SEARCH · PubMed Health

Results for “SOS Response, Genetics”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed1995

recA gene involvement in oxidative and thermal stress in Lactococcus lactis.

The recA gene is best known for its effects on homologous recombination and DNA repair via SOS induction. There is gathering evidence that recA also affects expression of genes associated with different types of stress. We studied recA properties in Lactococcus lactis by generating a recA-disrupted mutant of MG1363 and comparing it with the wild type strain. recA appears to have an important role in cell survival upon oxygen or thermal stress, in addition to its conserved role in DNA repair. Oxygen toxicity appears to be due to the production of hydroxyl radicals via the Fenton reaction; recA would be involved in the repair of DNA damage generated by these radicals. Surprisingly, the recA strain stops growing at elevated temperature (37 degrees C). Immunological tests indicate that amounts of three heat shock proteins are reduced in the recA strain compared to the wild type strain. In contrast, the amount of heat shock regulator HflB is markedly increased, even at low temperature. HflB is known to degrade heat shock transcription factor sigma 32 in Escherichia coli. We propose that heat shock response is reduced in the recA mutant due to overproduction of HflB.

ATP-Dependent Proteases↗

PubMed2006

Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae.

Natural transformation is a widespread mechanism for genetic exchange in bacteria. Aminoglycoside and fluoroquinolone antibiotics, as well as mitomycin C, a DNA-damaging agent, induced transformation in Streptococcus pneumoniae. This induction required an intact competence regulatory cascade. Furthermore, mitomycin C induction of recA was strictly dependent on the development of competence. In response to antibiotic stress, S. pneumoniae, which lacks an SOS-like system, exhibited genetic transformation. The design of antibiotherapy should take into consideration this potential of a major human pathogen to increase its rate of genetic exchange in response to antibiotics.

Aminoglycosides↗

PubMed2002

Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay.

Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly used fluorophores--fluorescein, DDAO and resorufin--are evaluated. Experimental evidence is given that S9 liver homogenate contains a heat-labile, reversible fluorophore-binding activity and therefore, significantly reduces fluorescence intensities. We have worked out simple solutions to overcome the S9 related interference in order to be able to establish a robust bacterial genotoxicity assay.

Bacteria↗

PubMed1999

Synergistic lethal effect between hydrogen peroxide and neocuproine (2,9-dimethyl 1,10-phenanthroline) in Escherichia coli.

Despite 2,9-dimethyl 1,10-phenanthroline (NC) has been extensively used as a potential inhibitor of damage due to oxidative stress in biological systems, the incubation of E. coli cultures with the copper ion chelator NC prior to the challenge with hydrogen peroxide caused a lethal synergistic effect. The SOS response seems to be involved in the repair of the synergistic lesions through the recombination pathway. Furthermore, there is evidence for the UvrABC excinuclease participation in the repair of the synergistic lesions, and the base excision repair may also be required for bacterial survival to the synergistic effect mainly at high concentrations of H2O2, being the action of Fpg protein an important event. Incubation of lexA (Ind-) cultures with iron (II) ion chelator 2,2'-dipyridyl simultaneously with NC prevented the lethal synergistic effect. This result suggests an important role of the Fenton reaction on the phenomenon. NC treatment was able to increase the number of DNA strand breaks (DNAsb) induced by 10 mM of H2O2 in lexA (Ind-) strain and the simultaneous treatment with 2,2'-dipyridyl was able to block this effect.

2,2'-Dipyridyl↗

PubMed2001

umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control.

The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.

Bacterial Proteins↗

PubMed1998

Characterization of RecA1332 in vivo and in vitro. A role for alpha-helix E as a liaison between the subunit-subunit interface and the DNA and ATP binding domains of RecA protein.

BACKGROUND: The RecA protein of Escherichia coli is essential for homologous recombination and induction of the SOS response. RecA has three cysteines located at positions 90, 116 and 129. Chemical modification of these residues abolishes ATP hydrolysis and repressor cleavage, and causes a reduction in the DNA strand exchange and DNA strand annealing activities. Several mutants at each of these positions were isolated and partially characterized. One of these, recA1332, replaces cysteine 129 with methionine. Although this is a relatively conservative mutation based on hydrophobicity, recA1332 was completely defective for DNA repair but the purified protein was active for ATPase in vitro. RESULTS: In vivo, strains containing this mutant allele were shown to be defective when assayed for all RecA-dependent activities. In vitro, RecA1332 protein possessed DNA-dependent ATP hydrolysis activity that showed an increased sensitivity to inhibition by monovalent cations, and whose k(cat) was reduced 3- to 12-fold. In addition, RecA1332 was unable to use oligodeoxyribonulceotides as ssDNA cofactors in the ATPase reaction. RecA1332 showed altered binding to single- and double-stranded DNA and, although it was able to perform DNA strand exchange, it was slowed in its ability to both form joint molecule intermediates and to convert these species to product. CONCLUSIONS: Our results are consistent with a defect in intermolecular interactions between RecA monomers. We propose that alpha-helix E (which includes C129M) is a liaison that connects the subunit-subunit interactions to DNA and ATP binding, thereby creating filament stability and cooperativity.

Adenosine Triphosphatases↗

PubMed1998

Bacterial stress responses to 1-megahertz pulsed ultrasound in the presence of microbubbles.

Members of a panel of stress-responsive biosensors have been used to study the effect of megahertz frequency ultrasound on Escherichia coli. Insonification causes acoustic cavitation, the collapse of oscillating microbubbles in solution, which can damage bacterial cells. A focused 1-MHz ultrasound transducer, capable of generating a spatial peak pulse average intensity of 500 W/cm2, was used to treat liquid bacterial cultures. Stress-responsive promoters fused to luxCDABE allowed the continuous measurement of light produced as a result of protein damage, DNA damage, oxidative stress, and membrane perturbation. A promoter responsive to ammonia limitation was not transcriptionally activated under test conditions. In contrast to bacteria in exponentially growing cultures, those in stationary-phase cultures were more resistant to the effects of ultrasound treatment. Quantification of the degree of acoustic cavitation due to symmetric bubble collapse was measured by a 20-MHz passive transducer, the output of which appears to be only partially correlated with cellular damage and survival. The methods and results summarized here provide the basis for further investigation into applications, including the purification of water samples.

Ammonia↗

PubMed1997

A recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP.

RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the alpha-helix H of the C-terminal subdomain of the protein. RecA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42 degrees C) in the absence of ATP resulted in inhibition at 42 degrees C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5 M) was shown to be another factor that can stabilize the RecAmut protein at 42 degrees C, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecA-mut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.

Adenosine Triphosphatases↗

PubMed2005

Construction of a ColD cda promoter-based SOS-green fluorescent protein whole-cell biosensor with higher sensitivity toward genotoxic compounds than constructs based on recA, umuDC, or sulA promoters.

Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 microM, 1.1 microM, and 141 microM, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.

Base Sequence↗

PubMed2000

lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli.

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.

ATP-Dependent Proteases↗

PubMedDate not supplied

EILATox-Oregon Workshop: blind study evaluation of Vitotox test with genotoxic and cytotoxic sample library.

In order to assess the robustness, sensitivity and specificity of a recently developed Vitotox test, 17 blind coded chemicals and three environmental water samples were tested at the EILATox-Oregon Workshop using the Thermo Electron Vitotox kit. The Vitotox test is a rapid geno- and cytotoxicity test using standard 96- or 384-well microtitre plates. The genotoxicity test is based on two genetically modified Salmonella typhimurium strains containing bacterial luciferase operon from Vibrio fisheri under the SOS inducible promoter. The SOS system is an inducible network in Escherichia coli that responds to DNA damage and activates DNA repair. The Vitotox genotoxicity test bacteria strain carries bacterial luciferase genes under the control of SOS inducible promoter and therefore any DNA damage inside the cells induces the production of bacterial luciferase. The luciferase expression is then followed with a microtitre plate luminometer for 3 h after mixing different dilutions of sample with the test bacteria. The genotoxicity index is calculated for each dilution and the genotoxicity of the sample is interpreted based on kinetic time curves and genotoxicity vs concentration/dilution curves. Cytotoxicity of the sample is determined simultaneously with another test strain containing the same luciferase operon controlled by the constitutive promoter. This bacterium produces constant bioluminescence and any decrease of the bioluminescence production is used as a marker for cytotoxicity. As a miniaturized microtitre plate assay the Vitotox test requires a very small quantity of the sample material. The samples used in the workshop were diluted 1 : 10 or 1 : 100 before testing. Genotoxicity and cytotoxicity data were collected at dilutions of 1 : 10-1 : 2000. When the samples of the EILATox-Oregon Workshop were tested using the Vitotox test, four coded chemicals out of 17 were determined to be genotoxic. Seven chemicals and one environmental sample were found to be cytotoxic. Three chemical samples were found to be both geno- and cytotoxic.

Aliivibrio fischeri↗

PubMed1991

Prevalence of recombinational versus mutational events in damaged plasmid DNA containing regions of homology with the chromosome.

Plasmid DNA modified by in vitro treatments was transformed in E. coli bacterial cells. A streptomycin-resistant strain, carrying the peculiar rpsL421 mutation, was used as a recipient for the cloning vector pNO1523, which carries the wild-type (streptomycin-sensitive) rpsL allele. Transformants were streptomycin-sensitive unless a change in plasmid sequence had occurred. The analysis of the MaeI restriction pattern of plasmids isolated from streptomycin-resistant transformants, together with the detection of the phenotype that they conferred to a streptomycin-dependent strain, allowed us to identify plasmids that had undergone recombination with the host chromosome. The number of these plasmids exceeded by far that of plasmids resulting from mutational events.

Chromosomes, Bacterial↗

PubMed1992

Effect of flash photoreactivation on Escherichia coli recA induction by ultraviolet light.

Excision-deficient Escherichia coli, carrying the gene for the photolyase on a multicopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.

Escherichia coli↗

PubMed1996

Overlapping functions for recF and priA in cell viability and UV-inducible SOS expression are distinguished by dnaC809 in Escherichia coli K-12.

The recF and priA genes have roles in DNA repair and homologous recombination. Mutations in these genes also cause decreases in cell viability and alterations in UV-inducible sulAp-lacZ (SOS) expression. To find out if the two genes are in the same or different pathways for viability and SOS expression, the phenotypes of the double mutant strains were studied. The recF priA double mutant showed a lower viability and SOS expression level than either of the single mutants. In the case of cell viability, recF missense mutations decreased viability of a priA2::kan strain two to five-fold whereas recF null priA2::kan double mutants were not viable at all. dnaC809, a mutation that suppresses the UV-sensitive (UVs and Rec- phenotypes of priA2::kan, restored cell viability, but not UV-inducible SOS expression, to a priA recF strain. Since recF is epistatic with recO and recR (recOR) for UV resistance, recOR mutations were also tested with priA2::kan. No overlap was found between recOR and priA for viability and SOS expression. It is concluded that priA and recF have two different overlapping functions in viability and SOS expression that are distinguishable by the effects of dnaC809. The role of recF in a priA2::kan strain in cell viability is a new function for recF and unlike recF's other roles in DNA repair and recombination, is independent of recOR. A new role for priA in UV-inducible SOS expression in a recF mutant is also defined.

Bacterial Proteins↗

PubMed1999

dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.

PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA's role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.

Bacterial Proteins↗

PubMed2004

Measurement of SOS expression in individual Escherichia coli K-12 cells using fluorescence microscopy.

Many recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp-lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the 'Uniform Expression Model (UEM)' where expression is evenly distributed in all cells or (ii) the 'Two Population Model (TPM)' where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter (sulAp) to the green fluorescent protein (gfp) reporter gene and inserting it at attlambda on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp-gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA, dnaT, recG, uvrD, dam, ftsK, rnhA, polA and xerC mutants were explained best by the TPM while only lexA (def), lexA3 (ind-) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.

Adenosine Triphosphatases↗

PubMed2001

Xanthomonas oryzae pv. oryzae recA is transcribed and regulated from multiple promoters.

Transcription regulation of Xanthomonas oryzae pv. oryzae recA was characterized. Primer extension experiments showed that recA is transcribed from three promoters designated P1, P2 and P3. The sequences of -10 and -35 regions of these promoters have moderate homology to the proposed consensus sequence for a Xanthomonas promoter. Putative SOS boxes were identified in the vicinity of P1 and P2 promoters. Deletion analysis and in vivo monitoring of promoter activity of these promoters revealed that the three promoters have different characteristics. P1 and P2 show stress-inducible high and low promoter strengths respectively. P3 is a non-inducible moderate promoter strength. These promoters are regulated by two SOS boxes. The multiplicity of promoters and SOS boxes provides back-up systems to ensure proper regulation of recA.

Base Sequence↗

PubMed1986

Induction of SOS functions by alkaline intracellular pH in Escherichia coli.

Alkalinization of intracellular pH (pHi) causes an increase in UV resistance in wild-type and pH-sensitive mutant (DZ3) cells of Escherichia coli. Utilizing cells transformed with a plasmid (pA7) which bears the uvrA promoter fused to galK galactokinase structural gene, it was shown that alkaline pHi leads to an increase in the specific activity of galactokinase. This effect was not displayed in a mutant bearing a recA-insensitive lexA gene, nor in cells harboring a plasmid (pA8) in which the galK is fused to a lexA-insensitive uvrA promoter. Hence, the effects of pHi on cells functions may involve the lexA product of the SOS system.

DNA Repair↗