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B Demple

Publications and source records attributed to B Demple.

At least 73 records · Page 4Linked to original sources
PubMed1995

Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli.

Exposure of cells to hydrogen peroxide (H2O2) mediates adaptive responses or oxidative damage, depending on the magnitude of the challenge. Determining the threshold for peroxide-mediated oxidative stress thus requires quantitation of the changes in endogenous H2O2 production. The intracellular steady-state concentrations of H2O2 were measured in intact Escherichia coli under different conditions. Compounds that block electron transport at NADH dehydrogenase (rotenone) or between ubiquinone and cytochrome b (antimycin) showed that univalent reduction of O2 can occur at these sites in vivo to form superoxide anion (O2-), in agreement with reports for mammalian mitochondria. Mutational inactivation of different components of the respiratory chain showed that H2O2 production also depended on the energy status of the cell and on the arrangement of respiratory chain components corresponding to particular growth conditions. Production rates for O2- and H2O2 were linearly related to the number of active respiratory chains that reached maximal values during exponential growth. In the strains defective in respiratory chain components, catalase activity was regulated to compensate for changes in the H2O2 production rates, which maintained intracellular H2O2 at 0.1-0.2 microM during aerobic growth over a wide range of cell densities. The expression of a katG'::lacZ fusion (reporting transcriptional control of the catalase-hydroperoxidase I gene) was increased by H2O2 given either as a pulse or as a steady production. This response not only depended on the type and severity of the stimulus but was also strongly influenced by the growth phase of the cells.

Aerobiosis↗
PubMed1995

Characterization of the promoter region of the human apurinic endonuclease gene (APE).

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.

Animals↗
PubMed1995

Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages.

Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This in vivo induction was almost completely eliminated by the NO. synthase inhibitor NG-monomethyl-L-arginine. The inhibitor increased the survival of a delta soxRS strain but not that of wild-type E. coli after phagocytosis, which suggests that induction of the soxRS regulon by NO. can counteract most of the cytotoxic effects of NO. production by the macrophages. We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages.

Animals↗
PubMed1995

Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein.

Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively. These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a approximately 100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC. We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes. Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate. The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake. Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology. Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA. Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at approximately 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10(-8) to 10(-7) M). Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.

Bacterial Proteins↗
PubMed1994

Interactions of Escherichia coli endonuclease IV and exonuclease III with abasic sites in DNA.

Duplex oligodeoxynucleotides with synthetic analogs of abasic sites were used to study the specificity of the abasic endonucleases of Escherichia coli. The apparent Km values of exonuclease III for the tetrahydrofuranyl, propanyl, and deoxyribosyl substrates varied only somewhat (20-140 nM) in either Mg2+ or Ca2+ and were similar to those for endonuclease IV. In Mg2+, exonuclease III had a turnover number 4-13-fold higher than measured for endonuclease IV (ranging 5.6-18 min-1), but was lowered in Ca2+ to values similar to those for endonuclease IV. The rate of cleavage of tetrahydrofuranyl (F) substrate by both enzymes was unaffected by the base in the opposite strand or its replacement by a tetrahydrofuranyl moiety. A C:C mismatch on the 5' but not the 3' side of F strongly inhibited cleavage by exonuclease III in Ca2+, while mismatches on both sides were required to diminish endonuclease IV cleavage significantly. A phosphorothioate ester linked 5' to the tetrahydrofuranyl moiety inhibited both enzymes, with the Rp stereoisomer most effective. Endonuclease IV bound stably to duplex substrates containing the Rp phosphorothioate in the presence of poly(dI-dC). Although the apurinic/apyrimidinic-cleaving activities of endonuclease IV and exonuclease III have some common features they also differ in their specific interactions with DNA containing abasic sites.

Base Sequence↗
PubMed1994

Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease.

The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions. The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases. Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule. Dissimilar results have been obtained by transfecting DNA into eukaryotic cells. It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transecting DNA. The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA. With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apn1, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced. The rate of transversion to a G.C pair was increased 10-fold in Apn1-deficient yeast, including a 59-fold increase in the rate of A.T-->C.G events. In contrast, the rate of transversion to an A.T pair was increased by only 3-fold. A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action. Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S. cerevisiae. Other results presented here show that AP endonuclease-deficient Escherichia coli exhibit a mutator phenotype consistent with the A-rule.

Base Composition↗
PubMed1994

A cluster of constitutive mutations affecting the C-terminus of the redox-sensitive SoxR transcriptional activator.

Activation of Escherichia coli oxidative stress regulon genes (sodA, zwf, fumC, nfo, etc.) is mediated by a two-stage regulatory system: the redox-sensitive SoxR protein transcriptionally activates the soxS gene, whose product then stimulates transcription of the regulon genes. Previous experiments showed that limited 3' truncation of soxR gene causes constitutive soxRS expression. DNA sequence analysis of the soxR genes from the soxRS-constitutive strains isolated originally (Greenberg et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6181-6185) revealed that three alleles encode amino acid substitutions or a chain termination clustered near the C-terminus of SoxR. Two other single-amino-acid substitutions in constitutive alleles mapped to the helix-turn-helix motif and to a region of unknown function in the center of the polypeptide, respectively. No constitutive mutation was found within the region encoding the cysteines of the SoxR FeS center, in the soxR or soxS promoters, or in the soxS structural gene. Since an in-frame deletion of just nine SoxR residues (136-144; full-length SoxR = 154 residues) gave rise to a powerful constitutive allele, it appears that a small segment of the SoxR C-terminus maintains the protein in the inactive state. Conservely, an intact C-terminus is evidently not required for gene activation by SoxR.

Alleles↗
PubMed1994

SoxS, an activator of superoxide stress genes in Escherichia coli. Purification and interaction with DNA.

A genetic response of Escherichia coli to nitric oxide or to superoxide-generating agents such as paraquat is controlled by the soxRS locus. The intracellular redox signals generated by these agents are sensed by the SoxR protein which, when activated, functions as a potent activator of soxS transcription. The resulting increased level of SoxS protein then activates approximately 10 genes that constitute the soxRS regulon. Although the SoxS protein is homologous to the COOH-terminal region of the AraC family of regulatory proteins, the mechanism by which SoxS protein activates the soxRS regulon promoters is unknown. We identified in extracts of cells expressing high levels of SoxS protein a DNA binding activity specific for fragments containing soxRS-regulated promoters. This binding activity was purified to physical homogeneity and proved to be the SoxS protein, as confirmed by NH2-terminal amino acid sequencing. The purified SoxS protein bound specifically to the promoters of the micF, zwf, nfo, and sodA genes. Multiple DNA-protein complexes were formed by SoxS in a concentration-dependent fashion with each of these promoters. This binding of SoxS protein also facilitated the subsequent binding of E. coli RNA polymerase to both the micF and the nfo promoters. The binding sites of SoxS in the zwf and micF promoters were identified by DNase I footprinting, which revealed an extended protected region immediately upstream of the respective -35 sites. These results indicate that the small SoxS protein (M(r) of only 12,900) is a direct transcriptional activator of the oxidative stress genes of the soxRS regulon, although the possible involvement of other proteins in transcription activation by SoxS has not been ruled out.

Bacterial Proteins↗
PubMed1994

Recognition of oxidized abasic sites by repair endonucleases.

The recognition of 'regular' and 'oxidized' sites of base loss (AP sites) in DNA by various AP endonucleases was compared. Model substrates with regular AP sites (resulting from mere hydrolysis of the glycosylic bond) were produced by damaging bacteriophage PM2 DNA by exposure to low pH; those with AP sites oxidized at the C-4'- and C-1'-position of the sugar moiety by exposure to Fe(III)-bleomycin in the presence of H2O2 and to Cu(II)-phenanthroline in the presence of H2O2 and ethanol, respectively. The results confirmed that AP sites-together with single-strand breaks-are indeed the predominant type of DNA modification in all three cases. For the recognition of 4'-oxidized AP sites, a 400-fold higher concentration of Escherichia coli exonuclease III and between 5-fold and 50-fold higher concentrations of bacteriophage T4 endonuclease V, E. coli endonuclease III and E. coli FPG protein were required than for the recognition of regular AP sites. In contrast, the recognition of 4'-oxidized AP sites by E. coli endonuclease IV was effected by 4-fold lower concentrations than needed for regular AP sites. 1'-oxidized AP sites (generated by activated Cu(II)-phenanthroline) were recognized by endonuclease IV and exonuclease III only slightly (3-fold and 13-fold, respectively) less efficiently than regular AP sites. In contrast, there was virtually no recognition of 1'-oxidized AP sites by the enzymes which cleave at the 3' side of AP sites (T4 endonuclease V, endonuclease III and FPG protein). The described differences were exploited for the analysis of the DNA damage induced by hydroxyl radicals, generated by ionizing radiation or Fe(III)-nitrilotriacetate in the presence of H2O2. The results indicate that both regular and 1'-oxidized AP sites represent only minor fractions of the AP sites induced by hydroxyl radicals.

Bleomycin↗
PubMed1994

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

Amino Acid Sequence↗
PubMed1994

An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein.

The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.-) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe-SoxR contained non-heme iron (up to 1.6 atoms per monomer); apo-SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe-SoxR indicated that it contains a redox active iron-sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe-SoxR and apo-SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe-SoxR stimulated transcription initiation at soxS in vitro > 100-fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe-SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress.

Bacterial Proteins↗
PubMed1994

Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli.

Resistance to multiple antibiotics and certain oxidative stress compounds was conferred by three independently selected mutations (marR1, soxQ1, and cfxB1) that mapped to 34 min on the Escherichia coli chromosome. Mutations at this locus can activate the marRAB operon, in which marR encodes a putative repressor of mar transcription and marA encodes a putative transcriptional activator of defense genes against antibiotics and oxidants. Overexpression of the wild-type MarR protein reversed the phenotypes (antibiotic resistance and increased antioxidant enzyme synthesis) of all three mutants. DNA sequence analysis showed that, like marR1, the other two mutations were alterations of marR: a 285-bp deletion in cfxB1 and a GC-->AT transition at codon 70 (Ala-->Thr) in soxQ1. All three mutations cause increased amounts of mar-specific RNA, which supports the hypothesis that MarR has a repressor function in the expression of the marRAB operon. The level of mar RNA was further induced by tetracycline in both the marR1 and soxQ1 strains but not in the cfxB1 deletion mutant. In the cfxB1 strain, the level of expression of a truncated RNA, with or without tetracycline exposure, was the same as the fully induced level in the other two mutants. Overproduction of MarR in the cfxB1 strain repressed the transcription of the truncated RNA and restored transcriptional inducibility by tetracycline. Thus, induction of the marRAB operon results from the relief of the repression exerted by MarR. The marRAB operon evidently activates both antibiotic resistance and oxidative stress genes.

Adaptation, Physiological↗
PubMed1994

Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli.

Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.

Bacterial Proteins↗
PubMed1993

Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages.

Nitric oxide is a free radical (NO) formed biologically through the oxidation of L-arginine by nitric oxide synthases. NO is produced transiently in mammalian cells for intercellular signaling and in copious quantities to cause cytostasis and cytotoxicity. In the latter situation, NO is a deliberate cytotoxic product of activated macrophages, along with other reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide (O2-). Escherichia coli has a complex set of responses to H2O2 and O2- that involves approximately 80 inducible proteins; we wondered whether these bacteria might induce analogous defenses against nitric oxide. We show here that a multigene system controlled by the redox-sensitive transcriptional regulator SoxR is activated by NO in vivo. This induction confers bacterial resistance to activated murine macrophages with kinetics that parallel the production of NO by these cells. Elimination of specific SoxR-regulated genes diminishes the resistance of these bacteria to the cytotoxic macrophages. The required functions include manganese-containing superoxide dismutase, endonuclease IV (a DNA-repair enzyme for oxidative damage), and micF, an antisense regulator of the outer membrane porin OmpF. These results demonstrate that SoxR is a sensor for cellular exposure to NO, and that the soxRS response system may contribute to bacterial virulence.

Animals↗
PubMed1993

Intracellular localization of the Apn1 DNA repair enzyme of Saccharomyces cerevisiae. Nuclear transport signals and biological role.

The Apn1 DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages. Apn1 is homologous to the repair endonuclease IV of Escherichia coli, but the yeast protein is approximately 80 residues longer at the C terminus. The Apn1 C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus. Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus. Elimination of just the last 12 residues from Apn1 (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme. Greater truncation of Apn1 produced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability. Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm. These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a delta apn1 strain. Apn355 and Apn334 complemented repair-deficient E. coli as effectively as did wild-type Apn1. Resistance to these DNA-damaging agents in yeast was restored if Apn355 and Apn334 (but not Apn315 or Apn293) were overproduced approximately 20-fold, which suggests either weak active transport or passive diffusion of these derivatives into the nucleus. Replacement of the C-terminal 12 residues of Apn1 with the nuclear targeting sequence of SV40 T-antigen did not restore effective function or nuclear localization in yeast.

Amino Acid Sequence↗
PubMed1993

Potent intracellular oxidative stress exerted by the carcinogen 4-nitroquinoline-N-oxide.

Oxidative stress exerted by superoxide-generating (redox-cycling) agents such as paraquat triggers the soxRS regulon of Escherichia coli. In this system, SoxR protein is the redox-sensitive activator of the soxS gene, the product of which then activates the approximately 10 promoters of this regulon. We found that 4-nitroquinoline-N-oxide (4NQO) is a powerful inducer of soxS, > 10-fold more potent than paraquat. The transcriptional induction of the soxS gene by 4NQO was tightly dependent on a functional soxR gene and on the presence of molecular oxygen, as found previously for several well characterized redox-cycling agents. Two 4NQO-related compounds were also shown to induce soxS:4-nitropyridine-N-oxide, with an efficiency only slightly less than 4NQO, and 4-hydroxyaminoquinoline-N-oxide, at approximately 50-fold lower potency than 4NQO. E. coli strains that are hypersensitive to oxidative stress (owing to deficiency in either superoxide dismutases or oxidative DNA repair enzymes) were hypersensitive to killing by 4NQO. Thus, considerable oxidative stress is induced in cells by 4NQO, which might contribute to the carcinogenic potency of this compound.

4-Nitroquinoline-1-oxide↗
PubMed1993

Negative autoregulation by the Escherichia coli SoxS protein: a dampening mechanism for the soxRS redox stress response.

The soxRS redox stress regulon of Escherichia coli is triggered in two stages, with the activated SoxR protein stimulating the soxS gene, whose product then triggers transcription of approximately 10 promoters. Genetic and biochemical experiments presented here show that SoxS protein also limits soxS transcription in vivo and binds the soxS promoter in vitro.

Bacterial Proteins↗