Changes in Protein Synthesis as a Consequence of Heme Depletion in Escherichia coli

Two-dimensional gel electrophoresis and N-terminal amino acid sequence determination were used to compare the protein synthesis of exponentially growing Escherichia coli with heme-deficient cells. Mutation of the E. coli hemA gene encoding glutamyl-tRNA reductase resulted in the absence of detectable amounts of heme. As a consequence of heme deficiency, the induction of tryptophanase (trpA), citrate synthase (gltA), and aldehyde dehydrogenase (aldA) and the repression of enolase (eno) and phosphoglycerate kinase (pgk) were observed. All induced genes are under the control of the catabolite repressor protein Crp. The observed changes in gene expression as a consequence of heme depletion are discussed. Received: 19 March 1998 / Accepted: 28 April 1998     

Polymorphism and selection of rpoS in pathogenic Escherichia coli

Background  

Though RpoS is important for survival of pathogenic Escherichia coli in natural environments, polymorphism in the rpoS gene is common. However, the causes of this polymorphism and consequential physiological effects on gene expression in pathogenic strains are not fully understood.     

Role of rpoS in Acid Resistance and Fecal Shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator ς^S and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10¹ to 10⁴ CFU, we found the wild-type strain in feces of mice given lower doses (10² versus 10³ CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10⁴ CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P ≤ 0.05). Thus, ς^S appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.     

Impact of rpoS deletion on the proteome of Escherichia coli grown planktonically and as biofilm

To investigate the role of rpoS in gene expression of Escherichia coli cells grown as biofilms, we compared the proteomes of a rpoS mutant and the wild-type strain. Experiments were performed on planktonic cells (in exponential or stationary growth phase) and biofilms developed on glass wool. Spot-by-spot comparison of gels obtained from biofilm and planktonic wild-type organisms showed that the intensity of between 22 and 30% of detected spots was affected by the growth mode, depending of the control used. Principal component analysis, used to interpret the variations in protein spot densities, discriminated exponential-phase cells (wild-type and mutant) from the other incubation conditions and secondarily 72-old cultures. The statistical analysis demonstrated that the rpoS mutation did not significantly modify the proteome of exponential-growth phase cells, the differences involving only 3% of the proteome. However, increasing the incubation time from 8 to 72 h noticeably increased the number of changed proteins. A cluster analysis showed that RpoS plays a role in the special nature of the gene expression of biofilm cells but lower than in stationary-phase bacteria. We identified 35 rpoS-regulated proteins that were already or not described as controlled by this sigma factor. For some of them, the mode of regulation by RpoS was obviously dependent on the culture condition (planktonic vs biofilm).     

Loss of Colicinogeny in Escherichia coli Strains Infected by Certain Resistance Factors

The original observation that in wild-type colicinogenic Escherichia coli strains the introduction of some R factors abolish their colicin production was studied in certain col(+) strains bearing well-defined col factors. Two resistance (R) factors were used and introduced by conjugation in these strains, namely the 222 factor of Watanabe and a Salmonella typhimurium ST factor (coding for resistance to streptomycin and tetracycline only). The introduction of above mentioned R factors abolished the colicin production of col(+) strains most probably by elimination of col factors. All col factors, however, were not equally susceptible to elimination by the R factors tested, since colicin production in ML strains was abolished by infection by the 222 factor but not by the R factor of S. typhimurium ST, which is able to eliminate other col factors.     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.     

Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Naturalized soil Escherichia coli populations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soil E. coli strains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day⁻¹) than that of MG1655 (0.85 day⁻¹). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among the E. coli strains. All E. coli strains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman''s ρ = −1.0; P = 0.02). De novo trehalose synthesis was further determined for 15 E. coli strains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. Most E. coli strains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soil E. coli strains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).     

Role of rpoS in the regulation of glyoxalase III in Escherichia coli

Methylglyoxal is an endogenous electrophile produced in Escherichia coli by the enzyme methylglyoxal synthase to limit the accumulation of phosphorylated sugars. In enteric bacteria methylglyoxal is detoxified by the glutathione-dependent glyoxalase I/II system, by glyoxalase III, and by aldehyde reductase and alcohol dehydrogenase. Here we demonstrate that glyoxalase III is a stationary-phase enzyme. Its activity reached a maximum at the entry into the stationary phase and remained high for at least 20 h. An rpoS- mutant displayed normal glyoxalase I and II activities but was unable to induce glyoxalase III in stationary phase. It thus appears that glyoxalase III is regulated by rpoS and might be important for survival of non-growing E. coli cultures.     

Identification and Classification of Two-component Systems That Affect rpoS Expression in Escherichia coli

The rpoS-encoded σ^S subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.     

Identification and classification of two-component systems that affect rpoS expression in Escherichia coli

The rpoS-encoded sigmaS subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.     

Variation in Resistance to High Hydrostatic Pressure and rpoS Heterogeneity in Natural Isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Genotype-by-environment interactions influencing the emergence of rpoS mutations in Escherichia coli populations

Polymorphisms in rpoS are common in Escherichia coli. rpoS status influences a trade-off between nutrition and stress resistance and hence fitness across different environments. To analyze the selective pressures acting on rpoS, measurement of glucose transport rates in rpoS+ and rpoS bacteria was used to estimate the role of F(nc), the fitness gain due to improved nutrient uptake, in the emergence of rpoS mutations in nutrient-limited chemostat cultures. Chemostats with set atmospheres, temperatures, pH's, antibiotics, and levels of osmotic stress were followed. F(nc) was reduced under anaerobiosis, high osmolarity, and with chloramphenicol, consistent with a reduced rate of rpoS enrichment in these conditions. F(nc) remained high, however, with alkaline pH and low temperature but rpoS sweeps were diminished. Under these conditions, F(sp), the fitness reduction due to lowered stress protection, became significant. We also estimated whether the fitness need for the gene was related to its regulation. No consistent pattern emerged between the level of RpoS and the loss of rpoS function in particular environments. This dissection allows an unprecedented view of the genotype-by-environment interactions controlling a mutational sweep and shows that both F(nc) and F(sp) are influenced by individual stresses and that additional factors contribute to selection pressure in some environments.     

Plasmid DNA supercoiling and gyrase activity in Escherichia coli wild-type and rpoS stationary-phase cells

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.