Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water.

Some effects of visible light on the survival of Escherichia coli in waters of the Butrón river were studied by comparing illuminated and nonilluminated systems. The following count methods were used: CFU on a selective medium (eosin-methylene blue agar), CFU on a medium of recuperation (Trypticase soy agar with yeast extract and glucose), number of metabolically active cells by reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, and total number of E. coli cells as determined by the acridine orange direct-count method. In the illuminated systems, decreases in CFU of E. coli and in the number of metabolically active cells were observed. However, no decline of the total number of E. coli cells was observed. By count methods, different stages of progressive dormancy of E. coli cells were determined to exist in illuminated systems. Culturable and recoverable cells were defined as viable cells, and metabolically active cells and morphologically intact cells were defined as somnicells. Indirect activity measurements were also done by using [14C]glucose. In illuminated systems, a decrease of glucose uptake by E. coli cells was observed throughout the experiments. The assimilated fraction of [14C]glucose decreased faster than the respired fraction in illuminated systems. The percentage of respired [14C]glucose (14CO2 production) with respect to the total glucose uptake increased throughout the experiments, and the percentage of assimilated glucose decreased. Therefore, the visible light was also responsible for an additional inhibition of biosynthetic processes.     

Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems

Some effects of visible light on Escherichia coli and Enterococcus faecalis in natural freshwater and seawater were studied by plate counts, colony area measurements, and direct counts. A large number of somnicells (non-culturable cells) were noted in illuminated systems as compared with non-illuminated ones. Colony areas were significantly smaller in illuminated systems. Indirect activity measurements were used to test the effects of visible light on the ability of E. coli and Ent. faecalis to metabolize substrates ([14C]glucose) in natural waters. In illuminated systems, a decrease of glucose uptake was observed. When percentages of assimilation and respiration with respect to the total glucose uptake were analysed a decrease of assimilation percentages and an increase of respiration percentages were observed. In addition, differences in glucose uptake, assimilation and respiration by enteric bacteria were detected for E. coli at the beginning of the experiments between fresh- and seawater and these were interpreted as a toxic effect exerted by seawater on E. coli cells. Differences between species, natural waters and parameters studied (excepting glucose assimilation) were detected in the illuminated systems. We concluded, however, that enteric bacteria under visible light illumination show a general survival strategy characterized by reaching progressively a somnicell stage which can be defined in terms of their (1) inability to form colonies on standard bacteriological media, (2) inability to incorporate substrates, and (3) inactivation of biosynthetic processes.     

Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems

Some effects of visible light on Escherichia coli and Enterococcus faecalis in natural freshwater and seawater were studied by plate counts, colony area measurements, and direct counts. A large number of somnicells (non-culturable cells) were noted in illuminated systems as compared with non-illuminated ones. Colony areas were significantly smaller in illuminated systems. Indirect activity measurements were used to test the effects of visible light on the ability of E. coli and Ent. faecalis to metabolize substrates ([¹⁴C]glucose) in natural waters. In illuminated systems, a decrease of glucose uptake was observed. When percentages of assimilation and respiration with respect to the total glucose uptake were analysed a decrease of assimilation percentages and an increase of respiration percentages were observed. In addition, differences in glucose uptake, assimilation and respiration by enteric bacteria were detected for E. coli at the beginning of the experiments between fresh-and seawater and these were interpreted as a toxic effect exerted by seawater on E. coli cells. Differences between species, natural waters and parameters studied (excepting glucose assimilation) were detected in the illuminated systems. We concluded, however, that enteric bacteria under visible light illumination show a general survival strategy characterized by reaching progressively a somnicell stage which can be defined in terms of their (1) inability to form colonies on standard bacteriological media, (2) inability to incorporate substrates, and (3) inactivation of biosynthetic processes. and accepted 8 June 1989     

Heterogeneity of Escherichia coli population during induced autolysis

Escherichia coli M-17 autolysis was induced by eliminating nutrition sources from the growth medium and exerting a shock with EDTA. The overall cell number, the optical density of the cell suspension, the number of colony-forming units (CFU), and [3H]uracil incorporation into the cells were analysed in the course of autolysis. The number of CFU was found to drop down faster than the overall cell number in the process of autolysis. The population of E. coli was shown to be heterogeneous in its sensitivity to the induction of autolysis, and some nonlysed cells were still metabolically active. When the rate of autolysis was highest in some cells of the population, the labeled precursor was found to be incorporated into the TCA-soluble and TCA-insoluble fractions of nonlysed cells. The overall cell number, the optical density of the cell suspension, and the number of CFU increased 96 h after the induction of autolysis. The authors discuss what is the role played by the heterogeneity of an E. coli population in its adaptation to EDTA-induced autolysis.     

Role of hydrogen peroxide in loss of culturability mediated by visible light in Escherichia coli in a freshwater ecosystem.

A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.     

Role of hydrogen peroxide in loss of culturability mediated by visible light in Escherichia coli in a freshwater ecosystem.

A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.     

Response of an antarctic lake heterotrophic community to high dissolved oxygen

The upper waters of Lake Hoare, Antarctica, contain dissolved oxygen at about three times the normal saturation (>/=42 mg liter). The response of the heterotrophic plankton community to this high dissolved oxygen was evaluated by the criteria of CFU and d-[U-C]glucose assimilated-respired. High dissolved oxygen was not inhibitory to d-[U-C]glucose assimilation-respiration compared with normal atmospheric dissolved oxygen in Lake Hoare water. The d-[U-C]glucose was assimilated and respired optimally at 12 degrees C in Lake Hoare. The d-[U-C]glucose assimilated-respired in the upper saturated atmospheric dissolved oxygen waters of Mountain Lake, Va., was inhibited in contrast to Lake Hoare (P < 0.05). CFU formation was inhibited in both lakes. CFU represent <1% of the fluorochrome-stained direct counts in Lake Hoare. Lake Hoare planktobacteria are smaller than the planktobacteria in Mountain Lake. ATP size fractionation revealed that 39% of the ATP biomass was <0.5 mum in Lake Hoare.     

Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin

The photodynamic activity of 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on agar surfaces. The results obtained for the tricationic A3B3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin p-tosylate (TTAP4+), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence. In both cases, a higher photostability was found for A3B3+ than for TTAP4+. Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0-14 nmol) and irradiated with visible light for 3h. The light source used was either a projector or midday sun. The A3B3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through an optical fiber. Under these conditions, A3B3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photodynamic activity of A3B3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces complete inactivation of E. coli cells on the agar surface. Therefore, tricationic A3B3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection.     

Living on a high sugar diet: the fate of sucrose ingested by a phloem-feeding insect,the pea aphid Acyrthosiphon pisum

The natural diet of aphids, plant phloem sap, generally contains high concentrations of sucrose. When pea aphids (Acyrthosiphon pisum) were fed on chemically defined diets containing sucrose radiolabelled in the glucose or fructose moiety, 2 to 12-fold and 87 to 110-fold more radioactivity was recovered from the tissues and honeydew, respectively, of aphids that ingested [U-(14)C-glucose]-sucrose than from those ingesting [U-(14)C-fructose]-sucrose. The total radioactivity recovered was 70% of the ingested [U-(14)C-glucose]-sucrose and <5% of ingested [U-(14)C-fructose]-sucrose. The dominant honeydew sugars produced by aphids feeding on 0.75 M sucrose diets were oligosaccharides comprising glucose. In vitro the guts of pea aphids had high sucrase activity, 1-5 U mg(-1) protein, generating equimolar glucose and fructose except at high sucrose concentrations where glucose production was inhibited (K(si)=0.1 M). These data suggest that the fructose moiety of ingested sucrose is assimilated very efficiently and may be preferentially respired by the aphid, and that the glucose moiety of sucrose is incorporated into oligosaccharides by the transglucosidase activity of the gut sucrase at high sucrose concentrations. These differences in the fate of sucrose-derived glucose and fructose are important elements in both the carbon nutrition and osmoregulation of aphids.     

Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.     

Inducible gene expression by nonculturable bacteria in milk after pasteurization

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.     

Osmotic stress drastically inhibits active transport of carbohydrates by Escherichia coli

In intact Escherichia coli cells, severe osmotic stress almost totally inhibited active transport of carbohydrate by all of the systems known to transport carbohydrates in E. coli: group translocation (glucose), binding-protein mediated transport (maltose), proton symport (lactose), and sodium cotransport (melibiose). Detailed study of glucose transport showed that this inhibition of transport was not secondary to the inhibition of growth by osmotic stress, but rather that the inhibition of transport of a source of carbon and energy was sufficient to cause the complete inhibition of growth observed during severe osmotic upshock. Transport and growth inhibition did not result from cell death; upshocked cells were viable and metabolically active.     

变性高效液相色谱检测食品中致泻性大肠杆菌

[目的]应用多聚酶链式反应(polymerase chain reaction,PCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中致泻性大肠杆菌的快速检测方法.[方法]分别根据4种致泻性大肠杆菌的特异性毒力因子基因序列设计引物,PCR扩增产物经变性高效液相色谱进行快速检测.以肠产毒性大肠杆菌等32株试验菌株做特异性检测;4种致泻性大肠杆菌标准菌株稀释成不同梯度,做灵敏度检测.[结果]试验结果表明该方法有很好的特异性,且灵敏度高,检测限可达到:肠产毒性大肠杆菌27 CFU/mL、肠致病性大肠杆菌33 CFU/mL、肠出血性大肠杆菌25 CFU/mL、肠侵袭性大肠杆菌42 CFU/mL.[结论]该方法可以快速、准确地检测食品中的致泻性大肠杆菌,是食品中病原菌检测的新技术和新方法.     

Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.     

Selective stimulation of in situ intermediary metabolism by free calcium in permeabilized rat adipocytes.

The hypothesis that ionized calcium [Ca2+]i may stimulate in situ rat adipocyte intermediary metabolism distal to glucose transport was tested. A metabolically active porous adipocyte model was employed in which pathway metabolism is exclusively pore-dependent using glucose 6-phosphate (G6P) as substrate. Cellular [Ca2+]i was, furthermore, directly adjusted to between 0-2.5 microM via the membrane pores. Three metabolic fluxes were examined, (1) glycolysis-Krebs ([6-14C]G6P oxidation), (2) glycolysis to lactate ([U-14C]G6P to [14C]lactate) and (3) pentose pathway ([1-14C]G6P oxidation). Glycolysis-Krebs oxidation was was found to be selectively (33% above basal P less than 0.001) stimulated by 0.625 microM free calcium. In contrast, there was no effect of [Ca2+]i on the other, exclusively cytoplasmic, pathways. The stimulation of glycolysis-Krebs by [Ca2+]i was inhibited by a mitochondrial calcium channel blocker (Ruthenium red) and persisted over a range of ATP/ADP ratios. Separate studies demonstrated that 2-[1-14C]ketoglutarate oxidation was also calcium-stimulated in the porous adipocytes (160% over baseline at 1 microM [Ca2+]i). These studies thus demonstrate that physiologically relevant increments in porous adipocyte [Ca2+]i enhance overall in situ glycolytic-Krebs pathway oxidation by a mechanism which entails mitochondrial calcium uptake. Methodologically, this metabolically active porous adipocyte model presents a novel experimental approach to investigations regarding the effects of ionized calcium on intermediary metabolism beyond glucose transport.     

Mode of antibacterial action by gramicidin S

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.     

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer.

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14C-labeled organic compounds, and the evolution of 14CO2 was measured over time. Gas chromatographic analyses were used to monitor CO2 production and O2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14CO2 was measured from [14C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [14C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14C label. [14C]benzene and [14C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO3, CO2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 mumol g-1 (dry weight) day-1 while oxygen concentration decreased at a rate of 0.124 mumol g-1 (dry weight) day-1. With no added nitrate, CO2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n-alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14C-labeled organic compounds, and the evolution of 14CO2 was measured over time. Gas chromatographic analyses were used to monitor CO2 production and O2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14CO2 was measured from [14C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [14C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14C label. [14C]benzene and [14C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO3, CO2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 mumol g-1 (dry weight) day-1 while oxygen concentration decreased at a rate of 0.124 mumol g-1 (dry weight) day-1. With no added nitrate, CO2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n-alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mode of condensation of aspartic acid and dihydroxyacetone phosphate in quinolinate synthesis in Escherichia coli.

Dihydroxy [3-14C]acetone phosphate was prepared enzymatically from [1-14C]glucose and use as a substrate in a partially purified quinolinate synthetase system prepared from Escherichia coli mutants. Carbon-by-carbon degradation of the resulting [14C]quinolinate showed that 96% of the 14C was located in carbon-4, indicating that carbon-3 of dihydroxyacetone phosphate condenses with carbon-3 of aspartate in quinolinate synthesis in E. coli.