Participation of rec genes of Escherichia coli K 12 in W-reactivation of UV-irradiated phage lambda.

The effect of the recombinational deficiency on W-reactivation of UV-damaged phage lambda was explored. In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment. Combination of these mutations in the recB21recF143 double mutant blocks W-reactivation completely after BM induction, but leaves residual W-reactivation ability after UV-irradiation, which is abolished by the introduction of uvrB deficiency (delta(uvrB-chlA]. W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role of the RecBC(D) enzyme in W-reactivation.     

tif-dependent induction of colicin E1, prophage lambda, and filamentation in Escherichia coli K-12.

To help understand how the tif-1 mutation of the recA gene of Escherichia coli confers adenine activability on the recA protein, we used the fact that cytidine plus guanosine inhibits induction of prophage lambda and cell filamentation in a tif-1 mutant, and that adenine reverses this inhibition. We varied the amount of adenine in agar plates containing a fixed amount of cytidine and scored for survivors of three different tif-dependent lethal induction processes. Much more adenine was required for cell killing when cytidine was present than when it was absent. Therefore adenine does not override cytidine inhibition, but instead appears to compete with it for a site of action which may be on the recA protein. The competition is not at the cell transport level. Our results lead to a model in which the tif form of the recA protein is an allosteric enzyme that binds both negative and positive modulators. By varying the adenine-cytidine ratio of the medium it is possible to control the degree of induction in a tif-1 cell. For the three different tif-dependent inductions studied here, least adenine was required for lambda induction and most for lethal filamentation, presumably reflecting requirements for different amounts of activated recA protein in each process. Varying the adenine-cytidine ratio revealed two stable intermediate stages in lambda induction, as well as a stage of colicin E1 induction in which the cells produced colicin without cell death. The rate of filament formation could be similarly controlled. Experiments with tif (ColE1, lambda) gave evidence of a competition between colicin repressor and lambda repressor for activated recA protein.     

DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations.

Summary In this article we describe some in vivo properties of a coldsensitive ribosomal mutant from Escherichia coli. The mutation affects the rplV gene which is the structural gene of ribosomal protein L22.Our work shows that at 22°C, the biosynthesis of both ribosomal subunits and the maturation processing of 16S and 23S ribosomal RNA are impaired. Integration of our results in a general model of in vivo ribosomal assembly in E. coli is presented.     

A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase.

A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing lambda phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (Mr 78,000) is still unidentified.     

OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.     

Effect of bacteriophage P1 lysogeny on lipopolysaccharide composition and the lambda receptor of Escherichia coli.

The outer membrane of Escherichia coli was altered as a consequence of lysogeny by bacteriophages P1 and P1 cmts. The predominant change was a reduction in the size of lipopolysaccharide to a heptose-deficient form. P1 cmts lysogens were still sensitive to several bacteriophages but were resistant to lambda vir. Neither whole cells nor solubilized outer membranes from P1 cmts lysogens were able to inactivate lambda vir, and 32P-labeled lambda vir was unable to adsorb to P1 cmts lysogens. P1 cmts lysogens were also affected in maltose transport. The level of periplasmic maltose-binding protein was reduced somewhat, but there was no significant reduction in the level of the outer membrane lambda receptor (LamB). These membrane abnormalities were all corrected in strains cured of P1 cmts. It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequently the function of the lambda receptor.     

The phage P.E1 isolated from hospital sewage reduces the growth of Escherichia coli

This study involves partial characterisation of a lytic bacteriophage P.E₁ against a multi drug-resistant clinical isolate of Escherichia coli, isolated from hospital sewage supply. The phage P.E₁ has showed a narrow host range suitable for its use in phage therapy. Phage showed lytic activity up to 70°C and at alkaline conditions, but at higher acidic conditions its activity decreased. Latent period and burst size of P.E₁ estimated from single-step growth curve was 40 min and 185 plaque-forming units per cell, respectively. The phage P.E₁ reduced the growth of host bacteria during the initial 12?h of infection; however, the host bacteria developed resistance afterwards. During the 24-hour observation period, the bacteriophage could still reduce the growth of its host bacteria evident by lower optical density in the phage-treated samples compared with control. The phage genome was double-stranded DNA and larger than 12?kb in size. Further manipulations of genome and proteins may help to unveil the unique aspects of this phage, to use it in phage therapy against E. coli.     

The need for protein synthesis in UV-irradiated Escherichia coli cells for fixation of induced Str mutations

The kinetics of accumulation of fixed Str mutations was determined during incubation in nutritional medium of Escherichia coli WP2 irradiated with 6.8 J/m2 either at log growth phase or after completion of DNA replication. Those Str mutations which lost ability for photoreactivation (fixation I) or susceptibility to antimutagenic activity of mfd-type (fixation II) were considered as fixed mutations. It was shown that both fixations occurred synchronously, starting in about 10 min after irradiation and being over in 40-50 min. In cells irradiated after completion of replication, fixation depended on protein synthesis de novo: chloramphenicol added to irradiated culture blocked fixation. An attempt to study the effect of chloramphenicol on fixation in a culture irradiated at the log phase failed, because of high lethal action of the antibiotic on such cells. Fixation could proceed in the presence of acriflavine. Possible mechanisms for fixation of Str mutations are discussed in connection with the fact of its dependence on protein synthesis.