Chaperone Hsp31 contributes to acid resistance in stationary-phase Escherichia coli

Hsp31, the product of the sigmaS - and sigmaD -dependent hchA gene, is a heat-inducible chaperone implicated in the management of protein misfolding at high temperatures. We show here that Hsp31 plays an important role in the acid resistance of starved Escherichia coli but that it has little influence on oxidative-stress survival.     

Escherichia coli xth mutants are hypersensitive to hydrogen peroxide.

Escherichia coli mutants lacking exonuclease III (xthA) are exceptionally sensitive to hydrogen peroxide. They are killed by H2O2 at 20 times the rate of wild-type bacteria and at 3 to 4 times the rate of recA cells. This is the first clear phenotypic sensitivity reported for xth- E. coli and should aid in clarifying peroxide-induced lethality and the in vivo role of exonuclease III.     

Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli.

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.     

NAD(P)H oxidation by hydrogen peroxide in Escherichia coli

A protein fraction from Escherichia Coli soluble extracts contain a NAD(P)H:hydrogen peroxide oxidoreductase activity. This activity is compared to and found to be distinct from well-known E. Coli enzymes involved in the protection from peroxides: hydroperoxidase I (HPI) and its o-dianisidine peroxidase component and the alkyl hydroperoxide reductase.     

Repair response of Escherichia coli to hydrogen peroxide DNA damage.

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.     

Factors affecting catalase level and sensitivity to hydrogen peroxide in Escherichia coli.

Composition of the culture medium, growth phase, and temperature play important roles in the sensitivity of Escherichia coli to H2O2. The medium and growth phase affected the sensitivity of the cells to H2O2 by modifying the amount of catalase synthesized by them, whereas the effect of temperature was due to the thermolability of the enzyme. Since catalase is unstable in the presence of its substrate, the correlation between the catalase level in the cells and their sensitivity to H2O2 could be observed only when the H2O2 concentration was not excessive in proportion to the amount of catalase.     

Transfer of the delta (argF-lac)U169 mutation between Escherichia coli strains by selection for a closely linked Tn10 insertion

To facilitate construction of mutants harboring delta lac for use in gene fusion studies, strains were constructed that carry the transposon Tn10 next to the well defined lac deletion U169. This deletion can now be moved to other Escherichia coli strains in transductional or conjugational crosses by selecting resistance to tetracycline.     

Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli.

The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Two sources of endogenous hydrogen peroxide in Escherichia coli

Mechanisms of hydrogen peroxide generation in Escherichia coli were investigated using a strain lacking scavenging enzymes. Surprisingly, the deletion of many abundant flavoenzymes that are known to autoxidize in vitro did not substantially lessen overall H₂O₂ formation. However, H₂O₂ production diminished by 25–30% when NadB turnover was eliminated. The flavin‐dependent desaturating dehydrogenase, NadB uses fumarate as an electron acceptor in anaerobic cells. Experiments showed that aerobic NadB turnover depends upon its oxidation by molecular oxygen, with H₂O₂ as a product. This reaction appears to be mechanistically adventitious. In contrast, most desaturating dehydrogenases are associated with the respiratory chain and deliver electrons to fumarate anaerobically or oxygen aerobically without the formation of toxic by‐products. Presumably, NadB can persist as an H₂O₂‐generating enzyme because its flux is limited. The anaerobic respiratory enzyme fumarate reductase uses a flavoprotein subunit that is homologous to NadB and accordingly forms substantial H₂O₂ upon aeration. This tendency is substantially suppressed by cytochrome oxidase. Thus cytochrome d oxidase, which is prevalent among anaerobes, may diminish intracellular H₂O₂ formation by the anaerobic respiratory chain, whenever these organisms encounter oxygen. These two examples reveal biochemical and physiological arrangements through which evolution has minimized the rate of intracellular oxidant formation.     

Hypochlorous acid stress in Escherichia coli: resistance, DNA damage, and comparison with hydrogen peroxide stress.

We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions. HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase. Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H2O2). In stationary phase, a mutation in rpoS, which controls the expression of starvation genes including those which protect against oxidative stress, renders the cells hypersensitive to killing by HOCl. RpoS-regulated genes responsible for this sensitivity were dps, which encodes a DNA-binding protein, and, to a lesser extent, katE and katG, encoding catalases; all three are involved in resistance to H2O2. In exponential phase, induction of the oxyR regulon, an adaptive response to H2O2, protected against HOCl exposure, and the oxyR2 constitutive mutant is more resistant than the wild-type strain. The genes involved in this oxyR-dependent resistance have not yet been identified, but they differ from those primarily involved in resistance to H2O2, including katG, ahp, and dps. Pretreatment with HOCl conferred resistance to H2O2 in an OxyR-independent manner, suggesting a specific adaptive response to HOCl. fur mutants, which have an intracellular iron overload, were more sensitive to HOCl, supporting the generation of hydroxyl radicals upon HOCl exposure via a Fenton-type reaction. Mutations in recombinational repair genes (recA or recB) increased sensitivity to HOCl, indicative of DNA strand breaks. Sensitivity was visible in the wild type only at concentrations above 0.6 mg/liter, but it was observed at much lower concentrations in dps recA mutants.     

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.     

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.     

Function of the sigma(E) regulon in dead-cell lysis in stationary-phase Escherichia coli

Elevation of active sigma(E) levels in Escherichia coli by either repressing the expression of rseA encoding an anti-sigma(E) factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a sigma(E)-dependent fashion. We propose there is a novel physiological function of the sigma(E) regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.     

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli.

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.     

Structural and catalytic properties of a deletion derivative (delta 133-157) of Escherichia coli adenylate kinase

Escherichia coli adenylate kinase (AKe) as well as the enzyme from yeast and mitochondria differs from the muscle cytosolic variant (AK1) by an insertion of 25 amino acid residues that are missing in AK1. The extra sequence, highly homologous in "large" size variants, is situated between residues 133 and 157 in AKe. Removal of 25 codons in the corresponding adk gene resulted in expression of a modified form of adenylate kinase (delta 133-157 AKe) which still conserved 7% of the maximal activity of the wild-type protein. The apparent Km for nucleotide substrates was increased by a factor of 4.6 (ADP), 23 (ATP) or 43 (AMP) in delta 133-157 AKe when compared with the wild-type enzyme. The secondary structure of delta 133-157 AKe, as well as its thermal stability were very similar to the parent protein. However, the deleted protein was much more sensitive than the wild-type enzyme to inactivation by trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of trypsin digested delta 133-157 AKe revealed accumulation of several well defined fragments which were not observed in the case of wild-type enzyme. We conclude that the additional sequence, although necessary for expression of full activity in AKe, is not critical for catalysis. It is perhaps responsible for interaction of enzyme with other cellular components although a different mechanism of water shielding for large and small size variants of AK can be also envisaged.