OBSERVATIONS ON THE ISOLATION OF SALMONELLAE FROM SELENITE BROTH

SUMMARY: Studies of growth curves of enterobacteria in selenite broth containing different carbohydrates showed that whereas mannitol and lactose brought about a steep fall in coli-aerogenes bacteria, mannitol improved the growth of Salm. typhi-murium . With mixed cultures of Cit. freundii I and Salm. typhi-murium the presence of lactose, utilizable by the former, adversely affected the viable count of the latter.
Comparative studies with routine faeces specimens showed that selenite broth was an efficient selective medium with MacConkey's agar; but much better results were obtained when it was combined with deoxycholate-citrate agar. Gentian violet introduced into selenite broth improved its selectivity for most Salmonella types when MacConkey's agar was used for final isolation.     

Comparison of Methods for the Isolation of Salmonella from Bone Meal

The efficiency of some methods for the isolation of Salmonella from bone meal was studied. Grinding of the granulated, industrial product was found to increase Salmonella recovery. An improvement in the efficiency of the pre-enrichment method was obtained by adding deoxycholate (1%) to mannitol broth. Selective enrichment in selenite cystine medium gave better results than in Kauffmann-Müller medium, when small inocula were used. Experimental data showed that, in the presence of large numbers of coliforms and very small numbers of Salmonella, the Kauffman-Müller medium offers better results, but, in the presence of smaller numbers of coliforms, selenite cystine is preferable. As solid selective medium, Brilliant Green Agar gave, in our laboratory, better results than S S medium.     

Detection of salmonellas in animal feeds by electrical conductance

A comparison was made between standard culture methods and electrical conductance using a Malthus AT Microbiological Analyser for the examination of animal feeds for salmonella. Conductance testing with a selenite cystine/trimethylamine-N-oxide/dulcitol medium resulted in the detection of salmonellas in 49 of 55 known positive animal feeds, 13 of 19 spiked feed samples and 36 of 47 salmonella cultures. Testing with a lysine decarboxylase/glucose medium gave significantly better results (P less than 0.05) than with selenite cystine medium but five lysine decarboxylase negative strains of salmonella were undetected. When both media were used in parallel all salmonella positive samples were detected. No difference was found between preenrichment in buffered peptone water containing trimethylamine/mannitol and that containing lysine/glucose. Positive detection criteria for selenite medium of conductance peak at greater than or equal to 500 microsiemens (microS) with a rate of change of greater than or equal to 60 microS/h or 400-499 microS with a rate of change of greater than or equal to 40 microS/h and for lysine medium with a peak of greater than or equal to 100 microS have been established. The method offers savings in media and operating costs over conventional standard culture methods, provides results within 48 h and is recommended for statutory feed monitoring purposes.     

Effect of carbohydrate source in selenite cystine trimethylamine oxide broth on the detection of salmonellas using the Bactometer

Ninety-five salmonellas and 40 non-salmonellas were screened in the Bactometer using the standard formulation for Easter and Gibson's selenite cystine trimethylamine oxide dulcitol broth and versions in which dulcitol was replaced by mannitol or deoxyribose. More strains of salmonellas exceeded the current detection criteria (magnitude 250, rate 25) when dulcitol was replaced by either mannitol or deoxyribose as carbohydrate source. Using mannitol, more non-salmonella strains exceeded the detection criteria than with either dulcitol or deoxyribose.     

Fluorescent light-induced chromosome damage in human IMR-90 fibroblasts. Role of hydrogen peroxide and related free radicals

Exposure of human fibroblasts (IMR-90) to cool-white fluorescent light causes chromatid breaks and exchanges. This chromatid damage is caused largely by the production of hydrogen peroxide (H2O2) since it can be prevented almost completely by the addition of catalase. In support of this conclusion, exogenous H2O2 is shown to induce chromatid breaks. The clastogenic amounts of H2O2 generated during light exposure are formed within the cell since cells illuminated in saline showed the same extent of damage as cells in culture medium. Addition of selenite to the cultures during light exposure significantly decreases the chromatid damage in a dose-related manner and may be necessary to maintain sufficient activity of glutathione peroxidase. The free hydroxyl radical, . OH, appears to be partially responsible for the light-induced chromatid damage. Of the free-radical scavengers tested, i.e., mannitol, vitamin E, and dimethyl sulfoxide, only mannitol, which scavenges . OH, significantly decreases the light-induced chromatid damage. Thus, both . OH and H2O2 formed within the cell during light exposure are agents that directly or indirectly cause chromatid damage.     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Catabolism of Fructose and Mannitol in Clostridium thermocellum: Presence of Phosphoenolpyruvate: Fructose Phosphotransferase, Fructose l-Phosphate Kinase, Phosphoenolpyruvate: Mannitol Phosphotransferase, and Mannitol l-Phosphate Dehydrogenase in Cell Extracts

Fructose and mannitol are fermented by Clostridium thermocellum in a medium containing salts and 0.5% yeast extract. The initial reaction in the catabolism of fructose was found to be the formation of fructose l-phosphate by phosphoenolpyruvate (PEP):fructose phosphotransferase which resembles the Kundig-Roseman phosphotransferase system. The phosphorylation of fructose l-phosphate to form fructose-1, 6-diphosphate is catalyzed by fructose l-phosphate kinase. Fructose-1, 6-diphosphate can be further metabolized by the Embden-Meyerhof pathway. The formation of both PEP:fructose phosphotransferase and fructose l-phosphate kinase is induced by growth in fructose medium. Mannitol catabolism was found to proceed by the phosphorylation of mannitol by PEP:mannitol phosphotransferase to form mannitol l-phosphate. Mannitol l-phosphate is converted to fructose 6-phosphate by a nicotinamide adenine dinucleotide-specific mannitol l-phosphate dehydrogenase. The fructose 6-phosphate formed in the reaction can enter the glycolytic scheme. The formation of both PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase is induced by growth in mannitol medium. Evidence is presented for the induction by mannitol of PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase in suspensions of fructose-grown cells.     

The uptake of mannitol by higher plants

Experiments with youngHordeum sativum andHelianthus annus plants showed that in the excretion of mannitol in the guttation liquid observed byGroenewegen andMills (1960) after uptake by the root system of plants, the osmotic concentration of mannitol in the nutrient medium and the temperature are significant. The beginning of mannitol excretion during guttation is accelerated considerably by the increase of the osmotic concentration of mannitol in the nutrient medium and the rising temperature. The osmotic concentration of mannitol is also important for the duration of mannitol excretion in the guttation liquid after transfer of the plants into a nutrient medium without mannitol. In the presence of mannitol in the nutrient medium water uptake by the root system and growth are inhibited and the tissues of the organs above ground and of the root system are dehydrated. The inhibitory effect of mannitol on the water uptake by the root system is immediate.     

Genetic and biochemical evidence for the involvement of a molybdenum-dependent enzyme in one of the selenite reduction pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.     

Genetic and Biochemical Evidence for the Involvement of a Molybdenum-Dependent Enzyme in One of the Selenite Reduction Pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.     

Isolation of Salmonellae From Food Samples: VI. Comparison of Methods for the Isolation of Salmonella From Egg Products

The recovery of salmonellae from egg products was studied, by use of three different enrichment procedures: (i) selenite broth, (ii) selenite broth containing 10% sterile feces, and (iii) the lactose pre-enrichment procedure. Brilliant Green Agar was used throughout as the recovery medium. Although the lactose pre-enrichment methodology promoted Salmonella recovery from samples containing small numbers of dormant organisms, the efficiency of this enrichment method is adversely affected by unfavorable coliform-Salmonella ratios. Under such conditions, early subculture of lactose broth into selenite broth is indicated. Selenite broth containing 10% sterile feces was more efficient than the lactose pre-enrichment methodology in promoting the growth of “dormant” salmonellae. Albumen adversely affected recovery of salmonellae from selenite broth, whereas whole egg and egg yolk enhanced Salmonella recovery from this medium. The selenite-feces medium presents a solution to the major problems encountered in the detection of salmonellae in egg products and offers an approach to a single medium in which food-borne salmonellae will manifest themselves with a minimum of laboratory manipulation.     

Enhancing Effect of Feces on Isolation of Salmonellae From Selenite Broth

The addition of feces to selenite broth significantly enhanced the ability of this medium to select for salmonellae in an environment initially containing overwhelming numbers of coliform bacteria. Either heat-sterilized or Seitz-filtered feces produced this effect. In most experiments, the selectivity of selenite broth was unaffected by unsterile feces. Human blood and plasma markedly reduced selenite efficiency. In a base medium supporting both coliform and Shigella growth, heat-sterilized feces imposed a measure of selectivity for Shigella.     

The amino acid sequences of two putative copper-site peptides from the "blue" copper protein, stellacyanin.

Mouse neuroblastoma cells grown in medium containing 10 percent fetal bovine serum have negligible amounts of glutathione peroxidase activity. Introduction of selenite to the medium to produce a concentration of 600 nM resulted in a 30-fold increase in the enzyme activity. This increase is directly proportional to the concentration below 60 nM and levels off at concentrations above this value. Selenate produces no increase in enzyme activity when present alone nor does it inhibit induction when present with selenite. Tellurite produces no increase in enzyme activity when present alone but does inhibit induction when present with selenite.     

Sulphydryl groups involved in Na+-Li+ exchange in human erythrocytes

Oxidative stress causes cellular injury that is thought to be due to increased cytosolic cation levels. Disturbances of a variety of mechanisms which normally maintain intracellular anion/cation homeostasis, occur during oxidative stress. Reactivity of the SH- groups essential for oubain-resistant Na(+)-Li(+) exchange by N-ethylmaleimide (NEM) and selenite was studied in human erythrocytes. In addition, the reactivity of the substances on SH- groups and Li(+) influx have been studied as a function of pH of the medium. The results show that NEM induces an irreversible inhibition of Li(+) influx. It diminishes progressively with the increasing pH of the medium. Whereas we obtain increasing intracellular Li(+) concentration with the rising selenite concentration in the medium. The maximum effect with this substance is reached at about pH 8.0. We can state that the -SH reagents (NEM and selenite) studied behave differently: NEM inhibits Li(+) influx by modifying the essential SH-groups of the membrane proteins in such a way that the exchange is reduced, whereas it maintains the Na(+) permeability almost unaltered. The slight increase in intracellular Na(+) induced by selenite suggests that the oxidative changes in the intracellular sulphydryl groups may constitute an important mechanism for the regulation of the intracellular cations.     

Biosynthesis of fumiquinazolines by the fungus Penicillium thymicola

Biosynthesis of fumiquinazolines F and G (FQs), PC-2, and pigments by the fungus P. thymicola VKM FW-869 is directly dependent on the content of carbon substrate (mannitol) in the medium. Pigment production prevailed at all of the tested mannitol concentrations. The necessary conditions for predominant FQ biosynthesis by the fungus P. thymicola are carbon source (mannitol) limitation and presence of NaCl in the cultivation medium. NaCl has a regulatory effect on the formation of secondary metabolites by enhancing FQ biosynthesis and reducing pigment formation. The maximum values of FQ biosynthesis and inhibition of pigment production are obtained at a mannitol concentration of 20 g/l and 2.5% NaCl in the medium.     

Toxicity of Selenium to the Blue-green Algae, Anacystis nidulans and Anabaena variabilis

Selenate, selenite, selenomethionine, and selenopurine are toxicto Anacystis nidulans and Anabaena variabilis. These compoundsare less toxic in culture medium containing sulphate than insulphur-free medium, thus suggesting a protective role of sulphuragainst selenium toxicity Selenite is more toxic in agar plate cultures and less toxicin liquid cultures than selenate. Cells grown in the highestgrowth-permitting concentration of selenite in liquid mediumform a variable number of red granules No such granulation occursin cells treated with other seleno-compounds, indicating therebythat the mode of selenite action in blue-green algae differsfrom that of other selenium compounds     

Effects of medium osmolarity on the release of amino acids from isolated cotyledons of developing pea seeds

The release of endogenous amino acids from isolated, immature pea (Pisum sativum L. cv. Marzia) cotyledons was investigated in relation to their developmental stage and the osmolarity of the bathing medium. The water potential of the cotyledons was about-1.1 MPa from which it could be inferred that the osmolarity of their apoplastic fluids will be approximately 450 mosmol·l^?1. The time course of amino-acid release conformed to an exponential function. Rate constants of the release were in the range 0.3 to 0.9 · h ^?1. No indication was found for increased permeability of the plasmamembrane for amino acids at low medium osmolarity. Rate constants were even 1.5-fold lower in 0 mM mannitol than in medium with 400 mM mannitol. This effect could be ascribed to reduced protein synthesis in hypotonic media. In the presence of 400 mM mannitol the release was nearly proportional to the total amino-acid pool of the cotyledons and ranged from 12% to 8% for the various developmental stages. Amino-acid release was stimulated by incubation in a hypotonic medium (< 400 mM mannitol), up to fourfold in a medium without mannitol where as much as 45% of the cotyledonary amino-acid content could be released. The extra aminoacid release induced by the hypotonic condition declined during development and eventually vanished completely. Release of amino acids into a medium with 400 mM mannitol was more selective than into a medium without mannitol. For instance, arginine was one of the main constituents of the cotyledonary amino-acid pool (19%) as well as of the released amino-acid mixture when the medium contained no mannitol (10%), whereas it was virtually absent when the medium contained 400 mM mannitol. As an overall interpretation of these results, it is proposed that the hypotonic condition greatly enhances the permeability of the tonoplast (not that of the plasmalemma) for amino acids so that the otherwise well-sequestered amino acids in the vacuole become available for release into the bathing medium.     

Biosynthesis of fumiquinazolines by the fungus Penicillium thymicola

Biosynthesis of fumiquinazolines F and G (FQs), PC-2, and pigments by the fungus P. thymicola VKM FW-869 is directly dependent on the content of carbon substrate (mannitol) in the medium. Pigment production prevailed at all of the tested mannitol concentrations. The necessary conditions for predominant FQ biosynthesis by the fungus P. thymicola are carbon source (mannitol) limitation and presence of NaCl in the cultivation medium. NaCl has a regulatory effect on the formation of secondary metabolites by enhancing FQ biosynthesis and reducing pigment formation. The maximum values of FQ biosynthesis and inhibition of pigment production are obtained at a mannitol concentration of 20 g/l and 2.5% NaCl in the medium.     

Mobilization of selenite by Ralstonia metallidurans CH34

Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.     

Proteomic profiling of L-cysteine induced selenite resistance in Enterobacter sp. YSU

Background  

Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway.