Transducing lambda phages with Escherichia coli genes

The paper presents data on transducing lambdoid phages containing Escherichia coli genes. The major genetic techniques for isolating transducing phages (in vivo) are outlined. A combined table of best-studied transducing phages obtained by the methods of molecular genetics and genetic engineering lists phages genotype & basic literature references for the phages and their derivatives. The chromosome fragments of E. coli inserted in phage DNA are separately specified. Another table presents information about phages carrying E. coli fused operons and genes. The paper also provides detailed physical maps of three regions of the E. coli chromosome. The bibliography contains 300 items.     

质粒在大肠杆菌对噬菌体抗性中的作用

The introduction of the ColV, I-K94 or R124 plasmid into Escherichia coli K12 resulted in resistance to certain phages. Derivatives of E. coli carrying the plasmid R124 and ColV, I-K94 were resistance to the phages T4, Mel comparing with the plasmid-free parent and the plasmid ColV, I-K94 conferred resistance to the phage Tull*. It suggested that an envelope change caused by the plasmids might be responsible for the resistance because most of the phages fell to absorb to the plasmid-bearing E. coli cells.     

Insertion of DNA carrying ribosomal protein genes of Escherichia coli into Charon vector phages.

DNA fragments from lambdaspc1 and lambdafus2, carrying ribosomal protein genes from Escherichia coli, were inserted into lambda phage vectors Charon 3 and Charon 4. Eight of the resulting clones were characterized by agarose gel electrophoresis of EcoRI digests, analytical CsCl equilibrium centrifugation, and electron micrographic analysis of heteroduplexes. In each case, the identity, order, and orientation of each cloned fragment was determined. In all, 8 of the 12 EcoRI fragments of lambdafus2 were cloned in various arrangements. In the accompanying paper, genes for 15 ribosomal and related proteins and three bacterial promoters were detected in these phages. In addition, four of the hybrid phages carried fragments of lambda-DNA including the phage origin of replication (ori), the late promoter, PR', and the cohesive ends (cos site) in both orientations. The latter phages yield a circularly permuted collection of DNA molecules.     

Construction of recombinant lambda phages that carry the E. coli recB and recC genes

Summary A fragment of the E. coli chromosome including the recC gene has been cloned by in vitro recombinant DNA techniques into a phage vector to give the recombinant phage drecC. This was used to derive the phage drecBC by in vivo recombination. On lysogenisation of recB and recC mutants with drecBC wild type levels of UV-resistance and RecBC DNase activity were restored. Infection of E. coli with drecBC led to the synthesis of phage-coded proteins of 125 kilodaltons (kd) and 135 kd that were not synthesised on infection with the original vector, whereas a 125 kd protein but not a 135 kd protein was synthesised in similar experiments with drecC. The recombinant phages, which are unable to form plaques, presumably due to the deletion of essential phage genes during their construction, provide useful starting points from which to subclone the recB, recC, and the neighbouring thyA and argA genes individually into multiple copy plasmid vectors.     

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli

Summary UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage , _i21, _i434 and 80. Maximal induction in lexA3 lysogens needed less UV than in lexA ⁺ bacteria and gave 25–100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA ⁺ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.     

Lytic phages obscure the cost of antibiotic resistance in Escherichia coli

The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.     

Detection of Escherichia coli with fluorescent labeled phages that have a broad host range to E. coli in sewage water

Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e-/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E. coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.     

Isolation and characterization of lambda transducing phages for the E. coli genes ksgA and pdxA

Summary Lambda phages carrying the Escherichia coli genes ksgA and pdxA were isolated from secondary site lysogens in araB. 1) The phage genomes were characterized by genetic complementation tests, restriction endonuclease digestion and electron microscopy. 2) A 6.3 kilobasepair (kb) EcoRI restriction fragment carrying both ksgA and pdxA was cloned in a lambda vector; this fragment has proven useful in further characterization of the ksgA gene (Andrésson and Davies, 1980a, b). The ksgA and pdxA genes are about 14 and 12–13 kb, respectively, counterclockwise of the arabinose operon and 1.5 and 2.5–3.5 kb clockwise of folA.