The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

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Determination of Antibiotic Hypersensitivity among 4,000 Single-Gene-Knockout Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01982-07 ◽

2008 ◽

Vol 190 (17) ◽

pp. 5981-5988 ◽

Cited By ~ 143

Author(s):

Cindy Tamae ◽

Anne Liu ◽

Katherine Kim ◽

Daniel Sitz ◽

Jeeyoon Hong ◽

...

Keyword(s):

Escherichia Coli ◽

High Throughput Screening ◽

Gene Knockout ◽

Single Gene ◽

E Coli ◽

Knockout Mutants ◽

Data Points ◽

Increased Sensitivity ◽

Gene Knockouts

ABSTRACT We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of “codrugs” that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's “intrinsic resistome,” which provides innate resistance to antibiotics.

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Exploring Synergy between Classic Mutagens and Antibiotics To Examine Mechanisms of Synergy and Antibiotic Action

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02485-15 ◽

2015 ◽

Vol 60 (3) ◽

pp. 1515-1520 ◽

Cited By ~ 3

Author(s):

Lisa Yun Song ◽

Sara D'Souza ◽

Karen Lam ◽

Tina Manzhu Kang ◽

Pamela Yeh ◽

...

Keyword(s):

Escherichia Coli ◽

Interaction Network ◽

Mechanisms Of Action ◽

Double Strand Breaks ◽

The Other ◽

Gram Negative ◽

Content Type ◽

Strand Breaks ◽

Test Models ◽

Dose Effects

We used classical mutagens in Gram-negativeEscherichia colito study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. We used 4-nitroquinoline 1-oxide (4NQO), zebularine (ZEB), 5-azacytidine (5AZ), 2-aminopurine (2AP), and 5-bromodeoxyuridine (5BrdU) as mutagens (with bactericidal potency of 4NQO > ZEB > 5AZ > 2AP > 5BrdU) and vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL) as antibiotics. We detected the strongest synergies with 4NQO, an agent that oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics VAN, TMP, CPR, and GEN, but no synergies with the bacteriostatic antibiotics TET, ERY, and CHL. Each of the other mutagens displays synergies with the bactericidal antibiotics to various degrees that reflect their potencies, as well as with some of the other mutagens. The results support recent models showing that bactericidal antibiotics kill bacteria principally by ultimately generating more double-strand breaks than can be repaired. We discuss the synergies seen here and elsewhere as representing dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. We also used the results of pairwise tests to place the classic mutagens into functional antibacterial categories within a previously defined drug interaction network.

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An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Applied and Environmental Microbiology ◽

10.1128/aem.00371-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3481-3492 ◽

Cited By ~ 3

Author(s):

Tina Netzker ◽

Volker Schroeckh ◽

Matthew A. Gregory ◽

Michal Flak ◽

Mario K. C. Krespach ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Deletion ◽

Gene Knockout ◽

Plasmid Transfer ◽

Gene Deletions ◽

Content Type ◽

Efficient Generation ◽

Knockout Mutants ◽

Starter Unit ◽

Targeted Gene Knockout

ABSTRACTStreptomyces iranensisHM 35 is an alternative rapamycin producer toStreptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation inS. rapamycinicus.The approach depends upon efficient conjugational plasmid transfer fromEscherichia colitoStreptomyces, and the failure of this step has frustrated its application toStreptomyces iranensisHM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions inS. iranensisHM 35. Among them wererapK, which generates the starter unit for rapamycin biosynthesis, andhutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants ofStreptomycesspecies that are genetically difficult to manipulate.IMPORTANCEThe developed protocol of conjugational plasmid transfer fromEscherichia colitoStreptomyces iranensismay allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable,Streptomycesspecies.

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

Infection and Immunity ◽

10.1128/iai.00716-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3704-3711 ◽

Cited By ~ 31

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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Escherichia coli Induces DNA Double-Strand Breaks in Eukaryotic Cells

Science ◽

10.1126/science.1127059 ◽

2006 ◽

Vol 313 (5788) ◽

pp. 848-851 ◽

Cited By ~ 487

Author(s):

J.-P. Nougayrede

Keyword(s):

Escherichia Coli ◽

Double Strand Breaks ◽

Eukaryotic Cells ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽

10.1093/genetics/151.2.439 ◽

1999 ◽

Vol 151 (2) ◽

pp. 439-446 ◽

Cited By ~ 2

Author(s):

Masaaki Onda ◽

Katsuhiro Hanada ◽

Hirokazu Kawachi ◽

Hideo Ikeda

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Hydrogen Peroxide ◽

Dna Damage ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Recombination Analysis ◽

Wild Type ◽

Strand Breaks ◽

In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

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Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

Applied and Environmental Microbiology ◽

10.1128/aem.01704-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 6953-6963 ◽

Cited By ~ 5

Author(s):

Zhe Zhao ◽

Lauren J. Eberhart ◽

Lisa H. Orfe ◽

Shao-Yeh Lu ◽

Thomas E. Besser ◽

...

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Disulfide Bonds ◽

Gene Knockout ◽

Single Gene ◽

Site Directed Mutagenesis ◽

Content Type ◽

E Coli ◽

Synthase Inhibitor ◽

Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Content Type ◽

Strand Breaks ◽

Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

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