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 Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous 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.

RecF, UvrD, RecX and RecN proteins suppress DNA degradation at DNA double-strand breaks in Escherichia coli

Biochimie ◽  
2018 ◽  
Vol 148 ◽  
pp. 116-126 ◽  
Author(s):  
Isidoro Feliciello ◽  
Davor Zahradka ◽  
Ksenija Zahradka ◽  
Siniša Ivanković ◽  
Nikolina Puc ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Degradation ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recn Proteins

scholarly journals Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

PLoS Genetics ◽  
2017 ◽  
Vol 13 (10) ◽  
pp. e1006895 ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Adeline Durand ◽  
Jean-Michel Desfontaines ◽  
Ielyzaveta Iurchenko ◽  
Hélène Auger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Repair Enzyme ◽  
Strand Breaks

scholarly journals An end-joining repair mechanism in Escherichia coli

2010 ◽  
Vol 107 (5) ◽  
pp. 2141-2146 ◽  
Author(s):  
Romain Chayot ◽  
Benjamin Montagne ◽  
Didier Mazel ◽  
Miria Ricchetti
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Antibiotic Resistance Gene ◽  
Bacterial Genome ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
E Coli ◽  
Alternative End Joining

Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.

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.

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