli , one widely accepted model of persister formation holds that stochastic accumulation of ppGpp causes activation of the Lon protease that degrades antitoxins; active toxins then inhibit translation, resulting in dormant, drug-tolerant persisters. We found that various stresses induce toxin-antitoxin (TA) expression but that induction of TAs does not necessarily increase persisters. The 16S rRNA promoter rrnB P1 was proposed to be a persister reporter and an indicator of toxin activation regulated by ppGpp. Using fluorescence-activated cell sorting (FACS), we confirmed the enrichment for persisters in the fraction of rrnB P1 -gfp dim cells; however, this is independent of toxin-antitoxins. rrnB P1 is coregulated by ppGpp and ATP. We show that rrnB P1 can report persisters in a relA / spoT deletion background, suggesting that rrnB P1 is a persister marker responding to ATP. Consistent with this finding, decreasing the level of ATP by arsenate treatment causes drug tolerance. Lowering ATP slows translation and prevents the formation of DNA double-strand breaks upon fluoroquinolone treatment. We conclude that variation in ATP levels leads to persister formation by decreasing the activity of antibiotic targets. IMPORTANCE Persisters are a subpopulation of antibiotic-tolerant cells responsible for the recalcitrance of chronic infections. Our current understanding of persister formation is primarily based on studies of E. coli . The activation of toxin-antitoxin systems by ppGpp has become a widely accepted model for persister formation. In this study, we found that stress-induced activation of mRNA interferase-type toxins does not necessarily cause persister formation. We also found that the persister marker rrnB P1 reports persister cells because it detects a drop in cellular ATP levels. Consistent with this, lowering the ATP level decreases antibiotic target activity and, thus, leads to persister formation. We conclude that stochastic variation in ATP is the main mechanism of persister formation. A decrease in ATP provides a satisfactory explanation for the drug tolerance of persisters, since bactericidal antibiotics act by corrupting energy-dependent targets.

scholarly journals Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Manon Oliero ◽  
Annie Calvé ◽  
Gabriela Fragoso ◽  
Thibault Cuisiniere ◽  
Roy Hajjar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ferrous Sulfate ◽  
Chromosomal Abnormalities ◽  
Double Strand Breaks ◽  
Luciferase Reporter ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
The Impact

Abstract Background Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. Methods Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 μM, 25 μM and 125 μM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. Results Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 μM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. Conclusion Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.

scholarly journals Evolution of Chi motifs in Proteobacteria

2020 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif

AbstractHomologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway in E.coli

2019 ◽  
Author(s):  
Xuefeng Pan ◽  
Li Yang ◽  
Nan Jiang ◽  
Xifang Chen ◽  
Bo Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Dna Replication ◽  
Replication Fork ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Differential Binding

AbstractFaithful duplication of genomic DNA relies not only on the fidelity of DNA replication itself, but also on fully functional DNA repair and homologous recombination machinery. We report a molecular mechanism responsible for deciding homologous recombinational repair pathways during replication dictated by binding of RecO and RecG to SSB in E.coli. Using a RecG-yfp fusion protein, we found that RecG-yfp foci appeared only in the ΔrecG, ΔrecO and ΔrecA, ΔrecO double mutants. Surprisingly, foci were not observed in wild-type ΔrecG, or double mutants where recG and either recF or, separately recR were deleted. In addition, formation of RecG-yfp foci in the ΔrecO::kanR required wildtype ssb, as ssb-113 could not substitute. This suggests that RecG and RecO binding to SSB is competitive. We also found that the UV resistance of recO alone mutant increased to certain extent by supplementing RecG. In an ssb-113 mutant, RecO and RecG worked following a different pattern. Both RecO and RecG were able to participate in repairing UV damages when grown at permissive temperature, while they could also be involved in making DNA double strand breaks when grown at nonpermissive temperature. So, our results suggested that differential binding of RecG and RecO to SSB in a DNA replication fork in Escherichia coli.may be involved in determining whether the SDSA or DSBR pathway of homologous recombinational repair is used.Author summarySingle strand DNA binding proteins (SSB) stabilize DNA holoenzyme and prevent single strand DNA from folding into non-B DNA structures in a DNA replication fork. It has also been revealed that SSB can also act as a platform for some proteins working in DNA repair and recombination to access DNA molecules when DNA replication fork needs to be reestablished. In Escherichia coli, several proteins working primarily in DNA repair and recombination were found to participate in DNA replication fork resumption by physically interacting with SSB, including RecO and RecG etc. However the hierarchy of these proteins interacting with SSB in Escherichia coli has not been well defined. In this study, we demonstrated a differential binding of RecO and RecG to SSB in DNA replication was used to establish a RecO-dependent pathway of replication fork repair by abolishing a RecG-dependent replication fork repair. We also show that, RecG and RecO could randomly participate in DNA replication repair in the absence of a functional SSB, which may be responsible for the generation of DNA double strand breaks in an ssb-113 mutant in Escherichia coli.

scholarly journals CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli

2019 ◽  
Author(s):  
Chaoyong Huang ◽  
Tingting Ding ◽  
Jingge Wang ◽  
Xueqin Wang ◽  
Jialei Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Genetic Engineering ◽  
Double Strand Breaks ◽  
Effective Strategy ◽  
End Joining ◽  
Strand Breaks ◽  
E Coli ◽  
Non Homologous End Joining ◽  
Genomic Editing

AbstractUnlike eukaryotes, bacteria are less proficient in homologous recombination (HR) and non-homologous end joining (NHEJ). All existing genomic editing methods for Escherichia coli rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in chromosomal engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient knocking-out of E. coli genomic sequence of up to 83 kb. Moreover, the positive rate and editing efficiency is independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9 complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.SignificanceThe application in prokaryotes is difficult because of the weak homologous recombination and non-homologous end joining systems. E. coli, as the most-used prokaryote in metabolic engineering, has no NHEJ system. All existing genomic editing methods for E. coli rely on exogenous HR or NHEJ systems to repair double-strand breaks introduced by CRISPR/Cas9. In this report, we firstly demonstrate that the weak and previously ignored end-joining mechanism in E. coli can be used for efficient large-scale genetic engineering assisted by CRISPR/Cas9. Our efforts greatly simplify the genomic editing procedure of E. coli and provide an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.

scholarly journals Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

2008 ◽  
Vol 191 (4) ◽  
pp. 1152-1161 ◽  
Author(s):  
Lyle A. Simmons ◽  
Alexi I. Goranov ◽  
Hajime Kobayashi ◽  
Bryan W. Davies ◽  
Daniel S. Yuan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Ionizing Radiation ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Strand Breaks ◽  
E Coli ◽  
Sos Induction

ABSTRACT DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

scholarly journals Repair System for Noncanonical Purines in Escherichia coli

2003 ◽  
Vol 185 (10) ◽  
pp. 3101-3110 ◽  
Author(s):  
Nicholas E. Burgis ◽  
Jason J. Brucker ◽  
Richard P. Cunningham
Keyword(s):  
Escherichia Coli ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Repair System ◽  
Purine Base ◽  
Strand Breaks ◽  
E Coli ◽  
Endonuclease V ◽  
Sos Induction ◽  
Deoxynucleotide Triphosphate

ABSTRACT Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.

scholarly journals The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection

2015 ◽  
Vol 83 (9) ◽  
pp. 3704-3711 ◽  
Author(s):  
Alex J. McCarthy ◽  
Patricia Martin ◽  
Emilie Cloup ◽  
Richard A. Stabler ◽  
Eric Oswald ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Virulent Strain ◽  
Systemic Infection ◽  
Double Strand Breaks ◽  
Neonatal Rat ◽  
Very High Frequency ◽  
Content Type ◽  
Strand Breaks ◽  
E Coli

Escherichia colistrains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34E. coliK1 neonatal isolates carriedclbAandclbP, genes contained within thepkspathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation ofclbAandclbPinE. coliA192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation ofclbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation ofclbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity ofE. coliK1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate.

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