Osmotic stress drastically inhibits active transport of carbohydrates by Escherichia coli

In intact Escherichia coli cells, severe osmotic stress almost totally inhibited active transport of carbohydrate by all of the systems known to transport carbohydrates in E. coli: group translocation (glucose), binding-protein mediated transport (maltose), proton symport (lactose), and sodium cotransport (melibiose). Detailed study of glucose transport showed that this inhibition of transport was not secondary to the inhibition of growth by osmotic stress, but rather that the inhibition of transport of a source of carbon and energy was sufficient to cause the complete inhibition of growth observed during severe osmotic upshock. Transport and growth inhibition did not result from cell death; upshocked cells were viable and metabolically active.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Abstract

Singlet oxygen (¹O₂) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe³⁺/O₂) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Osmotic deformation of red blood cell ghosts induced by carbohydrates

The osmotic response of bovine red blood cell ghosts to a series of sugars is studied by light scattering. The sealed and right-side-out ghosts are prepared by the procedure of Steck and Kant (Steck, T.L. and Kant, J.A. (1974) Methods Enzymol. 31, 172–180), swollen in a hypotonic phosphate-buffered saline solution and their size and shape determined by elastic and quasielastic light scattering. Different carbohydrates are then added to the suspending medium in order to examine the osmotic responses, and the osmotic deformation of ghosts is shown to be spherically symmetric. Having thus established the deformation behavior, we then rank the osmotic activity of a carbohydrate relative to a standard, i.e., raffinose. It is found that the osmotic response of the ghosts to sucrose is about the same as that to raffinose, and the response to the smaller carbohydrates simply follows the number of carbons in various sugars; glucose and fractose are about 1.7 times less effective than raffinose, and pentaerythritol and meso-erythritol are 2.3 times less effective. Glyceraldehyde, which is 3.6 times less effective than raffinose, is the least effective sugar analog among those that we have tested.     

Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners

Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.     

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.     

Mutator effects in Escherichia coli caused by the expression of specific foreign genes

Certain genes from Lactococcus lactis and Pseudomonas aeruginosa, including the nfxB gene, generate a mutator phenotype in Escherichia coli. The results of this study, together with those of a previous study, support conservation of regulatory sequences in E. coli and P. aeruginosa and suggest that some efflux pumps prevent mutagenicity by exporting mutagenic products of metabolism.     

Factors affecting the expression of foreign proteins inEscherichia coli

Summary A variety of factors affect the expression of foreign proteins inEscherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein inE. coli, location of the foreign protein inE. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene. This paper contains a critical review of these factors with the idea that a detailed understanding of them is the key to the development of strategies for the efficient large-scale production of foreign proteins inE. coli.     

Elevated Fis expression enhances recombinant protein production in Escherichia coli

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.     

Recombinant expression of selectively sulfated proteins in Escherichia coli

Although tyrosine sulfation is a post-translational modification widespread across multicellular eukaryotes, its biological functions remain largely unknown. This is in part due to the difficulties of synthesizing selectively sulfated proteins. Here we report the selective incorporation of sulfotyrosine into proteins in bacteria by genetically encoding the modified amino acid in response to the amber nonsense codon TAG. Moreover, we show that this strategy enables direct expression in Escherichia coli of sulfo-hirudin, previously inaccessible through recombinant methods. The affinity of sulfo-hirudin toward human thrombin is enhanced more than tenfold over that of desulfo-hirudin, suggesting that sulfo-hirudin may offer clinical advantages for use as an anticoagulant. This general approach to the biosynthesis of sulfated proteins should facilitate further study and application of tyrosine sulfation.     

Osmotic shock induces expression of Vibrio fischeri lux genes in Escherichia coli cells

The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells of Escherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC-RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.     

L-glutamate enhances the expression of Thermus maltogenic amylase in Escherichia coli

Escherichia coli BL21 (DE3) transformed with a thermostable Thermus maltogenic amylase (ThMA), isolated from a Gram-negative bacterium Thermus strain IM6501, grew well and efficiently produced ThMA in a complex medium but not in a chemically defined medium (DM). By supplementing L-glutamate to DM medium, both the specific growth rate and ThMA expression significantly increased. Alterations in the cellular responses of recombinant E. coli to L-glutamate were analyzed at the protein level by two-dimensional gel electrophoresis and mass spectrometry. The ppGpp synthase (RelA) was significantly reduced in cells grown with L-glutamate and was consistent with the low level of ppGpp, an indicator of stringent response. On the other hand, protein chain elongation factor (EF-Tu) and manganese-containing superoxide dismutase (MnSOD), which protects cells against oxidative damage, was significantly elevated by L-glutamate supplementation. These results indicate that L-glutamate enhances ThMA expression and increases the E. coli growth rate not only by overcoming the stringent response but also by increasing the synthesis of EF-Tu and MnSOD.     

Identification of the polyamine induced proteins in Escherichia coli.

The polyamine (PA)-induced proteins were identified by SDS-PAGE, and by two dimensional gel analysis in Escherichia coli strains. A large number of the PA-induced proteins were acidic. The molecular weights of the most highly induced proteins were 40 and 82 kDa proteins in the wild type and PA-auxotrophic mutant, respectively. Although a part of the PA-induced proteins were induced both in the wild type and PA auxotrophic mutant, most of them seem to be induced either in the wild type or mutant. These features may provide a foundation for evaluating the role of the PA-induced proteins relative to the physiology and environmental stress of Escherichia coli.     

Osmotic Reversal of Temperature Sensitivity in Escherichia coli

Forty temperature-sensitive mutants, unable to grow on tryptone or nutrient agar at 42 C, were isolated from Escherichia coli K-12. When 0.5% NaCl was added to the medium, 32 grew at the nonpermissive temperature. Several were tested with different amounts of NaCl added to tryptone broth; all grew best when the osmolality of the medium was between 400 and 1,000 milliosmolal. One of the mutants was studied in more detail. Sucrose, inositol, KCl, and MgCl(2), as well as NaCl, permitted growth at 42 C. Glycerol, however, had no effect. When shifted from 30 to 42 C without osmotic protection, the mutant stopped growing but did not lyse, die, or leak significant amounts of intracellular material. In a similar shift experiment, a second mutant leaked all of its trichloroacetic acid-soluble pools into the medium. The majority of the mutants were hypersensitive to certain antibiotics, indicating possible cell envelope defects.     

Construction of an Escherichia coli export-affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin glucanotransferase.

A novel export-affinity fusion vector employing the gene encoding cyclomaltodextrin glucanotransferase (CGTase; cgt) from Bacillus circulans var. alkalophilus (ATCC 21783) is described. CGTase binds to various sugar polymers, which makes it simple to purify it to near homogeneity in a single step. The CGTase fusion protein vector was constructed by deleting the translational stop codons from the gene encoding CGTase (cgt) by in vitro mutagenesis. As models, genes encoding Escherichia coli alkaline phosphatase (APase; phoA) and Bacillus stearothermophilus (ATCC 12980) alpha-amylase (BStA; amy) were fused to cgt. Overexpression of wild type CGTase and the hybrid proteins under the control of the lac promoter caused a 'leaky phenotype' in E. coli, the outer membrane became permeable, which enabled the adsorption of the fusion proteins directly from the culture medium onto alpha-cyclodextrin (alpha-CD) coupled agarose. The hybrid proteins were eluted from the column with alpha-CD solution under mild conditions at pH 7.5. The CGTase-APase' fusion had a good in vivo stability, whereas the CGTase-BStA' was less stable. In the latter case, according to protein sequencing, the proteolytically sensitive site was on the BStA' side of the fusion. The C-terminus of CGTase was stable against proteolysis as shown by narrow pH range isoelectric focusing. The fused enzymes retained their biological activities.     

Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical.

Two-dimensional gel analyses were made of proteins synthesized in Escherichia coli during various O2- -generating conditions. Nine proteins were constitutively synthesized over wild-type levels in superoxide dismutase (sodA sodB) double mutants. Addition of redox cycling agents such as paraquat and plumbagin at various concentrations induced up to 13 proteins in wild-type cells. Among these 13 were 5 of the 9 constitutively synthesized in the sodA sodB double mutants. Addition of these agents to the superoxide dismutase mutants in low micromolar concentrations induced an additional set of 14 proteins. The proteins induced included only five proteins that have been previously associated with stress responses, consisting of endonuclease IV (Nfo), three oxyR-regulated proteins, and one heat shock protein. O2- -mediated induction of the superoxide inducible (Soi) proteins in the wild type was independent of the oxyR+ gene for all but the three oxyR-regulated proteins. Analyses of proteins from three soi::lacZ gene fusions previously isolated (T. Kogoma, S. B. Farr, K. M. Joyce, and D. O. Natvig, Proc. Natl. Acad. Sci. USA 85:4799-4803, 1988) indicated the specific loss of one of these induced proteins in each fusion strain and the constitutive expression of some Soi proteins.     

Temperature-Sensitive Osmotic Remedial Mutants of Escherichia coli

A collection of temperature-sensitive mutants of Escherichia coli K-12 was examined for ability to grow at the restrictive temperature when the osmotic pressure of the medium was increased. Five of the fourteen mutants were found to be osmotic remedial. Four strains containing temperature-sensitive, osmotic-remedial mutations affecting aminoacyl-transfer ribonucleic acid synthetases were found to have altered permeability characteristics which may be attributable to changes in the lipopolysaccharide layer of the cell envelope at restrictive temperatures.