Inhibitors of DNA synthesis cause excessive DNA synthesis in dnaA mutants of Escherichia coli K12

1978 ◽  
Vol 24 (10) ◽  
pp. 1250-1252
Author(s):  
Jukka Heinonen ◽  
Marja-Leena Pekola
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Relative Rate ◽  
Cell Mass ◽  
Chromosomal Dna ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Low Concentrations ◽  
Dna Synthesis Inhibitors

The relative rate of net DNA synthesis was stimulated when cells of dnaA mutants of Escherichia coli K12 were grown in the presence of low concentrations of DNA synthesis inhibitors. This led to a supernormal DNA/cell mass ratio. The excessive DNA was similar to the normal chromosomal DNA in size and stability in vivo. However, the cells did not divide but turned into long filaments. Excessive DNA synthesis in the presence of inhibitors of DNA synthesis was observed in the cultures of two independent dnaA mutants of E. coli, but dnaB and dnaC mutants behaved like the wild type in this respect.

scholarly journals Differential Effects of Myeloperoxidase-Derived Oxidants on Escherichia coli DNA Replication

1998 ◽  
Vol 66 (6) ◽  
pp. 2655-2659 ◽  
Author(s):  
Henry Rosen ◽  
Bryce R. Michel ◽  
Donald R. vanDevanter ◽  
James P. Hughes
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Sequences ◽  
Regression Line ◽  
Chromosomal Dna ◽  
Chloride System ◽  
E Coli ◽  
Total Dna ◽  
Cell Envelopes

ABSTRACT The microbicidal myeloperoxidase (MPO)-H2O2-chloride system strongly inhibitsEscherichia coli DNA synthesis. Also, cell envelopes from MPO-treated E. coli cells lose their ability to interact with hemimethylated DNA sequences of oriC, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of oriC-related functions (H. Rosen, et al. Proc. Natl. Acad. Sci. USA, 87:10048–10052, 1990). To evaluate whether origin-specific DNA sequences play a role in the MPO effect on E. coli DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication. Plasmid pCM700 replication, replicating from oriC, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication. A regression line describing this relationship had a slope of 0.90, and ther 2 was 0.89. In contrast, the replication activities of three of four non-oriC plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants. The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage. pSP102 replication declined similarly to that of total DNA synthesis. The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the r 2 was 0.92. The biochemical requirements for P1-mediated replication are strikingly similar to those for oriC-mediated replication. It is proposed that one of these requirements, common to oriC and the P1 origin but not critical to the replication of the other non-oriC plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in E. colichromosomal DNA synthesis.

scholarly journals Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

2005 ◽  
Vol 389 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Rajesh K. Soni ◽  
Parul Mehra ◽  
Gauranga Mukhopadhyay ◽  
Suman Kumar Dhar
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dnab Helicase ◽  
H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

Streptonigrin toxicity in Escherichia coli: oxygen dependence and the role of the intracellular oxidation–reduction state

1982 ◽  
Vol 28 (5) ◽  
pp. 545-552 ◽  
Author(s):  
John B. Harley ◽  
Caroline J. Fetterolf ◽  
Cesar A. Bello ◽  
Joel G. Flaks
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Superoxide Dismutase Activity ◽  
Lethal Concentration ◽  
Bacterial Physiology ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Oxidation Reduction

The bacterial physiology of streptonigrin toxicity was further investigated. An optimal oxygen concentration for toxicity was inferred from data showing that steptonigrin at 5 µg/mL was rapidly lethal to aerobic cultures of Escherichia coli K12 JF361, but was without effect on anaerobic cultures and was bacteriostatic to cultures incubated in 5 atm of oxygen plus 1 atm of air (5 atm O2 plus air) (1 atm = 101.325 kPa). Escherichia coli were protected from a potentially lethal concentration of streptonigrin during anaerobic incubation, whether previously grown anaerobically, aerobically, or in 5 atm O2 plus air. Superoxide dismutase activity increased with increasing oxygen tension in the medium, but was not significantly changed by a lethal concentration of streptonigrin. Although the superoxide dismutase activity was four times greater in E. coli grown in 5 atm O2 plus air than those grown in air alone, the aerobic survival in 5 µg/mL streptonigrin was identical, which suggested that superoxide dismutase was not rate limiting for toxicity. Escherichia coli K12 strains deficient in glutathione (KMBL54-129, AB1157-821, and AB1157-830) were protected from streptonigrin poisoning. Dithiothreotol (5.0 mM), diamide (1 mM), methyl viologen (1 mM), and cyanide (10 mM) protected aerobic E. coli from 5 µg/mL streptonigrin.These data are also consistent with a model of in vivo streptonigrin toxicity that requires a favorable intracellular oxidation–reduction state and an optimal concentration of molecular oxygen.

Deoxyribonucleotide Synthesis in an Escherichia coli Mutant (H 1491) which Lacks Ribonucleotide Reductase Subunit B2

1989 ◽  
Vol 44 (7-8) ◽  
pp. 715-718 ◽  
Author(s):  
Jens Harder ◽  
Hartmut Follmann ◽  
Klaus Hantke
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Sensitive Mutant ◽  
E Coli ◽  
Key Enzyme ◽  
Minimal Media

An iron-sensitive mutant of E. coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase. Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis. We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase- catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides. It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens.

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

scholarly journals Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit Gaurav ◽  
Varsha Gupta ◽  
Sandeep K. Shrivastava ◽  
Ranjana Pathania
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Nalidixic Acid ◽  
Bacterial Infections ◽  
Clinical Isolates ◽  
Hospital Environment ◽  
Infection Model ◽  
Drug Resistant ◽  
E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

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