scholarly journals Survival, DNA-breakdown and induction of prophage lambda in aEscherichia coliK12recAuvrBdouble mutant

1969 ◽  
Vol 14 (3) ◽  
pp. 291-307 ◽  
Author(s):  
I. M. Hertman
Keyword(s):  
Mitomycin C ◽  
Ultraviolet Light ◽  
Double Mutant ◽  
Similar Rate ◽  
Complete Medium ◽  
Wild Type ◽  
Single Mutant ◽  
Pyrimidine Dimers ◽  
Cellular Dna ◽  
K 12

Cellular functions of a double mutant ofEscherichia coliK 12 deficient in recombination (recA) and defective in excision of pyrimidine dimers (uvrB) have been compared to those of isogenicrecAoruvrBsingle mutants and ‘wild type’ bacteria. A combined effect of the two mutations on cell survival both under normal conditions of growth and after exposure to ultraviolet light or mitomycin C was demonstrated. The ratio of optical density to the number of colony formers in growing cultures of the double mutant is three times greater than in similar cultures of therecAsingle mutant and 9 times greater than in eitheruvrBor in ‘wild type’ cultures. The doubling time in growingrecA uvrBcultures is 90 min, compared to 60 min, for therecAsingle mutant and 40 min for theuvrBsingle mutant and ‘wild type’ bacteria. Growing cultures ofrecA uvrB(λcI857) bacteria contain a substantial fraction of cells which are unable to form colonies at 32 °C, but produce phage when heated to 42 °C. No such cells were found in cultures of the single mutants or the ‘wild type’ bacteria lysogenic for λc1857. The double mutant is 10 times more sensitive to ultraviolet light and twice more sensitive to mitomycin C than therecAsingle mutant. In contrast torecAbacteria, exposure of the double mutant to mitomycin C induces little additional breakdown of cellular DNA. Induction of the prophage by mitomycin C is, however, prevented in bothrecA uvrB(λ) andrecA(λ) bacteria. Exposure to mitomycin C creates conditions which render the prophage inducible by a newly transducedree Agene. This effect of mitomycin C persists and can be revealed in complete medium at 37 °C after 100 min of incubation. The decay of the prophage, in cells exposed to mitomycin C, proceeds at a similar rate in both the double mutant and therecAsingle mutant. The inability ofrecAlysogens to be induced to phage production is discussed in the light of the present findings.

scholarly journals An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 381-393
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Strand Break ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Chromatid Cohesion ◽  
K 12 ◽  
Structural Maintenance Of Chromosomes ◽  
Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

scholarly journals PtrB of Pseudomonas aeruginosa Suppresses the Type III Secretion System under the Stress of DNA Damage

2005 ◽  
Vol 187 (17) ◽  
pp. 6058-6068 ◽  
Author(s):  
Weihui Wu ◽  
Shouguang Jin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Damage ◽  
Mitomycin C ◽  
Type Iii Secretion ◽  
Double Mutant ◽  
Type Iii Secretion System ◽  
Sos Response ◽  
Secretion System ◽  
Wild Type ◽  
Type Iii

ABSTRACT In a search for regulatory genes of the type III secretion system (TTSS) in Pseudomonas aeruginosa, transposon (Tn5) insertional mutants of the prtR gene were found defective in the TTSS. PrtR is an inhibitor of prtN, which encodes a transcriptional activator for pyocin synthesis genes. In P. aeruginosa, pyocin synthesis is activated when PrtR is degraded during the SOS response. Treatment of a wild-type P. aeruginosa strain with mitomycin C, a DNA-damaging agent, resulted in the inhibition of TTSS activation. A prtR/prtN double mutant had the same TTSS defect as the prtR mutant, and complementation by a prtR gene but not by a prtN gene restored the TTSS function. Also, overexpression of the prtN gene in wild-type PAK had no effect on the TTSS; thus, PrtN is not involved in the repression of the TTSS. To identify the PrtR-regulated TTSS repressor, another round of Tn mutagenesis was carried out in the background of a prtR/prtN double mutant. Insertion in a small gene, designated ptrB, restored the normal TTSS activity. Expression of ptrB is specifically repressed by PrtR, and mitomycin C-mediated suppression of the TTSS is also abolished in a ptrB mutant strain. Therefore, PtrB is a new TTSS repressor that coordinates TTSS repression and pyocin synthesis under the stress of DNA damage.

Rescue of mitomycin C- or psoralen-inactivated Micrococcus radiodurans by additional exposure to radiation or alkylating agents

1982 ◽  
Vol 152 (3) ◽  
pp. 976-982
Author(s):  
M T Hansen
Keyword(s):  
Mitomycin C ◽  
Uv Radiation ◽  
Uv Light ◽  
Alkylating Agents ◽  
Repair System ◽  
Wild Type ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Cellular Dna ◽  
Constitutive Synthesis

The processing of damaged DNA was altered in a mitomycin C-sensitive mutant (mtcA) of Micrococcus radiodurans. Even though the mutant retained resistance to 254-nm UV radiation, it did not, in contrast to the wild-type strain, show any excessive DNA degradation or cell death when incubated with chloramphenicol after sublethal doses of either UV light or mitomycin C. The results suggest the constitutive synthesis of an enzyme system responsible for wild-type proficiency in the repair of mitomycin C-induced damage. An alternative system able to repair damage caused by mitomycin C was demonstrated in the mtcA background. In this strain, additional damage inflicted upon the cellular DNA effected a massive rescue of cells previously inactivated by mitomycin C. Rescue was provoked by ionizing radiation, by UV light, or by simple alkylating agents. Cells treated with psoralen plus near-UV radiation could be rescued only when inactivation was due primarily to psoralen-DNA interstrand cross-links rather than to monoadducts. The rescue of inactivated cells was prevented in the presence of chloramphenicol. These results can be interpreted most readily in terms of an alternative repair system able to overcome DNA interstrand cross-links produced by mitomycin C or psoralen plus near-UV light, but induced only by the more abundant number of damages produced by radiation or simple alkylating agents.

The reactivation of tabun-inhibited mutant AChE with Ortho-7: steered molecular dynamics and quantum chemical studies

2016 ◽  
Vol 12 (4) ◽  
pp. 1224-1231 ◽  
Author(s):  
Rabindranath Lo ◽  
Nellore Bhanu Chandar ◽  
Shibaji Ghosh ◽  
Bishwajit Ganguly
Keyword(s):  
Molecular Dynamics ◽  
In Silico ◽  
Quantum Chemical ◽  
Double Mutant ◽  
Steered Molecular Dynamics ◽  
Wild Type ◽  
Single Mutant ◽  
Chemical Studies ◽  
Quantum Chemical Studies

Tabun inhibited AChE can be reactivated more easily with a single mutant than with a wild-type or double mutant: an in silico study.

scholarly journals Helicobacter pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor TlpB

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2445-2455 ◽  
Author(s):  
Bethany A. Rader ◽  
Christopher Wreden ◽  
Kevin G. Hicks ◽  
Emily Goers Sweeney ◽  
Karen M. Ottemann ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Double Mutant ◽  
Soft Agar ◽  
Dependent Manner ◽  
Wild Type ◽  
Environmental Chemical ◽  
Single Mutant ◽  
Autoinducer 2 ◽  
Reduced Frequency ◽  
H Pylori

Helicobacter pylori moves in response to environmental chemical cues using a chemotaxis two-component signal-transduction system. Autoinducer-2 (AI-2) is a quorum-sensing signal produced by the LuxS protein that accumulates in the bacterial environment in a density-dependent manner. We showed previously that a H. pylori luxS mutant was defective in motility on soft agar plates. Here we report that deletion of the luxS gene resulted in swimming behaviour with a reduced frequency of stops as compared to the wild-type strain. Stopping frequency was restored to wild-type levels by genetic complementation of the luxS mutation or by addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Synthetic DPD also increased the frequency of stops in wild-type H. pylori, similar to the behaviour induced by the known chemorepellent HCl. We found that whereas mutants lacking the chemoreceptor genes tlpA, tlpC or tlpD responded to an exogenous source of synthetic DPD, the chemoreceptor mutant tlpB was non-responsive to a gradient or uniform distribution of the chemical. Furthermore, a double mutant lacking both tlpB and luxS exhibited chemotactic behaviour similar to the tlpB single mutant, whereas a double mutant lacking both tlpB and the chemotransduction gene cheA behaved like a nonchemotactic cheA single mutant, supporting the model that tlpB functions in a signalling pathway downstream of luxS and upstream of cheA. We conclude that H. pylori perceives LuxS-produced AI-2 as a chemorepellent via the chemoreceptor TlpB.

scholarly journals Interaction Between Ustilago maydis REC2 and RAD51 Genes in DNA Repair and Mitotic Recombination

Genetics ◽  
1997 ◽  
Vol 145 (2) ◽  
pp. 243-251 ◽  
Author(s):  
David O Ferguson ◽  
Michael C Rice ◽  
Mara H Rendi ◽  
Hidehito Kotani ◽  
Eric B Kmiect ◽  
...  
Keyword(s):  
Dna Repair ◽  
Ultraviolet Light ◽  
Double Mutant ◽  
Ustilago Maydis ◽  
Methyl Methanesulfonate ◽  
Multicopy Plasmid ◽  
Independent Action ◽  
Wild Type ◽  
Gene Encoding ◽  
Γ Radiation

A gene encoding a Ustilago maydis Rad51 orthologue has been isolated. rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and γ radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.

scholarly journals An msbB Homologue Carried in Plasmid pO157 Encodes an Acyltransferase Involved in Lipid A Biosynthesis in Escherichia coli O157:H7

2004 ◽  
Vol 72 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Sang-Hyun Kim ◽  
Wenyi Jia ◽  
Russell E. Bishop ◽  
Carlton Gyles
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Double Mutant ◽  
Lipid A ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
Single Mutant ◽  
Double Mutant Strain ◽  
Mutant Strains ◽  
Lipid A Biosynthesis

ABSTRACT Escherichia coli O157:H7 carries a chromosomal msbB1 and a plasmid-encoded msbB2 gene. We characterized msbB2 function as a homologue of msbB1 by examination of wild-type organisms and mutant strains that lacked functional msbB1, msbB2, and both msbB1 and msbB2. The msbB double-mutant strain generated pentaacyl lipid A, while the single-mutant strains synthesized hexaacyl lipid A. Complementation with overexpressed msbB2 converted pentaacyl into hexaacyl lipid A in the double-mutant strain. The transcription of both msbB genes occurred simultaneously. Lack of MsbB2 activity slightly increased the microheterogeneity of the lipid A species. These results suggest that the msbB2 gene plays a role not only in the routine generation of fully hexaacylated lipid A but also in suppressing the microheterogeneity of lipid A species, the endotoxic determinant of the organism.

scholarly journals Separate Ion Pathways in a Cl−/H+ Exchanger

2005 ◽  
Vol 126 (6) ◽  
pp. 563-570 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Walden ◽  
Wang Nguitragool ◽  
Hariharan Jayaram ◽  
Carole Williams ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Transport Mechanism ◽  
Double Mutant ◽  
Wild Type ◽  
Extracellular Solution ◽  
X Ray ◽  
Single Mutant ◽  
Cl Transport ◽  
Alternating Access

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

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