Isolation and Characterization of a New Generalized Transducing Bacteriophage Different from Pl in Escherichia coli

A new generalized transducing bacteriophage in the Escherichia coli system was isolated and characterized. This phage, designated D108, makes clear plaques on E. coli K-10, K-12, K-12(P1kc), K-12(D6), B/r, C, and 15 T(-), and Shigella dysenteriae. The plaque of phage D108 is larger in size than that of phage P1kc. Electron-microscopic observation revealed that phages D108 and P1kc are morphologically different from each other, suggesting that phage D108 belongs to a phage group different from phage P1. The fact that all of the 10 markers tested were transduced by phage D108 indicates that this phage is a generalized transducing phage in the E. coli system. The transduction frequency by phage D108 of chromosomal markers and of a drug resistance factor (R factor) ranged from 2 x 10(-6) to 3 x 10(-8) and 3 x 10(-9) to 6 x 10(-10) per phage, respectively. The cotransduction frequency of the thr and leu markers was 2.8% for phage P1kc and 1.5% for phage D108. The CM and TC markers (chloramphenicol-resistant and tetracycline-resistant markers, respectively) of the R factor were not cotransduced by phage D108, but the markers were generally cotransduced by phage P1kc. The results suggest that the transducing particle of phage D108 contains a smaller amount of host deoxyribonucleic acid than does phage P1kc.     

Isolation of Transducing Bacteriophages for the Histidine and Isoleucine-Valine Operons in Escherichia coli K-12

In vitro studies have been of great value in elucidating the mechanism of the regulation of several bacterial operons. To obtain a deoxyribonucleic acid preparation enriched for the histidine (his) and for the isoleucine-valine (ilv) operons, we have isolated bacteriophages carrying the his and the ilv regions of the Escherichia coli chromosome. Transposition of the his operon to a site close to the att80 region of the E. coli chromosome has been carried out selecting for integration of a temperature-sensitive F'his(+) in the tonB locus. This transposed strain has been lysogenized with phi80i(lambda). Upon induction of the lysogen, His(+) transductants have been isolated, which, on further induction give rise to HFT (high frequency of transduction) lysates. Preliminary characterization of the transducing phage is reported. The ilv operon, carried on an F' particle, has been fused to an episome carrying the att80 region. The fused episome has been lysogenized with phi80i lambdat68. Upon induction of the lysogen, Ilv(+) transductants have been isolated which on further induction give rise to HFT lysates.     

Isolation of a Specialized Lambda Transducing Bacteriophage Carrying the Beta Subunit Gene for Escherichia coli Ribonucleic Acid Polymerase

A heat-inducible lysis-defective lambda prophage has been integrated directly into the E. coli chromosome at a site (bfe) very closely linked to the ribonucleic acid polymerase mutation rif(d), a dominant rifampin resistance allele. This unusual lysogen has facilitated the isolation of specialized transducing phages conferring rifampin resistance to sensitive cells, and carrying at least the beta subunit gene of RNA polymerase in intact form.     

大肠杆菌棉子糖操纵子

大肠杆菌(Escherichiacoli)棉子糖(raf)操纵子位于质粒,它编码能够使大肠杆菌吸收和利用三糖棉子糖的蛋白,即一个主动运输系统(Raf透性酶),α半乳糖苷酶和蔗糖水解酶。raf操纵子包括启动子rafP,调节基因rafR,操纵基因rafO以及rafA,rafB,rafD三个结构基因。这个操纵子由一个阻遏蛋白RafR负控制,同时以环腺苷代谢降解物基因激活蛋白(cAMPCAP)为中介的正调控也参与调节。     

Derivation of an F-Merogenote and a φ80 High-Frequency Transducing Phage Carrying the Histidine Operon of Salmonella

An F' factor, FS400, carrying the his operon, the gnd gene, and the rfb gene cluster of Salmonella typhimurium was isolated. FS400 was introduced into an Escherichia coli strain having a lengthy deletion of the his gene region. From this strain, Hfr derivatives were isolated which had the F' factor integrated in the tonB locus near the attachment site of phi80. One of the Hfr strains was lysogenized with a heat-inducible, h mutant of phi80, and from this strain a high-frequency transducing phage carrying the his genes and the gnd gene of Salmonella was isolated.     

Effects of Dimethylsulfoxide on the Lactose Operon in Escherichia coli

Fowler, Audree V. (University of California, Los Angeles), and Irving Zabin. Effects of dimethylsulfoxide on the lactose operon in Escherichia coli. J. Bacteriol. 92:353-357. 1966.-Dimethylsulfoxide (DMSO) at a concentration of 5% (v/v) in the culture medium inhibits the growth of Escherichia coli to only a slight extent, and does not affect the differential rate of synthesis of beta-galactosidase. Resting cells remain viable after shaking in the presence of 20% DMSO for 3 hr at 37 C. Both beta-galactosidase and thiogalactoside transacetylase retain almost all activity after incubation in even higher concentrations of the solvent for many hours. DMSO decreases the permeability barrier. The rate of hydrolysis of o-nitrophenyl-beta-d-galactoside (ONPG) in whole cells containing beta-galactosidase but lacking permease is increased in cells treated with 5% DMSO. Several permeaseless strains preinduced for beta-galactosidase will grow on lactose in the presence, but not in the absence, of 5% DMSO. When permeaseless strains are grown on tetrazolium-lactose-agar, the presence of 5% DMSO causes a definite but not marked shift toward the lactose-positive character.     

Isolation and Characterization of a Generalized Transducing Bacteriophage for Acinetobacter

A series of bacteriophages which grow in various strains of Acinetobacter have been isolated. One of these, phage P78, which forms turbid plaques on Acinetobacter strain 78 is specific for this particular host and fails to attack 389 other independently isolated strains of Acinetobacter. Phage P78 appears to be a temperate phage which lysogenizes its host. Various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulfate, mitomycin C, and ultraviolet light are effective inducers of the lysogen. Phage lysates of wild-type cells are capable of transducing auxotrophs of strain 78 to prototrophy at frequencies ranging from 0.3 x 10(-7) to 34 x 10(-7) per plaque-forming unit adsorbed. To date, no linkage has been detected between any of the markers studied in two-factor crosses. Donor phage grown in one particular mutant, strain 78 (arg-1), has been shown to give rise to significantly higher transduction frequencies than when phage is grown on wild-type or other auxotrophic strains. Phage P78 is rapidly adsorbed to its bacterial host and has a latent period of 25 min, and infection results in a burst size of approximately 50. Some of the physical properties of phage P78 and its DNA are described.     

Deletion of the Escherichia coli crp Gene

Spontaneous crp mutants Escherichia coli were selected from a strain that does not require 3',5'-cyclic adenosine monophosphate for CAP activity. Several deletions of the crp gene were characterized. The crp gene was not essential for growth of E. coli. crp mutations reduced the donor ability of Hfr strains.     

大肠埃希菌trp operon的克隆与表达

色氨酸操纵子所表达酶的高效表达和酶活性的提高,从而构建高产色氨酸菌株.利用PCR的方法从大肠埃希菌基因组中直接克隆色氨酸操纵子,并将其连接到原核表达载体pBV220中,得到重组质粒pBV220-trp operon,转化大肠埃希菌DH5α,温度诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性.通过凝胶电泳观察PCR扩增产物大小约为7 kb.SDS-PAGE鉴定目的蛋白得到了高效表达,邻氨基苯甲酸合成酶和色氨酸合成酶的活性分别比对照提高了3.4倍和2.5倍.成功构建了重组质粒pBV220-trp operon,邻氨基苯甲酸合成酶和色氨酸合成酶的表达量和表达活性在大肠埃希菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础.     

Impact of rpoS Deletion on Escherichia coli Biofilms

Slow growth has been hypothesized to be an essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (ΔlacZ rpoS⁺) and its isogenic ΔrpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h⁻¹) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P < 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h⁻¹. Overall, these studies suggest that rpoS is important for biofilm physiology.     

Effects of Lac Operon Activation,Deletion of the Yhha Gene,and the Removal of Oxygen on the Ultra-Weak Photon Emission of Escherichia coli

We observed a relation between gene activity and ultra-weak photon emission (UPE). By comparing the UPEs of E. coli with the LacI gene present and deleted we found that more gene activity produced higher UPE. This relation was further confirmed by studying the UPE of the E. coli with and without the Yhha gene. We interpreted that a higher aminoacyl t-RNA synthetase activity, which used ATP from the respiratory chain, could increase the emission. Satisfying the increased need of ATP by the E. coli through an increase of respiratory chain activity, which has reactive oxygen species (ROS) as a byproduct, results in a higher rate of photon emission. To ensure that oxygen is at the origin of this emission, we replaced the air by pure nitrogen. After 30?min, it was observed that the emission levels equaled the emission levels of the sterile medium. We could therefore conclude that the source of the photon emission would be affected by genetic activity and is oxygen related.     

Escherichia coli Mutants Permissive for T4 Bacteriophage with Deletion in e Gene (Phage Lysozyme)

Escherichia coli mutants have been isolated that are permissive for the infection by T4 phage with deletion in the cistron for the phage lysozyme, the e gene. Some, but not all, of these mutants are simultaneously permissive for the infection by T4 phage defective in the t gene, the product of which has also been implicated in the release of progeny phages. Most of these mutants shared the following properties: temperature sensitivity in growth and cell division, increased sensitivity towards a number of unrelated antibiotics and colicins, and increased sensitivity towards anionic detergents (sodium dodecyl sulfate and sodium deoxycholate). The possible biochemical basis for these phenotypes is discussed.     

Threonyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli: Subunit Structure and Genetic Analysis of the Structural Gene by Means of a Mutated Enzyme and of a Specialized Transducing Lambda Bacteriophage

Threonyl-transfer ribonucleic acid synthetase (ThrRS) has been purified from a strain of Escherichia coli that shows a ninefold overproduction of this enzyme. Determination of the molecular weight of the purified, native enzyme by gel chromatography and by polyacrylamide gel electrophoresis at different gel concentrations yielded apparent molecular weight values of 150,000 and 161,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band of 76,000-dalton size. From these results an alpha(2) subunit structure can be inferred. A mutant with a structurally altered ThrRS, which had been obtained by selection for resistance against the antibiotic borrelidin, was used to map the position of the ThrRS structural gene (thrS) by P1 transductions. It was found that thrS is located in the immediate neighborhood of pheS and pheT, which are the structural genes for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase, the gene order being aroD-pheT-pheS-thrS. A lambda phage that was previously shown to specifically transduce pheS, pheT, and also the structural gene for the translation initiation factor IF3 can complement the defect of the altered ThrRS of the borrelidin-resistant strain. This phage also stimulates the synthesis of the 76,000, molecular-weight polypeptide of ThrRS in ultraviolet light-irradiated. E. coli cells. These results indicate that the genes for ThrRS, alpha and beta subunits of phenylalanyl-tRNA synthetase, and initiation factor IF3 are immediately adjacent on the E. coli chromosome.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.