Iron uptake and iron limited growth of Escherichia coli K-12

Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 M without any of the known iron ionophores present. The growth rate increased between 0.05 and 2 M iron. Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 M iron. Under certain conditions iron uptake preceded growth of cells by more than an hour. During logarithmic growth the rate of iron uptake matched the growth rate. The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight.After consumption of the iron in the medium cells continued to grow with high rate for 1–2 generations. The iron uptake activity was increased during iron starvation.     

Dynamics of glucose uptake by single Escherichia coli cells

The fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was used to measure rates of glucose uptake by single Escherichia coli cells. When cell populations were exposed to the glucose analog, 2-NBDG was actively transported and accumulated in single cells to a steady-state level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation rate. The dependence upon substrate concentration could be described according to Michaelis-Menten kinetics with apparent saturation constant KM = 1.75 microM, and maximum 2-NBDG uptake rate= 197 molecules/cell-second. Specificity of glucose transporters to the analog was confirmed by inhibition of uptake of 2-NBDG by D-glucose, 3-o-methyl glucose, and D-glucosamine, and lack of inhibition by L-glucose. Inhibition of 2-NBDG uptake by D-glucose was competitive in nature. The assay for 2-NBDG uptake is extremely sensitive such that the presence of even trace amounts of D-glucose in the culture medium (approximately 0.2 microM) is detectable. The rates of single-cell analog uptake were found to increase proportionally with cell size as measured by microscopy or single-cell light scattering intensity. The assay was used to identify and isolate mutant cells with altered glucose uptake characteristics. A mathematical model was developed to provide a theoretical basis for estimating single-cell glucose uptake rates from single-cell 2-NBDG uptake rates. The assay provides a novel means of estimating the instantaneous rates of nutrient depletion in the growth environment during a batch cultivation.     

Analysis of threonine uptake in Escherichia coli threonine production strains

Threonine uptake in Escherichia coli wild-type and in threonine-producing strains decreased throughout threonine production. In contrast to previously published results, the SstT uptake system is not the sole serine/threonine permease in E. coli, since a novel transport system was detected in an sstT deletion strain.     

Effects of iron-limitation ofEscherichia coli on growth,the respiratory chains and gallium uptake

The effects of iron limitation on growth, the composition and function of the respiratory chains, and gallium uptake inEscherichia coli have been studied. Decreasing the iron concentration in a defined medium using Chelex resin gave lower growth yields in both continuous culture and prolonged batch culture. In the former, ironlimited (entering [Fe]2.0 M) cells exhibited diminished respiration rates, respiration-driven proton translocation quotients, and levels of non-haem iron and cytochromes. The cellular concentration of haemoproteinb-590 (a cytochromea ₁-like hydroperoxidase) decreased 20-fold on iron limitation, whilst a CO-binding pigment with an absorption maximum in the dithionite-treated form near 500 nm appeared. Gallium(III) (9 M) added to iron-limited, but not iron-sufficient, cultures diminished growth yields further; cells grown with low entering concentrations of iron took up less gallium than iron-sufficient cells. These results are attributed to the interference by gallium(III) with siderophore-mediated metal uptake. Gallium also stimulated iron uptake and was itself accumulated by iron-sufficient cells, suggesting that gallium(III) also affects the iron transport system(s) of low affinity.     

Extracellular factors affecting the adhesion ofPseudomonas fluorescens cells to glass surfaces

Two factors affecting the adhesionof Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, antiadhesin (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.     

Relation of potassium uptake to proton transport and activity of hydrogenases in Escherichia coli grown at low pH

Escherichia coli grown under anaerobic conditions in acidic medium (pH 5.5) upon hyperosmotic stress accumulates potassium ions mainly through the Kup system, the functioning of which is associated with proton efflux decrease. It was shown that H⁺ secretion but not glucose-induced K⁺ uptake was inhibited by N,N′-dicyclohexylcarbodiimide (DCC). The inhibitory effect of DCC on the H⁺ efflux was stronger in the trkA mutant with defective potassium transport. The K⁺ and H⁺ fluxes depended on the extent of hyperosmotic stress in the absence or presence of DCC. The decrease in external oxidation/reduction potential and H₂ liberation insensitive to DCC were recorded. It was found that the atpD mutant with nonfunctional F₀F₁-ATPase produced a substantial amount of H₂, while in the hyc mutant (but not the hyf mutant defective in hydrogenases 3 (Hyd-3) and 4 (Hyd-4)) the H₂ production was significantly suppressed. At the same time, the rate of K⁺ uptake was markedly lower in hyfR and hyfB-R but not in hycE or hyfA-B mutants; H⁺ transport was lowered and sensitive to DCC in hyf but not in hyc mutants. The results point to the relationship of K⁺ uptake with the Hyd-4 activity. Novel options of the expression of some hyf genes in E. coli grown at pH 5.5 are proposed. It is possible that the hyfB-R genes expressed under acidic conditions or their gene products interact with the gene coding for the Kup protein or directly with the Kup system.     

Cell yields of Escherichia coli during anaerobic growth on fumarate and molecular hydrogen

Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y _fum ^max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y _ATP ^max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Y_fum value of 4.8 g dry weight per mol fumarate was determined.     

Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH

Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium. In our studies, glycine betaine stimulated the growth rate of Escherichia coli K 12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2). Betaine also caused a reduction in the intracellular pools of K⁺ and low molecular weight thiols in E. coli growing both in medium with high osmolarity and at alkaline pH. These effects of betaine were absent at pH 7.0. In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 M N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E. coli. It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E. coli to betaine.     

Extracellular factors responsible for the adaptation ofPseudomonas fluorescens batch cultures to unfavorable conditions

The culture liquid filtrate of an exponential-phasePseudomonas fluorescens batch culture added to anotherP. fluorescens culture at the moment of inoculation was found (1) to prevent or diminish cell adsorption of the flask walls, (2) to enhance the intensity of cell respiration, (3) to shorten the period of adaptation of LB-grown cells to growth in glucose-containing mineral M9 medium, (4) to stimulate bacterial growth at supraoptimum temperature (36°C) and pH values (4.8 and 9.2), and (5) to decrease the death rate of bacteria at the supraoptimum growth temperature. These results were interpreted as indicating thatP. fluorescens cultures produce two types of regulatory exometabolites similar to those revealed earlier inEscherichia coli andBacillus subtilis cultures: the direct-action adaptogenic factorX ₁ capable of increasing bacterial resistance to unfavorable growth conditions (temperature and pH) and factor of accelerated adaptation to new media. Both factors are presumably low-molecular-weight hydrophilic nonprotein compounds.     

Relationships between Escherichia coli cells and the surrounding medium during survival processes

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 10(4) cells ml(-1). The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.     

Plasmid distribution in Escherichia coli urinary isolates with special reference to aerobactin and colicin production

Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified cases of urinary tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed an association of aerobactin and colicin production with the distribution of plasmid bands having a median value of 2.33 and 1.72 (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For colicin producers, the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains.     

DNA synthesis by Escherichia coli B/r/l synchronized and grown under conditions of slow growth

Escherichia coli B/r/l was synchronized by a novel method and its growth was followed in a minimal salts medium containing glucose, acetate, aspartate or succinate as the sole carbon source. Thymine incorporation experiments showed agreement with the Cooper-Helmstetter model for DNA synthesis, during the division cycle, both in glucose grown culture with a doubling time 57.5 min and in acetate, aspartate and succinate where the doubling time was extended up to 90 min. The ratio C/C+D was identical or close to that predicted by the model. Prolonged growth of the synchronized cultures prior to each experiment was practised in order to ensure their physiological state without causing any considerable deterioration of synchrony.     

F0 cysteine,bCys21, in the Escherichia coli ATP synthase is involved in regulation of potassium uptake and molecular hydrogen production in anaerobic conditions

The single cysteine in the b subunit of the membranous F₀ sector and the 19 cysteines in extramembranous F₁ sector of the Escherichia coli ATP synthase were replaced by alanine. When cells were grown under anaerobic conditions on glucose, the k _cat for ATP hydrolysis of membrane vesicles containing the bCys21Ala mutant enzyme, but not enzymes with other cysteine replacements, was lower, while ATP-driven H⁺ pumping was unchanged. However, the ATP-dependent increase in the number of accessible thiol groups in membrane vesicles was negated. Furthermore, K⁺ uptake and molecular hydrogen production by whole cells and protoplasts was greatly decreased. These results indicate a role for the F₀ subunit bCys21 in the functionality of F₀F₁ and coupling to other membranous activities under fermentative conditions.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

Study of the thermokinetic properties of copper(II) on Escherichia coli growth

By using an LKB2277 Bioactivity Monitor, stop-flow mode, the power-time curves of Escherichia coli at 37°C affected by Cu(II) were determined. Some parameters, such as growth rate constants k, inhibitory ratio I, the heat output Q_log in the log phase, and the generation times G were obtained. According to these parameters, we found that a low concentration of Cu(II) (0–20 μg/mL) had an promoting action on the growth of E. coli, but a high concentration of Cu(II) (40–100 μg/mL) had an inhibitory action. The toxicity of Cu(II) can also be expressed as the half-inhibitory concentration IC₅₀; the value is 69.7 μg/mL. The assay is quantitative, inexpensive, and versatile.     

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.