Minimal reaction sets for Escherichia coli metabolism under different growth requirements and uptake environments

A computational procedure for identifying the minimal set of metabolic reactions capable of supporting various growth rates on different substrates is introduced and applied to a flux balance model of the Escherichia coli metabolic network. This task is posed mathematically as a generalized network optimization problem. The minimal reaction sets capable of supporting specified growth rates are determined for two different uptake conditions: (i) limiting the uptake of organic material to a single organic component (e.g., glucose or acetate) and (ii) allowing the importation of any metabolite with available cellular transport reactions. We find that minimal reaction network sets are highly dependent on the uptake environment and the growth requirements imposed on the network. Specifically, we predict that the E. coli network, as described by the flux balance model, requires 224 metabolic reactions to support growth on a glucose-only medium and 229 for an acetate-only medium, while only 122 reactions enable growth on a specially engineered growth medium.     

The mechanism of carbonate killing of Escherichia coli

AIMS: To define the mechanism of carbonate killing in Escherichia coli. METHODS AND RESULTS: Sodium carbonate (150 mM) and ethylenediaminetetracetic acid (EDTA, 60 mM) both killed E. coli K-12 when the pH was 8.5, but ammonium chloride (150 mM) was ineffective. EDTA was a 5-fold more potent agent than carbonate, but some of this difference could be explained by ionization. At pH 8.5, only 1.6% of the carbonate is CO(-2), but nearly 100% of the EDTA is EDTA(-2). CONCLUSION: As carbonate and EDTA had similar effects on viability, cellular morphology, protein release and enzymatic activities, the antibacterial activity of carbonate seems to be mediated by divalent metal binding. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle manure is often used as a fertilizer, and E. coli from manure can migrate through the soil into water supplies. Previous methods of eradicating E. coli were either expensive or environmentally unsound. However, cattle manure can be treated with carbonate to eliminate E. coli, and the cost of this treatment is less than $0.03 per cow per day.     

Membrane damage to Escherichia coli and bactericidal kinetics by the alternative complement pathway of channel catfish

1. Increased permeability of cytoplasmic membranes in Escherichia coli was a consequence of alternative complement pathway (ACP) activity of serum of channel catfish, Ictalurus punctatus. Evidence was provided by beta-galactosidase activity extracellularly when E. coli was incubated with catfish serum. 2. Lesions were detected on outer membranes of E. coli following exposure to catfish serum. 3. Catfish ACP induced a temporal sequence of pre-killing and killing phases. 4. Loss of cell viability, killing rate and cytoplasmic enzyme release increased with increasing serum concentrations. 5. By incubating E. coli with sera treated to remove complement, both release of cytoplasmic enzyme and bactericidal activity were eliminated. 6. Lethal activity associated with channel catfish ACP against Gram-negative bacteria was functionally comparable to that seen in mammalian and reptilian systems.     

A stochastic killing system for biological containment of Escherichia coli.

Bacteria with a stochastic conditional lethal containment system have been constructed. The invertible switch promoter located upstream of the fimA gene from Escherichia coli was inserted as expression cassette in front of the lethal gef gene deleted of its own natural promoter. The resulting fusion was placed on a plasmid and transformed to E. coli. The phenotype connected with the presence of such a plasmid was to reduce the population growth rate with increasing significance as the cell growth rate was reduced. In very fast growing cells, there was no measurable effect on growth rate. When a culture of E. coli harboring the plasmid comprising the containment system is left as stationary cells in suspension without nutrients, viability drops exponentially over a period of several days, in contrast to the control cells, which maintain viability nearly unaffected during the same period of time. Similar results were obtained with a strain in which the killing cassette was inserted in the chromosome. In competition with noncontained cells during growth, the contained cells are always outcompeted. Stochastic killing obtained by the fim-gef fusion is at present relevant only as a containment approach for E. coli, but the model may be mimicked in other organisms by using species-specific stochastic expression systems.     

The bactericidal effect of ultraviolet and visible light on Escherichia coli

The bactericidal radiation dosages at specific wavelengths in the ultraviolet (UV)-visible spectrum are not well documented. Such information is important for the development of new monochromatic bactericidal devices to be operated at different wavelengths. In this study, radiation dosages required to cause mortality of an Escherichia coli strain, ATCC 25922, at various wavelengths between 250 and 532 nm in the UV and visible spectrum were determined. Radiation at 265 nm in the UV region was most efficient in killing the E. coli cells and 100% mortality was achieved at a dose of 1.17 log mJ/cm(2). In the visible spectrum, the radiation dosages required for a one-log reduction of the E. coli cell density at 458 and 488 nm were 5.5 and 6.9 log mJ/cm(2), respectively. However, at 515 and 532 nm, significant killing was not observed at radiation dosage up to 7 log mJ/cm(2). Based on the cell survival data at various radiation dosages between 250 and 488 nm, a predictive equation for the survival of E. coli cells is derived, namely log(S/S(0)) = -(1.089 x 10(7) e(-0.0633lambda))D. The symbols, S(0), S, lambda, and D, represent initial cell density, cell density after irradiation, wavelength of the radiation and radiation dosage, respectively. The proportion of the surviving E. coli cells decreases exponentially with the increase in radiation dosage at a given wavelength. In addition, the radiation dose required for killing a certain fraction of the E. coli cells increases exponentially as the wavelength of radiation increases.     

The 6-phosphogluconate dehydrogenase reaction in Escherichia coli.

This study is an attempt to relate in vivo use of the 6-phosphogluconate dehydrogenase reaction in Escherichia coli with the characteristics of the enzyme determined in vitro. 1) The enzyme was obtained pure by affinity chromatography and kinetically characterized; as already known, ATP and fructose-1,6-P2 were inhibitors. 2) A series of isogenic strains were made in which in vivo use of thereaction might differ, e.g. a wild type strain versus a mutant lacking 6-phosphogluconate dehydrase, as grown on gluconate; a phosphoglucose isomerase mutant grown on glucose or glycerol. 3) The in vivo rate of use of the 6-phosphogluconate dehydrogenase reaction was determined from measurements of growth rate and yield and from the specific activity of alanine after growth in 1-14C-labeled substrates. 4) The intracellular concentrations of 6-phosphogluconate, NADP+, fructose-1,6-P2, and ATP were measured for the strains in growth on several carbon sources. 5) The metabolite concentrations were used for assay of the enzyme in vitro. The results allow one to calculate how fast the reaction would function in vivo if ATP and fructose-1,6-P2 were its important effectors and if the in vitro assay conditions apply in vivo. The predicted in vivo rates ranged down to as low as one-tenth of the actual rates, and, accordingly, one cannot yet draw firm conclusions about how the reaction is actually controlled in vivo.     

Genetic and biochemical requirements for chemotaxis to L-proline in Escherichia coli.

Chemotaxis to L-proline was examined by the capillary assay, using a set of Escherichia coli strains bearing well-defined defects in the enzymes of proline transport and utilization. Aspartate taxis was measured as a constitutive, control activity whose receptor and transducer requirements are known. Proline chemotaxis showed a pattern of induction more analogous to that of proline dehydrogenase than of that of proline transport, but chemotaxis to proline was eliminated by mutations eliminating either or both of these activities. No response to proline was observed in the absence of a proline concentration gradient or when succinate was provided as an oxidizable carbon source. These data suggest that the chemotactic response to proline results from a direct impact of proline oxidation on the energy metabolism of the cell.