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An osmoduric-saccharophilic fungus, identified as a strain of Eurotium rubrum Konig, Speikermann and Bremer and isolated from a bottle of syrup, showed optimum growth (i.e., increase in colony diameter) on Sabouraud's agar amended with 60% (w/v) sucrose (calculated aw = 0.964) and still grew near optimally at 110% (aw = 0.927). On glucose, fructose or arabinose, optimum growth occurred at 40% (w/v) (aw = 0.962, 0.962, and 0.954, respectively), but glucose supported better growth than did fructose or arabinose. In the presence of glycerol, optimum growth (i.e., increase in dry weight of mycelium) occurred at a 10% (v/v) concentration (aw = 0.972) and no growth occurred above 35% (aw = 0.878). In general, growth was better with 12-C > 6-C > 5-C > 3-C compounds. The fungus did not grow on concentrations of inorganic salts above 30%; growth on salts was best with (on a w/v basis) 10% KCl (aw = 0.957), 5% NaCl (aw = 0.972) or 10% CaCl2, (aw = 0.965). In the absence of either organic or inorganic solutes, there was essentially no growth. When sucrose and either KCl or NaCl were added together, growth was greater on a salt/sugar mixture than on the same concentration of salt alone, and, at equivalent calculated osmotic pressures and aw, sucrose alone supported better growth than did any salt/sugar mixture. These data indicate that the fungus has a requirement for, and a tolerance to, high solute concentrations. At equivalent osmotic pressures and aw, however, sugars supported greater growth than did inorganic salts.

This chapter reviews the state of the art of the general and specific criteria used to determine microbiological activity in soil and to emphasize the exact meaning of each measurement and, therefore, to facilitate its correct use. A plethora of living species inhabit soil and conduct almost all known metabolic reactions. Plant roots and soil fauna and microbiota contribute to the overall soil respiration; therefore, the measurement of soil respiration represents an index of the overall biological activity. Dehydrogenase activity is increased by flooding and by the addition of nutrients to soil. Valid measurements of the activities of enzymes accumulated in soil can be obtained when both the enzyme production and the assimilation of reaction products by growing microorganisms are excluded during the assay period. The interpretation of measurements of enzyme activity in soil is also limited by the difficulty of comparing data obtained by different methods.

The insecticidal toxins from Bacillus thuringiensis subspp. kurstaki (antilepidopteran), morrisoni strain tenebrionis (anticoleopteran), and israelensis (antidipteran) did not affect the growth of a variety of bacteria (8 gram-negative, 5 gram-positive, and a cyanobacterium), fungi (2 Zygomycetes, 1 Ascomycete, 2 Deuteromycetes, and 2 yeasts), and algae (primarily green and diatoms) in pure and mixed culture, as determined by dilution, disk-diffusion, and sporulation assays with purified free and clay-bound toxins. The insecticidal crystal proteins from B. thuringiensis subspp. kurstaki and israelensis had no antibiotic effect on various gram-positive bacteria.Key words: insecticidal toxins, Bacillus thuringiensis, microbiostatic, microbicidal.

Insecticidal proteins produced by various subspecies of Bacillus thuringiensis and bacterial transforming DNA bind rapidly and tightly on clays, both pure mined clay minerals and soil clays, and on humic acids extracted from soil. This binding reduces the susceptibility of these biomolecules, which retain their biological activity when bound, to microbial degradation. The persistence of bound insecticidal toxins may enhance the control of target pests, constitute a hazard to nontarget organisms, and result in the selection and enrichment of toxin-resistant target insects. The persistence of bound DNA has relevance to horizontal gene transfer in soil. Because of the large differences in the chemical composition and structure between these proteins and DNA, as well as between clays and humic acids, these studies can serve as models for the potential fate and effects of other biomolecules that will be introduced to soil from “factories” of transgenic plants and animals genetically engineered to produce vaccines, hormones, antibodies, toxins, pharmaceuticals, and other bioactive compounds.

The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle–toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC–Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstaki. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC–Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.Key words: flow cytometry, soil, insecticidal toxins, Bacillus thuringiensis, clay, silt.

The insecticidal toxins produced by Bacillus thuringiensis subspp. kurstaki and tenebrionis were resistant when bound on clays, but not when free, to utilization by pure and mixed cultures of microbes as sources of carbon and carbon plus nitrogen, and their availability as a nitrogen source was reduced. The bound toxins retained insecticidal activity both before and after exposure to microbes or pronase. The insecticidal activity of the toxins persisted for 40 days (the longest time evaluated) in nonsterile soil continuously maintained at the -33-kPa water tension and room temperature, alternately air dried and rewetted to the -33-kPa water tension, or alternately frozen and thawed, although alternate drying and wetting reduced the activity.

Bacillus subtilis and Agrobacterium radiobacter remained viable when exposed to Ni (1 × 10−4 M; ionic strength (μ) = 3 × 10−4) at pH values known to cause a change of the net negative charge of the cells to a net positive charge (charge reversal). The gross morphology, as determined by scanning electron microscopy, of these and other bacteria and of Saccharomyces cerevisiae was not altered in the presence of Ni, Cu, and Zn (1 × 10−4 M; μ = 3 × 10−4), which caused a charge reversal at pH values between 6.0 and 9.0. Similar results were obtained in the presence of Na and Mg, which did not cause charge reversal at the same μ and pH values. These results confirmed that cells remain viable when their surface charge is changed in the presence of some heavy metals at high pH values.Key words: heavy metals, electrokinetic properties, survival of bacteria.

Bt corn has been genetically modified to express the Cry1Ab protein of Bacillus thuringiensis to kill lepidopteran pests. Fluorescence microscopy and staining with toluidine blue indicated a higher content of lignin in the vascular bundle sheaths and in the sclerenchyma cells surrounding the vascular bundle in all ten Bt corn hybrids, representing three different transformation events, studied than of their respective non-Bt isolines. Chemical analysis confirmed that the lignin content of all hybrids of Bt corn, whether grown in a plant growth room or in the field, was significantly higher (33-97% higher) than that of their respective non-Bt isolines. As lignin is a major structural component of plant cells, modifications in lignin content may have ecological implications.

The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (Mr=132 kDa) and toxins (Mr=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na2CO3, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni.

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase.

The dietary intake of selenium (Se) has been shown to influence the development and expression of various biologic processes. This study examined the immunologic competence of lymphocytes from C57BL/6J mice maintained for 8 weeks on Se-deficient (0.02 ppm Se), normal (0.20 ppm Se, as sodium selenite), or Se-supplemented (2.00 ppm Se) Torula yeast-based diets. The ability of the cells to recognize alloantigens, to proliferate in response to stimuli, and to produce interleukin 2 (IL-2) was determined. Se deficiency significantly inhibited the ability of the lymphocytes to proliferate in response to allogeneic stimulation in the mixed lymphocyte reaction or to mitogen stimulation by phytohemagglutinin, whereas Se supplementation significantly enhanced both responses. In contrast, the amounts of IL-2 and interleukin 1 (IL-1) produced by lymphocytes and macrophages, respectively, removed from Se-deficient or Se-supplemented animals did not differ significantly from the amounts of IL-2 and IL-1 produced by cells removed from animals maintained on the control diet. These results suggest that the mechanism(s) responsible for the observed effects of Se on lymphocyte proliferation are independent of the levels of IL-2 or IL-1.

Selenium (Se) is an essential nutritional factor with a chemopreventive potential. This study examined the ability of C57BL/6J mice, maintained for 8 weeks on Se-deficient (0.02 ppm Se), normal (0.20 ppm Se), or Se-supplemented (2.00 ppm Se) Torula yeast-based diets, to generate cytotoxic lymphocytes (CTL) and to destroy tumor cells. CTL were generated in vivo by intraperitoneal immunization with P815 cells and in vitro by allogeneic stimulation of cells from animals maintained on a normal diet in media supplemented with 1 x 10(-9) to 1 x 10(-6) M Se (as selenite). Lymphocytes from animals maintained on the Se-supplemented diet had a greater ability to destroy tumor cells than lymphocytes from animals maintained on the normal diet, whereas Se deficiency reduced the cytotoxicity. The effects on cytotoxicity were accompanied by parallel changes in the levels of lymphotoxin produced. The greatest enhancement of tumor cytodestruction occurred with supplementation of 1 x 10(-7) M Se, whereas with 1 x 10(-6) M there was inhibition of the cytotoxic responses. The stimulatory effect of Se occurred during the phase of CTL generation rather than during the lytic phase of cytotoxicity. These results indicated that Se supplementation enhances CTL generation and the ability of a host to destroy malignant cells, whereas Se deficiency has the opposite effect.

Sprays of commercial insecticidal preparations of the bacterium, Bacillus thuringiensis subsp. kurstaki (Btk), usually a mixture of cells, spores and parasporal crystals, have been used for the last 10 yr in Sardinia (Italy) to protect cork oak forests against the gypsy moth (Lymantria dispar L.). Until now, the protective antilepidopteran efficacies of each of the various spray treatments rather than their effects on the environment have been evaluated. Consequently, the persistence of Btk and its toxin, released in sprays (FORAY 48Bw), in soils of cork oak stands, located in Orotelli, Tempio Pausania and Calangianus (Sardinia), were investigated. In the Calangianus soil, the numbers of Btk remained essentially constant for 28 months (the longest time studied) after spraying, indicating that Btk was able to compete with the indigenous microbial community; the toxin was detected 28 months after spraying by immunological assay, but at a reduced concentration; and the larvicidal activity decreased essentially linearly to 14 months and then decreased markedly between 14 and 28 months. In the Tempio Pausania and Orotelli soils, cells of Btk were detected, whereas the toxin was not detected by immunological and larvicidal assays, 52 and 88 months (the longest times studied) after spraying, respectively. The numbers of Btk cells detected were probably too low to account for the presence of the toxin in all of the soils studied, as there was no correlation between numbers of Btk and toxin detected by immunological assays (correlation coefficient of20.66) in the Calangianus soil. Our results indicated that Btk and its toxin introduced into soils in sprays can persist for long periods (at least 88 months for Btk and at least 28 months for its toxin).

Publisher Summary Insecticidal proteins produced by various subspecies of Bacillus thuringiensis bound rapidly and tightly on clays and on humic acids extracted from soil. In the study explained in the chapter, this binding reduced the susceptibility of these proteins to microbial degradation and the bound proteins retained their insecticidal activity. Both purified proteins and proteins released from the biomass of transgenic Bt corn and in root exudates of growing Bt corn exhibited binding and persistence in soil. The toxin released in root exudates of Bt corn or from the degradation of the biomass of Bt corn is not toxic to a variety of organisms in soil. However, the presence of the toxin in the guts and casts of earthworms, grown with Bt corn and in soil amended with biomass of Bt corn, indicated again that the released toxin bound on surface-active particles in soil that protected the toxin from biodegradation, similar to the observation with purified toxins.

A plaque technique that uses antibiotic-resistant bacteria growing on antibiotic-containing agar for the assay lawn resulted in significantly better recovery of bacteriophages P1 of Escherichia coli and F116 of Pseudomonas aeruginosa from nonsterile soil than standard membrane filtration or centrifugation techniques. Adsorption of the phages on soil particles appeared to be involved in their recovery and survival in soil.Key words: bacteriophages, soil, antibiotic-resistant bacteria, enumeration, filtration, centrifugation.

This study examined the effect of dietary (2.00 ppm for 8 weeks) supplementation with selenium (as sodium selenite) on the ability of lymphocytes from aged (24-month-old), male, C57BL/6JNIA mice to respond to: (i) stimulation with mitogen (phytohemagglutinin) or alloantigen; (ii) develop into cytotoxic effector cells; and (iii) destroy tumor cells. Supplementation with selenium resulted in a significant increase in the ability of spleen lymphocytes from aged animals to undergo blastogenesis, as indicated by significantly higher amounts of nuclear incorporation of 3H-thymidine after stimulation with mitogen. The dietary regimen restored the age-related deficiency of the cells to respond to stimulation by nuclear DNA synthesis and cell proliferation, at least, to the level of cells from unsupplemented young adult animals. Furthermore, populations of in vivo, alloantigen-activated lymphocytes from Se-supplemented aged animals contained significantly higher numbers of cytotoxic lymphocytes than those from Se-normal aged animals, which resulted in an enhanced capacity to destroy tumor cells. The significant increase in the number of cytotoxic effector cells within these activated T-lymphocyte populations was probably the result of an enhanced clonal proliferation of cytotoxic precursors cells, followed by the differentiation of greater numbers of cytotoxic effector cells. This effect occurred in the absence of changes in the ability of the cells to produce IL-2, which confirmed our earlier observation that dietary supplementation with selenium does not affect the production of IL-2. The data suggested that selenium restores the age-related defect in cell proliferation through an increase in the number of high-affinity IL-2 receptors.

This study examined the effect of dietary (200 micrograms/d for 8 wk) supplementation with selenium (as sodium selenite) on the ability of human peripheral blood lymphocytes to respond to stimulation with alloantigen, develop into cytotoxic lymphocytes, and to destroy tumor cells, and on the activity of natural killer cells. The participants in the study were randomized for age, sex, weight, height, and nutritional habits and given selenite or placebo tablets; all participants had a selenium replete status as indicated by their plasma Se levels prior to supplementation. The data indicated that the supplementation regimen resulted in 118% increase in cytotoxic lymphocyte-mediated tumor cytotoxicity and 82.3% increase in natural killer cell activity as compared to baseline values. This apparently was related to the ability of the nutrient to enhance the expression of receptors for the growth regulatory lymphokine interleukin-2, and consequently, the rate of cell proliferation and differentiation into cytotoxic cells. The supplementation regimen did not produce significant changes in the plasma Se levels of the participants. The results indicated that the immunoenhancing effects of selenium in humans require supplementation above the replete levels produced by normal dietary intake.

This study examined the effect of dietary (200 μg/day for 8 weeks) supplementation with Se (as sodium selenite) on the ability of human peripheral blood lymphocytes to respond to stimulation with antigen, develop into cytotoxic lymphocytes, and to destroy tumor cells. Sixteen male and six female volunteers were matched by age (mean age = 26.2 years; range = 23–37), sex, weight, and nutritional habits, and given selenite or placebo tablets. Lymphocytes (2.5 × 106), isolated prior to and 8 weeks following supplementation, were activated by allogeneic stimulation with 8.3 × 105 mitomycin C-treated Ragi cells for 5 days. Lymphocyte mediated tumor cytotoxicity was evaluated using a 4h-51Cr release assay and effector: target cell ratios of 1.25–20:1. The results indicated that supplementation with selenite in humans results in a significant (118%) increase in the ability of a given lymphocyte population to destroy a fixed number of tumor cells, i.e., 45.57 ± 5.96 vs. 20.87 ± 2.62% cytotoxicity at 20:1 ratio. While the cytotoxic efficiency of both lymphocyte populations remained the same, the number of lymphocytes required to destroy 2×105 tumor cells decreased significantly after supplementation (1.53 × 106 ± 1.98 × 105 vs. 7.10 × 105 ± 1.49 × 105). The results indicate that supplementation with Se in humans results in a significant increase in the number of cytotoxic lymphocytes within a cell population.

Selenium (Se) is an essential nutritional factor that was shown previously by us to alter the kinetics of expression of high affinity (p55/p75) interleukin 2 receptors (IL-2R). This study shows that dietary (2 ppm for 8 weeks) or in vitro (1 x 10(-7) M) supplementation with Se (as sodium selenite) results in a significant upregulation of the expression of both the p55 and p70/75 IL-2 binding sites on the surface of concanavalin A-stimulated lymphocytes from C57BL/6J mice. This resulted in the formation of significantly higher numbers of high affinity IL-2R/cell with preservation of the normal ratio of high affinity to total IL-2 binding sites/cell. The high affinity IL-2R on cells from Se-supplemented animals functioned normally in terms of ligand binding and kinetics of IL-2 internalization, but their greater numbers/cell resulted in the internalization of significantly larger amounts of IL-2/cell. As Se supplementation results in an earlier expression of greater numbers of high affinity IL-2R, the presence of Se in the cell environment can result in an accelerated clonal expansion of activated lymphocytes.

Publisher Summary This chapter discusses some selected methods for the detection and assessment of ecological effects resulting from the release of genetically engineered microorganisms to the terrestrial environment. The chapter explains the methods and concepts developed to study the potential effects of genetically engineered microorganisms (GEMs) on microbial populations and microbe-mediated ecological processes in soil. The potential impacts of GEMs, unrelated to the purposes for which they are engineered, on the structure and function of the natural environments into which they are introduced constitute the bottom-line concern about the release of GEMs to the environment. The results in the chapter indicates that the introduction of GEMs into soil without the substrates on which the enzymatic products of the novel genes function or without the specific inhibitors to which the products confer resistance is insufficient to evaluate adequately the potential ecological effects of GEMs. In addition, the lack of appropriate theories and methodologies constitutes a major deficit in microbial ecology in general and, specifically, in risk assessment of the release of GEMS to the environment. The development of such theories and methodologies must be of high priority.

Selenium (Se) is an essential nutritional factor that has been shown to affect the development and expression of cell-mediated immune responses. This study shows that dietary (2 ppm for 8 weeks) or in vitro (1 x 10(-7) M) supplementation with Se results in a significant increase in the number of high affinity interleukin (IL) 2-binding sites (Kd of 10(-11) M) on the surface of concanavalin A-stimulated lymphocytes from C57BL/6J mice, whereas Se deficiency (0.02 ppm for 8 weeks) has the opposite effect. Se supplementation or deficiency apparently alters the kinetics of IL-2 receptor expression. Supplementation with Se in vivo or in vitro resulted in an earlier expression of high affinity IL-2 receptors, whereas Se deficiency resulted in a delayed expression of lower numbers of receptors. To exert its effect on IL-2 receptor expression, Se must be present or absent in the cell environment 8-24 hr after stimulation, and it most likely affects processes in the cytoplasmic and/or nuclear compartments of activated lymphocytes. Thus, in the presence of continuous immunologic stimulation, the presence or absence of Se in the cell environment can result in an accelerated or delayed clonal expansion of immunocompetent lymphocytes, respectively.

Selenium (Se) is an essential nutritional factor that affects the development and expression of cell-mediated immune responses directed toward malignant cells. These studies have shown that dietary (2 ppm for 8 wk) or in in vitro (1 x 10(-7)M) supplementation with Se (as sodium selenite) results in a significant enhancement of the proliferative responses of spleen lymphocytes from C57Bl/6J mice in response to stimulation with mitogen or antigen. Se deficiency (0.02 ppm for 8 wk) had the opposite effect. The alterations in the ability of the cells to proliferate, which occurred in the absence of changes in the endogenous levels of interleukin-2 (Il2) or interleukin 1, were apparently related to the ability of Se to alter the kinetics of expression of high-affinity Il2 receptors on the surface of activated lymphocytes. This resulted in an enhanced or delayed clonal expansion of the cells, and in an increased or decreased frequency of cytotoxic cells within a given cell population. The changes in tumor cytotoxicity were paralleled by changes in the amounts of lymphotoxin produced by the activated cells. Dietary Se modulations had a comparable effect on macrophage-mediated tumor cytodestruction. The results also suggested that Se exerts its effect 8-24 h after stimulation, and that it most likely affects processes in the cytoplasmic and/or nuclear compartments of activated lymphocytes.

A protein with a molecular mass of 66 kDa was isolated by a simple, rapid, and inexpensive method, using 3-N-morpholinopropanesulfonic acid, potassium thiocyanate, and dithiothreitol, from a mixture of spores, parasporal crystals, and cell debris of Bacillus thuringiensis subsp. kurstaki. The protein was active against the third instar larvae of Trichoplusia ni, was soluble in 19 mM Na2CO3, and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed as the insecticidal component of the 132-kDa protoxin of B. thuringiensis subsp. kurstaki by an enzyme-linked immunosorbent assay using antibodies prepared against the protoxin.

Publisher Summary This chapter discusses the method for studying bacterial gene transfer in soil by conjugation and transduction. Soil is unique among microbial habitats in that it is a structured environment with a high solid to water ratio. The discreteness of microhabitats in soil, the probability of genetic exchange may be less than the probability in ecosystems wherein water is continuous. Except for periods when soil is saturated with water, individual microhabitats are isolated by the surrounding pore space, movement of bacteria, transducing bacteriophages, and transforming DNA among the microhabitats. However, filamentous fungi appear to be able to bridge pore spaces between microhabitats, even when the pore spaces are not filled with water, as these fungi grow apically from mycelia that have a food and water base in a microhabitat and, therefore, are independent of the nutrient and water conditions surrounding the growing mycelia. Moreover, the extending mycelia probably have a surrounding water film in which bacteria or bacteriophages may be transported from one microhabitat to another.

Publisher Summary This chapter reviews and evaluates the relevance and quality of existing xenobiotic data bases and test methods for evaluating (1) direct and indirect effects (both adverse and beneficial) of xenobiotics on the soil microbial community, (2) direct and indirect effects of the soil microbial community on xenobiotics, and (3) “adequacy of test methods used to evaluate these effects and interactions. The distribution of a xenobiotic between environmental compartments depends on the chemodynamic properties of the compound and on the physicochemical properties of the soil, and it occurs across soil–water and soil–air interfaces and across biological membranes. In assessing the effects of xenobiotic compounds on soil microorganisms, it is necessary to decide which microbial processes or properties should be evaluated. When exposed to xenobiotic compounds, various segments of the soil microbial community are affected to different extents. The degree to which a xenobiotic affects microbial activities is largely dependent on the chemical, its dosage, method of application, and the particular physicochemical characteristics of the soil, such as soil type, temperature, water content, and pH. A systemic examination of those classes of xenobiotics that have not been evaluated for their effects on microorganisms needs to be performed to allow development of a predictive model for environmental risk assessment.

Selenium (Se) is an essential nutritional factor that was shown by us to alter the expression of the high affinity interleukin 2 receptor (Il2-R) and its subunits, cell proliferation, and clonal expansion of cytotoxic T-lymphocytes in mice. This study shows that dietary supplementation of Se-replete humans with 200 micrograms/d of sodium selenite for 8 wk, or in vitro supplementation with 1 x 10(-7) M Se (as sodium selenite), result in a significant augmentation of the ability of peripheral blood lymphocytes to respond to stimulation with 1 microgram/mL of phytohemagglutinin or alloantigen (mixed lymphocyte reaction) and to express high affinity Il2-R on their surface. There was a clear correlation between supplementation with Se and enhanced 3H-thymidine incorporation into nuclear DNA, preceded by enhanced expression of high affinity Il2-R. Supplementation with Se can apparently modulate T-lymphocyte mediated immune responses in humans that depend on signals generated by the interaction of interleukin 2 with Il2-R.

The adsorption and binding of amino acids (aspartic acid, cysteine, glycine, proline and arginine), ranging in molecular weight from 115 to 174 and in isoelectric point from pH 2.8 to 10.8, to montmorillonite [M] and kaolinite [K] homoionic to H, Na, Ca, Zn, La or Al were studied in unbuffered suspensions. Aspartic acid was adsorbed and bound on M homoionic to Ca or Zn but only adsorbed to K homoionic to Ca or Zn. Cysteine was only adsorbed on M homoionic to Al and adsorbed and bound on M homoionic to Zn and on K homoionic to H or Zn. Proline was adsorbed and bound on M homoionic to H, Ca or Zn and on K homoionic to H; it was adsorbed but not bound on both M and K homoionic to Al. Arginine was adsorbed and bound on M homoionic to H or Al and only adsorbed on K homoionic to Ca or Zn. Glycine was not bound on any of the clays. The amounts of amino acid adsorbed and bound (measured by the loss of amino acid from solution) and the class (Giles et al., 1974a and b) of both the adsorption and binding isotherms (retention against ultimate washing with water) were dependent on the type of amino acid, the type of clay, the type of cation predominant on the clays and the basicity or the additional function moiety (e.g. carboxyl, thiol, guanido) of the amino acid. The relative values of the non-standard free energy of clay-amino acid complexes that had isotherms of the Giles C-class were dependent on the type and molecular weight of the amino acid and on the exchangeable cation on the clay.

Studies were conducted to determine whether the differential toxicities of the two valence states of Cr (+3 and +6) occur with fungi as they do with bacteria as noted in previous studies. It was found that mycelial growth rates were inhibited more by Cr/sup 6 +/ than by Cr/sup 3 +/. Hexavalent Cr was also more toxic than equivalent trivalent Cr to spore formation and spore germination of the fungi tested. The greater toxicity of hexavalent Cr than of trivalent Cr to fungi is in agreement with the results observed with the bacterial studies. (JMT)

The respiration of three phenotypes of Histoplasma capsulatum, the causal agent of histoplasmosis in humans, was markedly reduced by low concentrations of montmorillonite but was reduced less by even higher concentrations of kaolinite or attapulgite (palygorskite). The reduction in respiration followed a pattern that suggested saturation-type kinetics: an initial sharp reduction that occurred with low concentrations of clay (0.01 to 0.5% [wt/vol]), followed by a more gradual reduction with higher concentrations (1 to 8%). Increases in viscosity (which could impair the movement of O2) caused by the clays were not responsible for the reduction in respiration, and the clays did not interfere with the availability of nutrients. Scanning electron microscopy after extensive washing showed that the clay particles were tightly bound to the hyphae, suggesting that the clays reduced the rate of respiration of H. capsulatum by adhering to the mycelial surface and, thereby, interfered with the movement of nutrients, metabolites, and gases across the mycelial wall.

In vivo exposure of mice to lidocaine (0.25 mg/10 g body weight 4 times a day for 7 days) resulted in impairment of immunocompetent cell function. Spleen lymphocytes removed from animals immediately and 3 days after lidocaine exposure showed changes in their surface charge properties, inhibition of blastogenesis in response to concanavalin A and lipopolysaccharide, and inhibition of antigen-stimulated activation as measured by the mixed lymphocyte reaction. Lymphocytes from animals sensitized to keyhole limpet hemocyanin showed a significantly lower capacity to produce macrophage migration inhibitory factor 8 days after termination of exposure to lidocaine. Animals exposed to the drug were unable to accumulate an adequate number of immunocompetent cells at the site of challenge with a foreign substance (i.e. dextran), and the ability of the animals to destroy tumor cells nonspecifically and specifically was also impaired. The results indicated that chronic exposure to lidocaine resulted in impairment of lymphocyte function, even in the subsequent absence of the drug, and in significant changes in the expression of the immune response.

Volatile compounds evolved from germinating seeds of slash pine, bean, cabbage, corn, cucumber, and pea were evaluated for their ability to support growth of microorganisms in liquid mineral salts media lacking a carbon source. Growth of eight bacteria was measured turbidimetrically and of six fungi as dry weight of mycelium.Volatiles caused increased growth of Pseudomonas fluorescens, Bacillus cereus, Erwinia carotovora, Agrobacterium tumefaciens, A. radiobacter, Rhizobium japonicum, Mucor mucedo, Fusarium oxysporum f. conglutinans, Trichoderma viride, and Penicillium vermiculatum but not of Sarcina lutea, Serratia marcescens, Chaetomium globosum, or Schizophyllum commune. Spores of Trichoderma viride showed higher germination in the presence of volatiles.Effects on growth were apparent only during the first 3 or 4 days after planting the seeds. Killed or dried seeds had no effect. The volatiles did not support microbial growth in the absence of nitrogen nor did they supply growth factors. Passing volatiles through KMnO4 or hydrazone reduced growth of the bacteria, indicating that oxidizable organic compounds, primarily aldehydes, were the active components. The volatiles were not absorbed by sterile soil, clay minerals, or water, but they were absorbed by non-sterile soil and activated charcoal.

The toxicity of a combination of nickel (Ni) and copper (Cu) toward growth of heterotrophic microorganisms was greater than the sum of the toxicity of each metal individually. This synergism between Ni and Cu was potentiated at acidic pH levels, with the potentiation of the toxicity being, for some organisms, an effect of the acidic pH on the toxicity of the Ni, rather than of the Cu, component. For others, however, the potentiation was an effect of acidic pH on both the Ni and Cu components. The potentiation of the Ni-Cu synergistic interaction at acidic pH levels has relevance to the deposition of acid precipitation into environments contaminated with these metals. Furthermore, the occurrence of such synergistic interactions and their mediation by pH should be considered in the methodologies used to establish criteria for tolerable levels of metals in the environment.

In vitro exposure of mouse lymphocytes and macrophages for 24 h to noncytotoxic doses of lidocaine (10(-4) to 10(-6)M) resulted in inhibition of random macrophage motility and in an interference with the production of macrophage migration inhibitory factor or with its interaction with the cell surface. The effects of lidocaine, membrane-stabilizing local anesthetic, were related to its concentration in the medium and to its ability to interact with the cell surface and cause changes in the ionic configuration of the plasma membrane. The drug conferred permanent changes on the surface of lymphocytes at all concentrations tested, but changes in the surface of macrophages induced by 10(-5) and 10(-6)M lidocaine were reversible. The presence of noncytotoxic doses of lidocaine in the cellular environment resulted in significant changes in cellular functions that appeared to be related to the ability of the drug to interact with cell membranes in a manner determined by the specific properties of the cell.

The clay minerals, kaolinite (K) and montmorillonite (M), suspended in either distilled water (DW) or a minimal medium (M-9), were better adsorbents for coliphages T1 or T7 than were bacteria (including early log, late log, or stationary phase cultures of the hosts), actinomycetes, and yeasts. Except for the host bacteria, the microbial cells (regardless of their type, phase of growth, viability, weight or number of cells, and volume of the suspension medium) adsorbed few or no coliphages. Although early log phase cultures (3 h) ofEscherichia coli B, suspended in DW, adsorbed an appreciable amount of T1 (94%), washing the cells with DW reduced the amount of T1 adsorbed (48%); 3-h cultures ofE. coli B/1,5, suspended in DW, adsorbed 15% of a T7 inoculum, and washing the cells with DW reduced the amount of T7 adsorbed to 1%. There was appreciable adsorption (35 to 97%) of both coliphages (with the exception of T1 on K) to 1 mg K or M suspended in either DW or M-9. These results suggest that clays are more important than microbes as adsorbents of viruses in environments of low ionic strength and that microbes do not inactivate coliphages T1 and T7.

The toxicity of lead (Pb) to fungi in pure culture was influenced by several abiotic factors: pH, inorganic anions, clay minerals, and particulate (humic acid) and soluble organic matter. The toxicity of Pb was potentiated under acidic conditions (pH 5 and 6), and phosphate or carbonate anions reduced the toxicity, apparently as a result of the formation of sparingly soluble Pb salts. Clay minerals (montmorillonite greater than attapulgite greater than kaolinite) and particulate humic acid protected against the toxicity of Pb, presumably as the result of sorption, by cation exchange of the Pb to the exchange complexes, which reduced its availability for uptake by the fungi. Soluble organics, such as tryptone, yeast extract, cysteine, succinic acid, and increasing concentrations of neopeptone, also reduced the toxicity of Pb.