Coexistence of Genetically Engineered Escherichia coliStrains and Natural Microorganisms in Experimental Aquatic Microcosms

In experimental aquatic microcosms (AMCs), the population of the Escherichia colistrain Z905 harboring the recombinant plasmid pPHL7 (Ap^rLux⁺) was found to gradually accumulate AMC-adapted cells, which retained the plasmid but differed from the original cells in some biochemical and physiological characteristics. Both the original and the AMC-adaptedE. colicells could coexist with the native AMC microflora for one year or longer. When introduced into AMCs together with native pseudomonads, the AMC-adapted E. coliZ905-33 (pPHL7) cells were more competitive than the nonadapted cells.     

Quantitative criteria for the estimation of the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria

Computation coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria P. leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in multicopy plasmid are suggested, and their use on the molecular, cell, and population levels was considered. It was shown that, on the population level, all transgenic variants got the better of natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. On the cell level, in the bright and dim variants of the transgenic strain, the effectiveness of bioluminescence expression increases by several orders. On the level of one lux-operon, the effectiveness of expression of the bright variant of transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar, and the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2 /pPHL7 is by two orders lower than that in the dark variant of P. leiognathi 54.     

Quantitative criteria for estimating the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria

Computational coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria Photobacterium leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in a multicopy plasmid are suggested and their use at molecular, cell, and population levels was considered. It was shown that at the population level, all transgenic variants have an advantage over natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. At the cell level, the effectiveness of bioluminescence expression in the bright and dim variants of the transgenic strain increased by several orders. At the level of one lux-operon, the effectiveness of expression in the bright variant of the transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar; the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2/pPHL7 is by two orders of magnitude lower than in the dark variant of P. leiognathi 54.     

Cloning and expression of genes of the luminescence system in Photobacterium leiognathi

The genes of Photobacterium leiognathi luminescence system were cloned in plasmid pUC18. Escherichia coli cells harboring a recombinant plasmid pPHL1 are luminescent. pPHL1 contains luciferase genes and genes responsible for aldehyde biosynthesis. The luminescence of Escherichia coli is subject to autoinductor regulation similar to the one existing in luminescent bacteria. The 2.7 kb fragment of Photobacterium leiognathi DNA containing the genes for alpha- and beta-luciferase subunits were cloned in pUC19.     

Dynamics of the population structure of the Escherichia coli recombinant strain during continuous culture]

The populational structure of the Escherichia coli strain Z905 containing the recombinant plasmid with the phenotype AprLux+ was studied in chemostat. It was shown that the stability of the ratio of plasmid containing cells and cells without plasmids depends in the first place on the presence of the selective factor (ampicillin) in the medium and on the sources of carbon and energy limiting growth.     

Mathematical modeling of population dynamics of unstable plasmid-containing bacteria during continuous cultivation in a chemostat

A structural approach to studying the regularities of the population dynamics of unstable recombinant bacterial strains in a chemostat was elaborated. The approach is based on the mathematical modeling of cell distribution in a population with different numbers of plasmid copies. The effect of decreased selective preference of plasmidless variants of the recombinant strain in the chemostat, which is related to a decrease in the number of plasmid copies in cells upon long-term incubation was analyzed. It is shown that the time of half-elimination of plasmids from the bacterial population in the steady state in the chemostat T1/2 does not depend on the maximum number of plasmid copies in cells N but is determined only by the mean time of generation g and the probability of the loss of one plasmid copy tau. The dependence of the preference of bacterial plasmidless variants on the efficiency of expression of genes cloned into plasmids in chemostat was analyzed using the recombinant strain E. coli Z905, whose plasmids pPHL-7 contain cloned genes for the luminescence system of marine luminescing bacteria Photobacterium leiognathi.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance.

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli

1. A plasmid for constitutive expression of the human interferon-alpha 1 (hIFN-alpha 1) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-alpha 1 gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-alpha 1 from E. coli LE392 cells transformed with the expression plasmid pJP1R9-hIFN-alpha 1 is evaluated to be 2-6 x 10(7) U/l bacterial culture for metabolic shaker and 6-8 x 10(7) U/l for fermentor.     

Site-specific recombination system from Tn1000(γδ) stabilises recombinant plasmids in Escherichi coli

The resolution system from Tn1000 was cloned into a pBR322 recombinant plasmid carrying E. coli threonine operon. The resulting plasmid displayed a significant increase in segregational stability with respect to the original plasmid, either in a rec or a rec E. coli strain (100% of plasmid-containing cells versus 64% and 1% respectively). The Tn1000 resolution system proved to be responsible for the stabilising effect exerted by the transposon when present in recombinant plasmids.     

Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli.

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.     

Inhibition of biological activities of the aerobactin receptor protein in rough strains of Escherichia coli by polyclonal antiserum raised against native protein

The aerobactin iron-uptake system of plasmid ColV-K30, genetically isolated from other plasmid determinants by molecular cloning, was sufficient to restore full virulence in a mouse peritonitis model to a clinical Escherichia coli isolate, D551 (O78:H-), whose resident aerobactin-encoding ColV plasmid had been lost by curing. Antiserum was raised in rabbits against live E. coli K12 cells expressing the outer-membrane aerobactin receptor protein and absorbed with an isogenic strain lacking the receptor. This antiserum inhibited binding of aerobactin, cloacin DF13 and bacteriophage B74K to the native protein in whole E. coli K12 bacteria expressing the receptor, or in membranes prepared from such organisms. However, it did not react with the native receptor protein in several wild strains unless lipopolysaccharide was first removed by treatment with trichloroacetic acid, nor did it protect mice in experimental infections with strain D551. Antisera raised in rabbits against partially or fully denatured forms of the aerobactin receptor reacted only in assays involving denatured protein; they showed no inhibition of the biological activities of the native receptor.     

肠出血性大肠杆菌毒力因子Z1444的原核表达及其丝/苏氨酸激酶活性验证

目的:在大肠杆菌中表达肠出血性大肠杆菌(EHEC)毒力岛上的毒力因子Z1444并纯化,对其丝/苏氨酸激酶活性进行初步检测。方法:根据GenBank中Z1444基因序列及pET-28a(+)载体的多克隆位点设计引物,以EHECO157∶H7全菌裂解液为模板,经PCR钓取1047 bp的目的片段,与表达载体pET-28a(+)连接,构建重组表达质粒pET-28a(+)-Z1444,将其转化至大肠杆菌BL21(DE3)中,IPTG诱导蛋白表达并经SDS-PAGE鉴定,利用体外反应体系鉴定重组蛋白的丝/苏氨酸激酶活性。结果:双酶切和测序鉴定表明,pET-28a(+)-Z1444原核表达质粒构建正确;诱导表达后经纯化,获得纯度在90%以上的可溶性重组Z1444,相对分子量约为38×103;体外酶活实验验证了Z1444的丝/苏氨酸激酶活性。结论:Z1444在大肠杆菌中获得高效可溶性表达,为后续功能验证奠定了基础。     

Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli

The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41 kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control. Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells. Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmission of plasmid Rldrd19 from E. coli to Hafnia]

Investigations carried out demonstrated a possibility of transmission of plasmid Rldrd19 from E. coli to Hafnia. The incidence of the plasmid transmission varied from 10(-7) to 10(-9) and depended on the properfies of Hafnia strains. Tra-operon of plasmid Rldrd19 in the Hafnia 614 strain functioned with the same efficacy as in E. coli. Plasmid Rldrd19 in Hafnia was unstable and was eliminated from the cells in case of storage at low temperatures. As shown, plasmid Rldrd19 was under strict replication control in Hafnia as in E. coli. Formation of the CCC-form of the plasmid Rldrd19 was suppressed in Hafnia at 29--30 degrees C and was not suppressed in E. coli.     

大肠杆菌菌蜕装载质粒DNA实验方法的优化

目的:优化大肠杆菌菌蜕装载质粒的效率,并将装载质粒的菌蜕转染抗原提呈细胞,以提高核酸疫苗的递送水平。方法:将质粒pHH43转化大肠杆菌DH5α,制备大肠杆菌菌蜕;优化菌蜕装载质粒时菌蜕、质粒和膜囊的比例,获得更高的装载效率,通过扫描及透射电镜、流式细胞术观察其形态变化及装载效率;将装载质粒的菌蜕与抗原提呈细胞——巨噬细胞RAW264.7和树突状细胞DC2.4共孵育,观察吞噬效果。结果:优化了大肠杆菌菌蜕装载质粒的效率,当菌蜕、质粒、膜囊的比例为7∶10∶4时效率达到最佳,装载DNA效率达98%以上;抗原提呈细胞吞噬装载了质粒的菌蜕,效率达100%。结论:大肠杆菌菌蜕可高效装载核酸疫苗,且高效被抗原提呈细胞捕获,有助于提高核酸疫苗的递送和免疫效果的提高。     

Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli

The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.     

A freeze-thaw method for disintegration of Escherichia coli cells producing T7 lysozyme used in pBAD expression systems

The pLysN plasmid containing the T7 lysozyme gene under control of the lac promoter was constructed to facilitate cell disintegration after expression of recombinant proteins in arabinose-induced expression systems. The usefulness of this plasmid was tested in Escherichia coli TOP10 and E. coli LMG194 cells carrying pBADMHADgeSSB plasmid containing Deinococcus geothermalis SSB protein gene under control of the araBAD promoter. The results showed that low-level expression of T7 lysozyme did not interfere with the target SSB protein production, and that the freezing-thawing treatment was sufficient for disruption of the E. coli cells producing low amounts of T7 lysozyme.