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.     

Adaptation of Escherichia coli to Elevated Sodium Concentrations Increases Cation Tolerance and Enables Greater Lactic Acid Production

Adaptive evolution was employed to generate sodium (Na⁺)-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na⁺ concentrations. The isolates also showed increased tolerance of K⁺, although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na⁺ tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na⁺. Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na⁺ tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.     

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 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.     

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.     

Synthetic reconstruction of extreme high hydrostatic pressure resistance in Escherichia coli

Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600–800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.     

High-pressure tolerance in Halobacterium salinarum NRC-1 and other non-piezophilic prokaryotes

In this study, we examined the high-pressure survival of a range of prokaryotes not found in high-pressure environments to determine the effects of adaptations to osmotic and oxidative stresses on piezo-resistance. The pressure survivals of Halobacterium salinarum NRC-1, Deinococcus radiodurans R1, and Chromohalobacter salexigens were compared to that of Escherichia coli MG1655. C. salexigens, which uses the compatible solute ectoine as an osmolyte, was as piezo-sensitive as E. coli MG1655, suggesting that ectoine is not a piezolyte. D. radiodurans R1 and H. salinarum NRC-1, both resistant to oxidative stress, were found to be highly piezo-resistant. H. salinarum NRC-1 showed nearly full survival after pressurization up to 400 MPa; a survival 3.5 log units higher than E. coli MG1655. This piezo-resistance was maintained in H. salinarum NRC-1 for pressurizations up to 1 h. We hypothesize that the high-pressure resistance of H. salinarum NRC-1 is due to a combination of factors including cell envelope structure and the presence of intracellular salts.     

Motility influences biofilm architecture in Escherichia coli

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).     

Bacterial Hemoglobins and Flavohemoglobins for Alleviation of Nitrosative Stress in Escherichia coli

Escherichia coli MG1655 cells expressing novel bacterial hemoglobin and flavohemoglobin genes from a medium-copy-number plasmid were grown in shake flask cultures under nitrosative and oxidative stress. E. coli cells expressing these proteins display enhanced resistance against the NO^· releaser sodium nitroprusside (SNP) relative to that of the control strain bearing the parental plasmid. Expression of bacterial hemoglobins originating from Campylobacter jejuni (CHb) and Vitreoscilla sp. (VHb) conferred resistance on SNP-challenged cells. In addition, it has been shown that NO^· detoxification is also a common feature of flavohemoglobins originating from different taxonomic groups and can be transferred to a heterologous host. These observations have been confirmed in a specific in vitro NO^· consumption assay. Protein extracts isolated from E. coli strains overexpressing flavohemoglobins consumed authentic NO^· more readily than protein extracts from the wild-type strain. Oxidative challenge to the cells evoked nonuniform responses from the various cell cultures. Improved oxidative-stress-sustaining properties had also been observed when the flavohemoglobins from E. coli, Klebsiella pneumoniae, Deinococcus radiodurans, and Pseudomonas aeruginosa were expressed in E. coli.     

Quantification and analysis of metabolic characteristics of aerobic succinate-producing Escherichia coli under different aeration conditions

The production of succinate by engineered Escherichia coli strains has been widely investigated. In this study, quantitative comparison of metabolic fluxes was carried out for the wild-type E. coli strain and a quintuple mutant strain QZ1111 that was designed for the production of succinate aerobically by knocking out five genes (ptsG, poxB, pta, sdhA, iclR) of the wild-type E. coli MG1655. Metabolic flux distributions of both strains were quantified by ¹³C-labeling experiments, together with the determination of physiological parameters and the expression level of key genes. The experimental results indicated that under the same aeration condition the fraction of oxaloacetate molecules originating from phosphoenolpyruvate was increased in E. coli QZ1111 compared to that in the wild-type E. coli MG1655. The glyoxylate shunt was likely activated in E. coli QZ1111 only under high aeration condition but repressed in other conditions, indicating that the deletion of the iclR gene could not completely remove the repression of the glyoxylate shunt with limited oxygen supply. Our results also suggested further genetic manipulation strategies to enhance the production yield of succinate.     

Comparative genomic hybridization detects secondary chromosomal deletions in Escherichia coli K-12 MG1655 mutants and highlights instability in the flhDC region

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655, we identified unintended secondary genomic deletions in the flhDC region in Δfnr, Δcrp, and ΔcreB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.     

Development of a Biofilm Production-Deficient Escherichia coli Strain as a Host for Biotechnological Applications

Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Δ(csgG-csgC); colanic acid biosynthesis and assembly, Δ(wcaL-wza); and type I pilus biosynthesis, Δ(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were ~16% and ~25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena.     

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.     

Effect of hydrostatic pressure on a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene

Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0–200 MPa. Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells. Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells. Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type. The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.     

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).

Results

Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.

Conclusions

Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.     

Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.