Determination of antibiotics using luminescent Escherichia coli and serum

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.     

Metal content of DNA polymerase I purified from overproducing and wild type Escherichia coli

DNA polymerase I purified from both E. coli strain B, and from an overproducing E. coli stain lysogenized with a lambda pol A phage were analyzed for metal content. After gel filtration to remove loosely bound metals, DNA polymerase I from both strains contained less than or equal to 0.2 gm atoms Zn2+/mole enzyme and 0.09 to 0.7 Mg2+/mole enzyme. Substoichiometric amounts of Fe, Co, Ni (less than or equal to 0.2 gm atoms), and Mn (less than or equal to 0.1 gm atoms) were detected. Since the metal content does not correlate with enzymatic activity, we conclude that DNA polymerase I is not a metalloenzyme.     

Dependence of the rate of aspartate ammonia-lyase reaction catalyzed by free and immobilized cells of E. coli on temperature and preliminary thermal treatment]

The effects of temperature (45--55 degrees) and duration of thermal treatment on the L-aspartase activity of free and immobilized on polyacrylamide gel cells of E. coli, strain 85 were studied. It was found that preliminary thermal treatment of the cells at 50 degrees for 40--60 min is optimal for a high aspartase activity. Within the temperature interval of 20--55 degrees the temperature dependence of effective rate constants of L-aspartate synthesis obeys the Arrhenius equation, whereas the effective energy of activation is decreased from 12,6 to 3,6 kcal/mole, when the "activation" of the cells shows an increase.     

Cell density dependent acid sensitivity in stationary phase cultures of enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7, the causative agent of hemorrhagic colitis and hemolytic uremic syndrome, can survive in a highly acidic environment. The acid resistance of this organism, as measured by its ability to survive in low pH, depended on the density of the cells present during the assay. At low cell densities (相似文献   

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

The whole cell tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) activity of Erwinia herbicola (ATCC 21434) was microen-capsulated. We studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 degrees C to 32 degrees C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 degrees C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42 degrees C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 +/- 100 mum, mean +/- std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of-the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K(M) of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K(M) for phenol is 0.5 mM. The apparent K(M) values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme. (c) 1995 John Wiley & Sons, Inc.     

Heterogeneity of human monocytes: differences in response to IFN-gamma

Human peripheral blood monocytes can be separated into two subpopulations which differ in the efficiency of their adherence to glass after 16 hours of incubation. The adherent subpopulation was found to be about twice as effective in binding mannose-resistant E. coli 0-124, mannose-sensitive E. coli 0-128 and opsonised E. coli than the nonadherent one. In addition, reduction of cytochrome C in response to E. coli binding or 12-myristate 13-acetate (PMA) stimulation was two fold higher in adherent cells. The binding of E. coli O-124 and the superoxide generation stimulated by E. coli were inhibited by the addition of mannose only in the adherent monocytes, indicating the presence of mannose receptors on the cell surface in the adherent subpopulation. The treatment of the nonadherent cells with 0.1-1000 U/ml of Interferon (IFN-gamma) for 24 hours resulted in a dose dependent increase in superoxide generation. After 72 hours of incubation with IFN-gamma (1000 U/ml) the amount of superoxide generated by the nonadherent cells was elevated to 20.5 +/- 1.4 nmoles/10(6) cells/15 min, similar to that of the adherent cells (24.5 +/- 1.2 nmoles/10(6) cells/15 min untreated adherent monocytes). The generation of superoxide in the IFN-gamma treated nonadherent monocytes stimulated by E. coli 0-128 was significantly reduced by addition of mannose.     

Melanin content and hydroperoxide metabolism in human melanoma cells

Human melanoma cells were grown to exponential and stationary phases showing melanin contents of 4.2 +/- 0.3 and 11.3 +/- 0.6 micrograms/10(6) cells, respectively. The cells were separated in four subpopulations by a Percoll gradient; the subpopulation of density 1.07 (g/ml) was the most enriched in pigmented cells and produced 28 and 58% of the cells in exponential and stationary phases, respectively. Melanoma cells had similar superoxide dismutase and glutathione peroxidase activities in exponential and stationary phases. Moreover melanoma cells exhibited a higher catalase activity in the stationary phase: whole homogenate and cytosol activities were 7.0 +/- 0.3 and 10.8 +/- 0.6 U/mg protein, whereas in exponential phase the activities were 4.9 +/- 0.1 and 7.6 +/- 0.3 U/mg protein for whole homogenate and cytosol, respectively. The intracellular H2O2 steady-state concentration was 3.3 +/- 0.2 and 2.1 +/- 0.2 microM H2O2 for exponential and stationary phases, respectively. The spontaneous chemiluminescence of the two culture phases was 169 +/- 27 cps/10(6) cells (exponential) and 78 +/- 24 cps/10(6) cells (stationary). The cytotoxicity of H2O2 generated extracellularly by glucose oxidase was determined after 60 min of exposure. IC50 values for exponential and stationary cell cultures were 0.9 and 2.4 mU/ml of glucose oxidase, respectively. The increased catalase activities in the stationary phase as compared with the exponential phase are consistent with the decreased intracellular H2O2, with the decreased spontaneous chemiluminescence, and with the increased resistance to exogenous H2O2.     

Maintenance of intracellular calcium in Escherichia coli

Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells. Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli. Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid. The concentration of free [Ca2+]i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium. Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium. In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration. In starved cells [Ca2+]i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM. Addition of glucose lowered the Ca2+ levels to 90 nM. Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux. Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool. These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+]i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.     

Comparison of the aspartase activity and its stability in Escherichia coli cells immobilized on polyacrylamide gel and carrageenan

The conditions for immobilization of Escherichia coli cells (Soviet strain 85) on the natural polysaccharide carrier carrageenan (Soviet-made) were investigated and kinetic regularities of the aspartase reaction catalysed by immobilized in carrageenan cells of E. coli 85 were established. The conditions for retaining a high aspartase activity and stability of biocatalysts based on the E. coli 85 cells immobilized in PAAG and carrageenan were determined using full-loaded tanks for continuous synthesis of L-aspartic acid. The time-stable aspartase activity of the biocatalyst can be increased by treating the beads of the catalyst with bifunctional reagents (hexamethylenediamine, glutaraldehyde), the most active catalyst for the biotechnological synthesis of L-aspartic acid being obtained when carrageenan is used.     

Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600

A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E. coli MRE-600 was developed. It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite). The mode of cell destroying was found to affect the process of the further enzyme purification. The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein. The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG. The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha). The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000. The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively.     

Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid

Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.     

Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide

The effects of carbon tetrachloride (CCl4) and paraquat on the growth of Escherichia coli were investigated. Paraquat at 10 mM caused some inhibition of growth of E. coli in trypticase-soy-yeast extract medium. CCl4 enhanced growth inhibition by paraquat in a concentration-dependent manner. In the absence of paraquat, CCl4 had no effect on growth rate or on surviving cell numbers at stationary phase. CCl4 did not prevent the induction of manganese-superoxide dismutase by paraquat. Under anaerobic conditions, CCl4 and paraquat exhibited no effect on E. coli. In the presence of Mn(II) and paraquat, intracellular superoxide dismutase was markedly induced and protected E. coli against the toxicity of CCl4 and paraquat. The reactive free radical CCl3OO-, which can be formed from the reaction of O2- with CCl4, may cause cell damage. The growth-inhibiting effects of polyhalides in the presence of paraquat followed the order CBrCl3 greater than CCl4 greater than CHCl3 greater than CH2Cl2, which is in accord with that of the reaction rates of these compounds with O2- and with their hepatotoxicities. These results suggest that O2- plays a role in the hepatotoxicity of polyhalides.     

Interactions of Escherichia coli strains of non-EPEC serogroups that carry eae and lack the EAF and stx gene sequences with undifferentiated and differentiated intestinal human Caco-2 cells

Escherichia coli strains of non-EPEC serotypes that carry eae and lack the EAF and the Shiga toxin (stx) gene sequences have been found in acute diarrhea. Both the cell association and the cell entry of these strains in human intestinal epithelial cells were studied as a function of cell differentiation and polarization. The eae+/EAF-/stx- non-EPEC E. coli strains invaded undifferentiated Caco-2 cells more efficiently than differentiated cells. In contrast, prototype EPEC strain E2348/69 did not show significative differences from invasion rates of undifferentiated and differentiated cells. The uptake of these strains was greatly enhanced by pretreatment of differentiated Caco-2 cells with EGTA. These results suggest that the eae+/EAF-/stx- non-EPEC E. coli invasion of intestinal cells may be dependent on receptors expressed on the surface of undifferentiated cells and the basolateral pole of differentiated cells.     

Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv

The enzyme peptidyl-tRNA hydrolase (Pth, EC 3.1.1.29) is essential for the viability of bacteria. The ORF Rv1014c of Mycobacterium tuberculosis H37Rv, designated as the mtpth gene, was cloned and over-expressed and the product was purified. Generation of polyclonal antibodies against the purified recombinant protein, termed MtPth, facilitated detection of endogenously expressed MtPth in M. tuberculosis H37Rv cell lysate. MtPth could release diacetyl-[(3)H]-lysine from diacetyl-[(3)H]-lysyl-tRNA(Lys) with Michaelis-Menten kinetic parameters of K (m)=0.7+/-0.2 microM and k (cat)=1.22+/-0.2 s(-1). Transformation of a pTrc99c/mtpth vector allowed the growth of E. coli thermosensitive Pth(ts) mutant strain AA7852 at the non-permissive temperature of 42 degrees C, demonstrating the in vivo activity of MtPth. In addition, at 39 degrees C, over-expression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 microg/ml erythromycin. A 3D fold based on NMR and a structural model based on the E. coli Pth crystal structure were generated for MtPth. The essential nature of conserved active-site residues N12, H22 and D95 of MtPth for catalysis was demonstrated by mutagenesis and complementation in E. coli mutant strain AA7852. Thermal and urea/guanidinium chloride (GdmCl)-induced unfolding curves for MtPth indicate a simple two-state unfolding process without any intermediates.     

Formation and Ultrastructure of Extra Membranes in Escherichia coli

A temperature-sensitive strain of Escherichia coli (strain 0111a(1)) was shown to accumulate membranous structures at 40 C. These "extra membranes" appeared as vesicles or whorls (or both), depending on the time of growth at 40 C. After 2 hr of growth at 40 C, only vesicles were observed in E. coli 0111a(1) cells; both vesicles and whorls were apparent after 6 hr. The number of cells which contained both types of extra membrane reached a maximum value (75%) after 10 hr of growth at 40 C. Extra membrane production was also studied by using temperature shifts. In shift-up experiments, cells grown at 30 C into early stationary phase accumulated extra membrane after a shift to 40 C. The percentage of E. coli 0111a(1) cells containing extra membrane decreased significantly after a shift from 40 to 30 C. Phase- and electron-microscopic observations indicated that E. coli 0111a(1) cells grown at 40 C were larger than E. coli 0111: B(4) cells grown at either temperature. The ratio of optical density per cell and cell measurements obtained from quantitative electron microscopy confirmed that E. coli 0111a(1) cells grown at 40 C were about twice as large. Microdensitometer traces indicated that the dimension of a single membrane of either whorls or vesicles was 5.4 nm in peak-to-peak distance (8.8 nm total thickness).     

New approach for specific determination of antibiotics by use of luminescent Escherichia coli and immune serum

This paper describes a possible application of luminescent Escherichia coli activated by blood serum for high-sensitivity and high-specificity assays of antibiotics in solutions. Antibiotics inhibited luminescence of a genetically engineered E. coli strain; the system sensitivity to some antibiotics grew notably after the cells had been preactivated by blood serum. The highest level of sensitivity (2.8 +/- 0.6 ng/ml) of luminescent cells was obtained for aminoglycoside antibiotics (gentamicin and streptomycin). It is feasible to create the specific biosensor for antibiotics on the basis of bioluminescent E. coli strains by applying sera containing antibodies against the antibiotic under assay. The presence of antibodies specific for gentamicin in serum affects inhibition of luminescent cells by gentamicin but not inhibition by other antibiotics.     

掺入大肠杆菌膜中的磷脂酰胆碱(PC)影响青霉素b-内酰胺酶的分泌

[目的]为了研究磷脂酰胆碱(PC)在原核生物细胞中的生物学作用,探讨PC对细菌膜系统的功能的影响.[方法]使用ptac 85质粒作载体,将螺旋菌pcs基因导入E.coli Top10细胞构建了E.coli Fop10 pcs 菌株,并在特定的条件下培养细菌,使细菌膜磷脂中合成30%左右的磷脂酰胆碱.然后再使用抗生素抗性分析、β-内酰胺酶的酶活测定以及Western blot杂交技术,分析质粒编码的β-内酰胺酶从细胞质到细胞问质的分泌情况.[结果]抗生素抗性分析发现,高浓度的氨苄青霉素抑制E.coliTop10 pcs 细菌的生长的氨苄青霉素剂量低于对照组,其半致死剂量IC50在700～800μg/mL之间.酶活检测显示E.coli Top10 pcs 细菌周质内β-内酰胺酶的酶活性只有对照菌株的1,5,Western blot进一步分析发现周质内β-内酰胺酶的含量也为对照菌株的1/5.由此可见,周质内低含量的β-内酰胺酶是导致E.coli Top10pcs 细菌氨苄青霉素抗性降低的原因.[结论]掺入细菌膜磷脂双分子层的PC影响p.内酰胺酶通过Sec转运途径从细胞质分泌到细菌周质空间内,提示细菌磷脂酰胆碱可能在调节蛋白转运和分泌方面起着重要的作用.     

Increased phenotypic stability and ethanol tolerance of recombinant Escherichia coli KO11 when immobilized in continuous fluidized bed culture

The recombinant Escherichia coli B strain KO11, containing chromosomally-integrated genes for ethanol production, was developed for use in lignocellulose-to-ethanol bioconversion processes but suffers from instability in continuous culture and a low ethanol tolerance compared to yeast. Here we report the ability cell immobilization to improve its phenotypic stability and ethanol tolerance during continuous culture on a 50 g/L xylose feed. Experiments conducted in a vertical tubular fermentor operated as a liquid-fluidized bed with the cells immobilized on porous glass microspheres were compared to control experiments in the same reactor operated as a chemostat without the support particles. Without cell immobilization the ethanol yield fell sharply following start-up, declining to 60% of theoretical after only 8-9 days of continuous fermentation. While immobilizing the cells did not prevent this decline, it delayed its onset and slowed its rate. With immobilization, a stable high ethanol yield (>85%) was maintained for at least 10 days, thereafter declining slowly, but remaining above 70% even after up to 40 days of fermentation. The ethanol tolerance of E. coli KO11 cells was substantially increased by immobilization on the glass microspheres. In ethanol tolerance tests, immobilized cells released from the microspheres had survival rates 2.3- to 15-fold higher than those of free cells isolated from the same broth. Immobilization is concluded to be an effective means of increasing ethanol tolerance in E. coli KO11. While immobilization was only partially effective in combating its phenotypic instability, further improvements can be expected following optimization of the immobilization conditions.