Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Induction of Shiga toxin-converting prophage in Escherichia coli by high hydrostatic pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20 degrees C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i). the expression of rpoH, encoding the heat shock-specific sigma factor sigma(32), was also induced by high pressure; (ii). heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii). basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.     

Evidence for an evolutionary antagonism between Mrr and Type III modification systems

The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.     

Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.     

Characterization of the extracellular polymeric substances produced by Escherichia coli using infrared spectroscopic, proteomic, and aggregation studies

The aim of this study was twofold: first, to characterize the free extracellular polymeric substances (EPS) and bound EPS produced by Escherichia coli during different growth phases in different media, and then to investigate the role of the free EPS in promoting aggregation. EPS was extracted from a population of E. coli MG1655 cells grown in different media composition (Luria-Bertani (LB) and Luria-Bertani with the addition of 0.5 w/v% glucose at the beginning of the growth phase (LBG)) and at different growth phases (6 and 24 h). The extracted EPS was characterized using Fourier transform infrared spectroscopy and further identified using one-dimensional gel-based electrophoresis and tandem mass spectrometry. E. coli MG1655 was found to produce significantly lower amounts of bound EPS compared to free EPS under all conditions. The protein content of free EPS increased as the cells progressed from the exponential to stationary phase when grown in LB or LBG, while the carbohydrate content only increased across the growth phases for cells grown in LBG. FTIR revealed a variation in the different functional groups such as amines, carboxyl, and phosphoryl groups for free EPS extracted at the different growth conditions. Over 500 proteins were identified in the free EPS, with 40 proteins common in all growth conditions. Proteins with functionality related to amino acid and carbohydrate metabolism, as well as cell wall and membrane biogenesis were among the highest proteins identified in the free EPS extracted from E. coli MG1655 under all growth and media conditions. The role of bound and free EPS was investigated using a standardized aggregation assay. Bound EPS did not contribute to aggregation of E. coli MG1655. The readdition of free EPS to E. coli MG1655 resulted in aggregation of the cells in all growth conditions. Free EPS extracted from the 24 h E. coli MG1655 cultures grown in LB had the greatest effect on aggregation of cells grow in LBG, with a 30% increase in aggregation observed.     

Sensitization of Escherichia coli cells to oxidative stress by deletion of the rpoH gene, which encodes the heat shock sigma factor.

A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress. The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal. Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin. sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type.     

Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli

AIMS: To investigate the influence of the source of tryptone in the growth medium on the resistance of Escherichia coli to various types of oxidative stress. METHODS AND RESULTS: Cultures of Escherichia coli MG1655 were grown in Luria-Bertani (LB) medium at 37 degrees C to stationary phase, harvested, and subsequently subjected to various types of oxidative stress. A marked difference in oxidative stress sensitivity was observed depending on the origin of the tryptone in the LB medium used to grow the cultures. Cells harvested from LB containing tryptone from source x (LBx) were more sensitive to inactivation by the superoxide generating compound plumbagin and by t-butyl peroxide, and to growth inhibition by the lactoperoxidase enzyme system, than cells harvested from LB containing tryptone from source y (LBy). By monitoring expression of a panel of stress gene promotors linked to the gfp (green fluorescent protein) gene, and using Delta2-22 alkaline phosphatase as a probe for disulphide bridge formation from protein sulphydryl groups, it was demonstrated that a greater cytoplasmic oxidative stress existed in cells during growth in LBy than in LBx. CONCLUSIONS: Depending on the source of tryptone, bacteria may experience different levels of oxidative stress in tryptone-containing nonselective growth media. Although these levels of oxidative stress are subinhibitory, they may trigger a stress response that makes the bacteria more resistant to a subsequent exposure to a lethal or inhibitory level of oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the importance of controlling very subtle differences in composition of nonselective growth media in studies on bacterial physiology.     

Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.     

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ³², was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

Type 1 fimbriation and fimE mutants of Escherichia coli K-12.

We reexamined the influence of fimE, also referred to as hyp, on type 1 fimbriation in Escherichia coli K-12. We found that one strain used previously and extensively in the analysis of type 1 fimbriation, strain CSH50, is in fact a fimE mutant; the fimE gene of CSH50 contains a copy of the insertion sequence IS1. Using a recently described allelic exchange procedure, we transferred the fimE::IS1 allele from CSH50 to our present wild-type strain, MG1655. Characterization of this IS1-containing strain (AAEC137), together with another fimE mutant of MG1655 (AAEC143), led to two conclusions about the role of fimE. First, the formation of phase variant colony types, reported widely in strains of E. coli, depends on mutation of fimE, at least in K-12 strain MG1655. Here we showed that this phenomenon reflects the ability of fimE to stimulate the rapid inversion of the fim invertible element from on to off when the bacteria are grown on agar. Second, our analysis of fimE mutants, which is limited to chromosomal constructs, provided no evidence that they are hyperfimbriate. We believe that these results, which are at odds with a previous study using fim-containing multicopy plasmids, reflect differences in gene copy number.     

Using genomic sequencing for classical genetics in E. coli K12

We here develop computational methods to facilitate use of 454 whole genome shotgun sequencing to identify mutations in Escherichia coli K12. We had Roche sequence eight related strains derived as spontaneous mutants in a background without a whole genome sequence. They provided difference tables based on assembling each genome to reference strain E. coli MG1655 (NC_000913). Due to the evolutionary distance to MG1655, these contained a large number of both false negatives and positives. By manual analysis of the dataset, we detected all the known mutations (24 at nine locations) and identified and genetically confirmed new mutations necessary and sufficient for the phenotypes we had selected in four strains. We then had Roche assemble contigs de novo, which we further assembled to full-length pseudomolecules based on synteny with MG1655. This hybrid method facilitated detection of insertion mutations and allowed annotation from MG1655. After removing one genome with less than the optimal 20- to 30-fold sequence coverage, we identified 544 putative polymorphisms that included all of the known and selected mutations apart from insertions. Finally, we detected seven new mutations in a total of only 41 candidates by comparing single genomes to composite data for the remaining six and using a ranking system to penalize homopolymer sequencing and misassembly errors. An additional benefit of the analysis is a table of differences between MG1655 and a physiologically robust E. coli wild-type strain NCM3722. Both projects were greatly facilitated by use of comparative genomics tools in the CoGe software package (http://genomevolution.org/).     

CorA Affects Tolerance of Escherichia coli and Salmonella enterica Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg²⁺ transporter and at least three other inducible open reading frames belonged to the Mg²⁺ regulon, repression of the Mg stimulon by Mg²⁺ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni²⁺, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni²⁺-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression

Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.