The use of gel-stabilized gradient plates to map the responses of microorganisms to three or four environmental factors varied simultaneously

The Szybalski wedge plate technique has been used to map the responses of bacteria to up to 4 simultaneously varying environmental factors. 2-dimensional NaCl-pH gradient plates were used throughout. Sets of the latter were incubated at a range of temperatures to provide a third dimension. A fourth variable, nitrate concentration, was investigated by incorporating it homogeneously in sets of NaCl-pH gradient plates.     

Detection of growth of Escherichia coli on two-dimensional diffusion gradient plates

One drawback of using two-dimensional diffusion gradient plates, the subjective visual assessment of microbial growth, has been overcome. Growth of Escherichia coli was detected with pH indicators in the medium or by staining growth with a biochemical stain, L-alanine- p -nitroanilide, or a respiratory dye, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride. Stained growth was scanned with a laser densitometer and traces combined in a computer to give a three-dimensional semi-quantitative representation of growth over the gradient plate.     

Use of gradient plates to study combined effects of temperature, pH, and NaCl concentration on growth of Monascus ruber van Tieghem, an Ascomycetes fungus isolated from green table olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35 degrees C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30 degrees C but not at 35 degrees C. Growth was better at 30 and 25 degrees C as judged by the larger extent of the plates covered by mycelium compared with that at 35 degrees C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35 degrees C, whereas at 35 degrees C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Method for Investigation of Competition between Bacteria as a Function of Three Environmental Factors Varied Simultaneously

Competition between microorganisms as affected by temperature, pH, and the sodium chloride (NaCl) concentration was investigated by selective replication from gradient plates. Salmonella typhimurium was inhibited by Pseudomonas putida at 20 and 23°C but not 30 and 35°C. P. putida no longer grew at the extremes of pH and NaCl concentration, particularly at 30 and 35°C.     

A steep thermal gradient thermotaxis assay for the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans with its well-described nervous system is one of the multicellular organisms of choice to study thermotaxis. The neuronal circuitry for thermosensation has been analyzed at the level of individual cells. Two methods have previously been described to study the behavior of C. elegans with respect to temperature: 1) isothermal tracking assays and 2) linear thermal gradients (Hedgecock and Russell, 1975). Here we present a short linear thermal gradient assay which is faster and which allows statistical evaluation of different populations using a thermotaxis index. Thin agar plates are used on which a temperature gradient from about 10 degrees to 30 degrees is induced over the distance of about 5 cm. The short linear thermal gradient uses inexpensive materials so that multiple tests can be performed in parallel in a short period of time.     

Comparison of the effects of sodium chloride, pH and temperature on the growth of Listeria monocytogenes on gradient plates and in liquid medium

Gradient plates provide a convenient means of assessing the effect of sodium chloride and pH in combination with different temperatures on the growth of microorganisms. The effects of these variables on the growth of Listeria monocytogenes in gradient plates at four temperatures are compared with growth in a micro-well system. The growth-limiting conditions in the two systems are in good agreement, and also compare favourably with data in the literature. The gradient plates gave a good illustration of the concentrations of sodium chloride required to inhibit growth at different temperatures and pHs, although incubation time is limited to 72 h.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

Use of Gradient Plates To Study Combined Effects of Temperature, pH, and NaCl Concentration on Growth of Monascus ruber van Tieghem, an Ascomycetes Fungus Isolated from Green Table Olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35°C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35°C, whereas at 35°C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Focusing of pepsin in strongly acidic immobilized pH gradients

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.     

Investigation of the effect of combined variations in temperature, pH, and NaCl concentration on nisin inhibition of Listeria monocytogenes and Staphylococcus aureus.

Gradient plates were used to investigate the effects of varying temperature, pH, and sodium chloride (NaCl) concentration on nisin inhibition of Staphylococcus aureus and Listeria monocytogenes, Nisin was incorporated into the plates of 0, 50, 100, 250, and 500 IU ml -1. Gradients of pH (3.7 to 7.92) at right angles to NaCl concentration (2.1 to 7% [wt/vol]) were used for the plates, which were incubated at 20, 25, 30 and 35 degrees C. Growth on the plates were recorded by eye and by image analysis. The presence of viable but nongrowing cells was revealed by transfer to nongradient plates. Lower temperatures and greater NaCl concentrations increased the nisin inhibition of S. aureus synergistically. Increasing the NaCl concentration potentiated the nisin action against L. monocytogenes; the effect of temperature difference was not so apparent. Between pH 7.92 and ca. pH 5, a fall pH appeared to increase nisin's effectiveness against both organisms. At more acid pH values (ca. pH 4.5 to 5), the organisms showed resistance to both nisin and NaCl at 20 and 25 degrees C. Similar results were obtained with one-dimensional liquid cultures.     

Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli.

A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified. The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon. The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity. It can mediate sodium uptake when a transmembrane pH gradient is applied. Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient. An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested. These findings suggest that the antiporter is electrogenic both at acid and alkaline pH. The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range.     

Isoelectric focusing using non-amphoteric buffers in free solution: II. Apparatus and measures of pH stability

Isoelectric focusing of amino acids or proteins requires a time-invariant pH gradient. The maintenance, in an electric field, of such profiles with simple non-amphoteric buffers is possible in multicompartment cells using well determined concentration profiles. The method for determining these concentration profiles has been proposed in a preceding paper. A multicompartment cell has been built for the purpose of verifying the assumptions made. The technical characteristics of this cell will be described here. The time-stability of pH profiles obtained with sodium acetate/acetic acid buffers has been measured for concentration profiles determined so as to keep constant the transport number of each ion throughout the cell. Measured over a time span of 10 h and with a current density of 96 mA cm-2, the pH shift is, in the worst case, 0.009 pH unit/h. This corresponds to a much better stability than the one obtained with a constant concentration of sodium acetate in all compartments, and justifies the chosen concentration profiles based on the condition of constant transport numbers. The above mentioned method for the computation of the buffer concentration assumed a constant relative mobility of the ions. The experiments have shown the limits of this assumption, i.e. the variation of ionic mobilities with the ionic strength and the temperature diminishes the stability of the pH gradient. However, slight adjustments of the concentration in the end compartments allow some improvement of the stability. If the temperature could be precisely controlled in each compartment, a better pH stability than what has been achieved in these experiments should be reached. Two methods of compensation of the migration of ions in the end compartments (buffer renewal method and external recycling method) have been tested and will be discussed.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]     

Isotope and thermal effects in chemiosmotic coupling to the flagellar motor of Streptococcus

The torque generated by the flagellar motor of Streptococcus strain V4051 has been determined from rates of rotation of cells tethered by a single flagellum in media of different isotopic composition and temperature. Starved cells were energized artificially with either a potassium diffusion potential or a pH gradient. The torque increased linearly with protonmotive force. Identical results were obtained in media made with D2O or H2O; there was no solvent isotope effect. At a fixed protonmotive force, the torque was approximately constant over a temperature range of 4 degrees -38 degrees C. In cells chemotactically inert to changes in cytoplasmic pH, the motor turned counterclockwise when protons moved inward and clockwise when they moved outward. We conclude that the motor is a reversible engine driven by simple acid-base dissociation. A detailed model is discussed.     

Use of pH Gradient Plates for Increasing the Acid Tolerance of Salmonellae

Several strains of salmonellae survived higher concentrations of lactic acid after streaking on the surface of pH gradient plates. Most strains increased their acid tolerance by about 0.8 to 1.0 pH unit (9-to 10-fold), with Salmonella madelia showing the greatest differential, pH 5.2 in the wild strain and pH 4.2 after conditioning. The increased acid resistance was quickly lost after transferring to normal tryptic soy agar. Tests for survival in a liquid medium at pH values lower than those giving visible growth indicated that these pH values were bactericidal rather than bacteriostatic for both the wild and acid-conditioned strains.     

Chromatographic fractionation of Escherichia coli transfer RNA on a new support, naphthoyl-Sepharose.

The noncharged naphthoyl-Sepharose CL-6B has been prepared. Escherichia coli tRNA binds to this new adsorbent in 0.75 M ammonium sulphate at neutral pH at room temperature. Using a negative salt gradient, the tRNAs are eluted in a defined order. The chromatographic pattern is clearly different from those of other commonly used tRNA separation techniques.     

The use of concanavalin A in the purification or separation of multiple forms of brain hydrolases

Concanavalin A bound to Sepharose has been used for the purification of brain β-galactosidase, α-L-fucosidase, α-D-mannosidase, arylsulphatase and β-glucuronidase.0 Several factorsviz pH, temperature and concentration of α-methyl glucoside influenced the binding and elution of these enzymes. A lysosomal acid α-mannosidase and a cytosolic neutral mannosidase were separable by concanavalin A-Sepharose chromatography. Similarly lysosomal and microsomal β-glucuronidases were separable using gradient elution with α-methyl glucoside. The results indicate the usefulness of this lectin for the isolation of wide variety of enzymes under specified experimental conditions.     

Design and oxygen-transfer potential of a pulsed continuous tubular fermentor

A new type of continuous fermentor has been developed which features an agitation-aeration system based on pulsed flow across perforated plates the use of plug flow to achieve a concentration gradient. The influence of the agitation-aeration parameters (in this case the pulsed speed) has been measured, and mathematical models have been produced for the gas hold-up, the power dissipated in agitation and aeration, the oxygen-transfer rate, and efficiency. The oxygen transfer is high, up to 600 mmol/liter hr with a transfer efficiency much higher than that published for any other technique.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.     

The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.