Localization of replication forks in wild-type and mukB mutant cells of Escherichia coli

2005 ◽  
Vol 274 (3) ◽  
pp. 264-271 ◽  
Author(s):  
Shun Adachi ◽  
Masamichi Kohiyama ◽  
Toshinari Onogi ◽  
Sota Hiraga
Keyword(s):  
Escherichia Coli ◽  
Replication Forks ◽  
Wild Type ◽  
Mutant Cells

scholarly journals Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli

2016 ◽  
Vol 198 (8) ◽  
pp. 1305-1316 ◽  
Author(s):  
Emily Helgesen ◽  
Solveig Fossum-Raunehaug ◽  
Kirsten Skarstad
Keyword(s):  
Escherichia Coli ◽  
Dna Content ◽  
Average Distance ◽  
Replication Forks ◽  
Wild Type ◽  
Content Type ◽  
Cellular Dna ◽  
Adjacent Segments ◽  
Mutant Cells

ABSTRACTThe architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of theEscherichia colichromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein. In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ∼100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic ofE. colicells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a “slow-growth” type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content.IMPORTANCEIt is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation inEscherichia coli. In this work, we findin vivoindications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is involved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such conditions.

scholarly journals TolC-Dependent Exclusion of Porphyrins in Escherichia coli

2008 ◽  
Vol 190 (18) ◽  
pp. 6228-6233 ◽  
Author(s):  
Ryoko Tatsumi ◽  
Masaaki Wachi
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
High Performance ◽  
Aminolevulinic Acid ◽  
Wild Type ◽  
E Coli ◽  
Chromatography Analysis ◽  
Increased Sensitivity ◽  
Mutant Cells ◽  
Reddish Brown Color

ABSTRACT We found that Escherichia coli tolC mutants showed increased sensitivity to 5-aminolevulinic acid (ALA), a precursor of porphyrins. The tolC mutant cells grown in the presence of ALA showed a reddish brown color under visible light and a strong red fluorescence under near-UV irradiation. Fluorescence spectrometry and high-performance liquid chromatography analysis showed that the tolC mutant cells grown in the presence of ALA accumulated a large amount of coproporphyrin(ogen) intracellularly. In contrast, the wild-type cells produced coproporphyrin extracellularly. The tolC mutant cells grown in the presence of ALA, which were capable of surviving in the dark, were killed by near-UV irradiation, suggesting that the intracellular coproporphyrin(ogen) renders these cells photosensitive. These results suggest that the TolC-dependent efflux system is involved in the exclusion of porphyrin(ogen)s in E. coli.

Unusual properties of a new division mutant of Escherichia coli

1983 ◽  
Vol 29 (6) ◽  
pp. 694-699 ◽  
Author(s):  
G. R. Drapeau ◽  
J. P. Chausseau ◽  
F. Gariépy
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
Pleiotropic Effect ◽  
Single Mutation ◽  
Permissive Temperature ◽  
Wild Type ◽  
Genetic Lesion ◽  
Host Cell Reactivation ◽  
Mutant Forms ◽  
Mutant Cells

The properties of a division mutant of Escherichia coli were investigated. At 42 °C, this mutant forms nonseptate, multinucleate, filamentous cells typical of division mutants, and at the permissive temperature, is sensitive to ultraviolet (UV) irradiation. Temperature and UV sensitivities are probably due to a single mutation. The mutant phenotype is dominant to wild type. The mutant cells make DNA nearly as effectively as control cells at 42 °C or following UV irradiation. They exhibit normal host-cell reactivation capacities and can express all manifestations of the SOS response with the exception of Weigle reactivation. The genetic lesion which mediates this pleiotropic effect is located very close to the leu locus of the linkage map.

scholarly journals Phenotypes of dnaXE145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Ingvild Flåtten ◽  
Emily Helgesen ◽  
Ida Benedikte Pedersen ◽  
Torsten Waldminghaus ◽  
Christiane Rothe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Replication Fork ◽  
Temperature Sensitive ◽  
Replication Forks ◽  
Clamp Loader ◽  
Topoisomerase Iv ◽  
Content Type ◽  
Stalled Replication Forks ◽  
Mutant Cells

ABSTRACT The Escherichia coli dnaXE145A mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation parE10. The gene for the clamp loader subunits τ and γ, dnaX, but not the mutant dnaXE145A , was found to suppress parE10(Ts) when overexpressed. Purified mutant protein was found to be functional in vitro, and few phenotypes were found in vivo apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at oriC never reach the terminus in dnaXE145A mutant cells. The SOS response was found to be induced, and a combination of the dnaXE145A mutation with recBC and recA mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of recBC and recA, respectively. The results indicate that the dnaXE145A mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the dnaX-encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination. IMPORTANCE The E. coli clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the dnaXE145A clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.

scholarly journals Glutathione and Transition-Metal Homeostasis in Escherichia coli

2008 ◽  
Vol 190 (15) ◽  
pp. 5431-5438 ◽  
Author(s):  
Kerstin Helbig ◽  
Corinna Bleuel ◽  
Gerd J. Krauss ◽  
Dietrich H. Nies
Keyword(s):  
Escherichia Coli ◽  
Transition Metal ◽  
Metal Tolerance ◽  
Metal Homeostasis ◽  
Metal Resistance ◽  
Wild Type ◽  
Strong Decrease ◽  
Trace Element Metabolism ◽  
P Type ◽  
Mutant Cells

ABSTRACT Glutathione (GSH) and its derivative phytochelatin are important binding factors in transition-metal homeostasis in many eukaryotes. Here, we demonstrate that GSH is also involved in chromate, Zn(II), Cd(II), and Cu(II) homeostasis and resistance in Escherichia coli. While the loss of the ability to synthesize GSH influenced metal tolerance in wild-type cells only slightly, GSH was important for residual metal resistance in cells without metal efflux systems. In mutant cells without the P-type ATPase ZntA, the additional deletion of the GSH biosynthesis system led to a strong decrease in resistance to Cd(II) and Zn(II). Likewise, in mutant cells without the P-type ATPase CopA, the removal of GSH led to a strong decrease of Cu(II) resistance. The precursor of GSH, γ-glutamylcysteine (γEC), was not able to compensate for a lack of GSH. On the contrary, γEC-containing cells were less copper and cadmium tolerant than cells that contained neither γEC nor GSH. Thus, GSH may play an important role in trace-element metabolism not only in higher organisms but also in bacteria.

scholarly journals Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (9) ◽  
pp. 4773-4781 ◽  
Author(s):  
D H Lee ◽  
M Y Sherman ◽  
A L Goldberg
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperone ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Unfolded Proteins ◽  
Ubiquitinated Protein ◽  
Large Defect ◽  
Selective Degradation ◽  
Mutant Cells ◽  
Beta Galactosidase

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.

scholarly journals Susceptibility to Antibiotics and β-Lactamase Induction in Murein Hydrolase Mutants of Escherichia coli

2005 ◽  
Vol 49 (4) ◽  
pp. 1404-1409 ◽  
Author(s):  
Dorota Korsak ◽  
Sylvia Liebscher ◽  
Waldemar Vollmer
Keyword(s):  
Escherichia Coli ◽  
Peptidoglycan Hydrolase ◽  
Wild Type ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Lytic Transglycosylase ◽  
Antibiotic Susceptibilities ◽  
Type Mutant ◽  
Murein Hydrolase ◽  
Mutant Cells

ABSTRACT The antibiotic susceptibilities and capabilities to induce β-lactamases were studied in multiple Escherichia coli murein (peptidoglycan) hydrolase mutants. E. coli mutants lacking either three amidases, three amidases and one lytic transglycosylase, or six lytic transglycosylases showed higher levels of susceptibility to bacitracin, erythromycin, gallidermin, and vancomycin than the wild type. Mutant cells without three amidases lost viability in the presence of vancomycin and gallidermin, whereas the wild type was resistant to both antibiotics. β-Lactamase induction was studied after introduction of a plasmid carrying the ampC and ampR genes. Upon addition of cefoxitin to the growth medium, the wild type as well as a mutant lacking all known amidases and dd-endopeptidases induced β-lactamase, whereas a mutant lacking all known lytic transglycosylases was unable to induce β-lactamase, showing that lytic transglycosylase activity is essential for β-lactamase induction. Consequently, cells lacking lytic transglycosylase activity lysed in the presence of penicillin, despite the presence of the inducible β-lactamase system. We discuss the potential of murein hydrolase inhibitors for antibiotic therapy.

scholarly journals Increased Episomal Replication Accounts for the High Rate of Adaptive Mutation in recD Mutants of Escherichia coli

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 15-30 ◽  
Author(s):  
Patricia L Foster ◽  
William A Rosche
Keyword(s):  
Escherichia Coli ◽  
Mutation Rate ◽  
Copy Number ◽  
Frameshift Mutation ◽  
High Rate ◽  
Adaptive Mutation ◽  
Lacz Gene ◽  
Exonuclease Activity ◽  
Wild Type ◽  
Mutant Cells

Abstract Adaptive mutation has been studied extensively in FC40, a strain of Escherichia coli that cannot metabolize lactose (Lac-) because of a frameshift mutation affecting the lacZ gene on its episome. recD mutants of FC40, in which the exonuclease activity of RecBCD (ExoV) is abolished but its helicase activity is retained, have an increased rate of adaptive mutation. The results presented here show that, in several respects, adaptive mutation to Lac+ involves different mechanisms in recD mutant cells than in wild-type cells. About half of the apparent increase in the adaptive mutation rate of recD mutant cells is due to a RecA-dependent increase in episomal copy number and to growth of the Lac- cells on the lactose plates. The remaining increase appears to be due to continued replication of the episome, with the extra copies being degraded or passed to recD+ recipients. In addition, the increase in adaptive mutation rate in recD mutant cells is (i) dependent on activities of the single-stranded exonucleases, RecJ and ExoI, which are not required for (in fact, slightly inhibit) adaptive mutation in wild-type cells, and (ii) enhanced by RecG, which opposes adaptive mutation in wild-type cells.

scholarly journals Differential Binding Profiles of StpA in Wild-Type and hns Mutant Cells: a Comparative Analysis of Cooperative Partners by Chromatin Immunoprecipitation-Microarray Analysis

2009 ◽  
Vol 191 (7) ◽  
pp. 2388-2391 ◽  
Author(s):  
Ebru Uyar ◽  
Ken Kurokawa ◽  
Mika Yoshimura ◽  
Shu Ishikawa ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Microarray Analysis ◽  
Chromatin Immunoprecipitation ◽  
Wild Type ◽  
Binding Profile ◽  
Binding Regions ◽  
Differential Binding ◽  
Mutant Cells

ABSTRACT We performed chromatin immunoprecipitation-microarray analysis to investigate differences in function between StpA and H-NS in Escherichia coli cells. StpA binding regions essentially overlap those of H-NS in wild-type cells, while they are reduced to one-third in the hns mutant. The H-NS binding profile was unaffected by stpA inactivation.

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