Fertility functions of resistance factors

Summary The isolation of transducing phages carrying the tolPAB cluster is described. These genes map between gltA and gal in Escherichia coli, and thus are relatively close to att. To isolate these transducing phages, it was necessary to use a strain deleted of most of the intervening genes (nadA to chlD) between tolPAB and att. Using a lysogen of such a deletion strain, several defective dtol phages were isolated that carry different amounts of the tolPAB cluster.All of these dtolPAB phages were defective in both lysogenization and vegetative growth, and in this respect were similar to dgal transducing phages.The usefulness of such specialized transducing phages in studying the cell surface is discussed.Research Fellow of the National Cancer Institute of Canada.     

Plasmid formation from bacteriophage lambda in Escherichia coli

Summary Among the survivors of Escherichia coli derivatives infected with phage c1 or vir that are unable to establish ordinal lysogeny, clones arise which perpetuate the nondefective phage genome. When the bacteria bears a mutation(s) that makes the cell tolerant to the phage multiplication, such clones appear readily.The bacteria- complex was studied genetically and chemically, and it was concluded that the intact phage genomes, about two to four circular copies per bacterial chromosome, are perpetuated in bacterial cytoplasm as plasmids or in lysogenic state in cytoplasm.Several lines of evidence suggests that the phage genome in the lysogenic state in cytoplasm is under a different regulatory system from that in the normal prophage state on chromosome.     

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element

Summary Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield trp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of trp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of -transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final ::Tn409 or ::Tn410 product.Abbreviations p.f.u. plaque-forming units - MIC minimal inhibitory concentration - LFT low frequency transducing - HFT high frequency transducing     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.     

lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22.

Summary Genetically marked and P22 phages were recombined in Escherichia coli-Salmonella typhimurium hybrid WR4028, a host sensitive to infection by both of these phages. Hybrid phages that acquired the immC region of P22, but retained the genes for the protein coat were selected on WR4027 (), a -immune, P22-resistant derivative of WR4028. In these immP22 hybrids, at least the c through P genes of were replaced with functionally related P22 genes. Phage recombinants with more extensive regions of the P22 genome were selected on the double lysogen WR4027 (, immP22). One such hybrid, immP22dis, was determined by heteroduplex analysis to contain approximately 40% of the P22 genome. Genetic studies established that immP22dis possesses the two widely separated immunity control regions of P22 (immC and immI) and that these loci are expressed in E. coli K-12 lysogenic for immP22dis. In addition, immP22dis contains the P22 a1 locus responsible for somatic 0–1 antigen conversion in Salmonella. Although the immP22dis phage particle has the head and tail, the phage genome also carries P22 tail gene 9 as evidenced by the production of free P22 tails. It also has the P22 att site as indicated by the integration of the immP22dis prophage near the proA locus on the bacterial chromosome.     

Isolation and characterization of lambda b221poriCasnA, a plaque-forming specialized transducing phage carrying the origin of replication of the Escherichia coli chromosome

Summary A specialized transducing phage, b221poriCasnA has been isolated carrying oriC the origin of chromosomal replication of Escherichia coli. All phage genes required for lytic growth are retained, thus the phage is capable of lytic growth. The presence of the oriC locus confers upon infecting phage DNA the ability to replicate as a plasmid using only host DNA replication functions. The presence of both oriC and asnA markers has allowed the development of a plaque assay for origin function which can be used to identify mutants at these loci. Comparison of restriction endonuclease cleavage sites present on b221poriCasnA DNA to those on tis parent, b221 rex::Tn10 suggests the steps involved in the formation of the transducing phage.     

In vitro insertion of the lambda attachment site into the plasmid RP4.

Summary The region of the phage lambda chromosome containing the attachment site (P · P) and the genes int and xis, excised by the action of endonuclease R.EcoRI, has been inserted into the unique site for that enzyme on the promiscuous conjugative plasmid, RP4, generating the recombinant plasmid RP4att. Transformants containing the hybrid plasmid were recognised by their ability to allow efficient lysogenization by phage b2 (Weil and Signer, 1968; Echols et al., 1968) containing the mutant attachment site · P. The construction and properties of the hybrid plasmid RP4att are described.     

Host specificity of DNA produced by Escherichia coli. XVI. Phage lambda DNA carries a single site of affinity for A-specific restriction and modification

Summary Bacteria with A-specific restriction plate unmodified phage with an efficiency of 10^-2. One mutational event can produce restriction insensitive (sA^o) mutants of . These differ from the original sA form of by no other property than their response to A-host specificity. Two-parental phage crosses involving sA and sA^o, respectively, as non-selective marker allowed to map sA between genes cII and O. These data indicate that sA is the only site on DNA with affinity for A-specific restriction. DNA is thus an interesting substrate in in vitro A-specific restriction and modification. Using an assay based on the infectivity of DNA on helper-infected bacteria, A-specific modification activity was found in partially purified sonicates of bacteria with A-host specificity. In parallel to modification, ³H-methyl label from s-adenosylmethionine, the only cofactor required for modification, was transferred to unmodified DNA. No association of radioactivity was observed in control experiments with DNA from either modified ·A or from asA^o mutant. These data suggest that A-specific modification is brought about by DNA methylation and that the sA^o mutation not only abolished the affinity for A-specific restriction, but also for A-specific modification.     

Construction in vitro of "phage-plasmid" chimerae: a new tool to analyse the mechanism of plasmid maintenance.

Summary In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Ap^r plasmid: pSC138 (Timmis et al., 1975). F recombinants were shown to be chimerae between the and the F replicons. By genetical tests, we have demonstrated that both and F replication mechanisms are functional: the F recombinant behaves as a non defective plaque forming phage on sensitive bacteria and establishes itself as a stable plasmid on recA F^- homoimmune bacteria. In the extra-chromosomal state, the F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.     

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function

Summary Escherichia coli mutants, called groNB, which block the growth of bacteriophage at the level of action of the gene N product, have been isolated as survivors at 42°C of bacteria carrying a) the defective prophage bio1 1 i cI857 H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage cI857. In addition, groNB bacterial mutants have been isolated at 37° C, as large colony formers in the presence of i cI h ⁴³⁴, i cI h , and i cI h ⁸⁰ phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42° C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro⁺ for growth. Lambda transducing phages carrying the groNB ⁺ bacterial gene have been isolated. GroNB mutant bacterial lysogenized by the transducing phage acquire the Gro⁺ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.     

Analysis of a temperature sensitive mutation in gene cII of bacteriophage lambda

Summary The mutation cIIts612 was found to map outside the immunity region of phage imm21 hybrid. As expected of a cII mutation, cIIts612 is unable to stimulate either cI repressor or Int synthesis during the establishment of lysogeny. These results indicate that part of the cII gene of is homologous to that of imm21 phage.     

Studies on lambda virulent mutants

Summary The clearish plaque mutants virC which were isolated from true-virulent, virLvirCvirR (virLCR), do not complement CI mutants but CII, CIII and mutant (c ₄₂) for lysogenization. No complementation for lysogenization was observed between virCR and any CI, CII, CIII or y mutants. No lysogen was obtained when virC or virC carrying susN, susO or susP was infected to -sensitive sup ^- host. This was also true for virCR. Infection of ind ^- lysogen with virCRsusNO(P) or virCsusNO(P) results in marked prophage induction. Effect of virCRsusNO(P) on prophage induction is stronger than that of virCsusNO(P). These results suggest the existence of gene(s) for anti-repressor. When virCsusNO(P) or virCRsusNO(P) was infected to W3350 sup ^- at high m.o.i., lysogen in anti-immune state and that in weak-immune state was obtained, respetively. Wild type phage forms clear plaque on virCsusNO(P) lysogen with e.o.p. of one and no plaque on virCRsusNO(P) lysogen. T4rII can plate on both lysogens. This weak-immunity caused by virCRsusNO(P) prophage is different from CI immunity and not abolished by irradiation of ultraviolet light (hereafter this is referred to as the vir-immunity). Action of anti-immunity and vir-immunity are almost specific. Possible functional sites for anti-and vir-immunity substances are suggested to be virL and virR regions. A hypothesis was presented that the vir-immunity may caused by the overproduced anti-immunity substance coded from x region.This material has been published as an abstract in Jap. J. Genetics 45, 479 (1970).     

Lambda transducing phages for the nalA gene of Escherichia coli and conditional lethal nalA mutations.

Summary Defective transducing phages for the nalA region of the Escherichia coli chromosome were isolated from a lysogen in which is inserted in the nearby glpT gene. The three classes of transducing phages designated nrdA, dubiG, and dnalA contained bacterial DNA extending from glpT through nrdA, ubiG, and nalA, respectively. The bacterial genes are in the left arm of the chromosome. Of the eleven polypeptides coded by dnalA that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate only one was not also specified by dubiG This 105,000 dalton polypeptide is the nalA gene product. The electrophoretic mobility and isoelectric point of this protein were unaffected by a nalA mutation (nalA48) that confers nalidixic acid resistance. Temperature-sensitive and amber mutations in the nalA gene were isolated using a dnalA48 lysogen which is heterodiploid for nalA. The conditional lethality of these mutations proves that nalA is an essential locus.     

lamB mutations in E. coli K12: Growth of λ host range mutants and effect of nonsense suppressors

Summary Over sixty EMS induced mutations affecting gene lamB, presumably the structural gene for the receptor in Escherichia coli K12, were examined for growth of host range mutants and effect of nonsense suppressors. By the first criterion the mutations could be grouped in three classes. Bacteria with class I mutations allow growth of mutants with extended host range (noted h) of the type already described (Appleyard, Mac Gregor and Baird, 1956). Bacteria with class II mutations allow growth of h mutants with still more extended host range (noted hh ^*). No host range mutants of could be found which would grow on bacteria with class III mutations. Using nonsense suppressors it was found that class I and II consist of missense mutations, while class III consists of nonsense mutations. Exceptions are likely to exist (especially in class III) but were not found among the mutations tested. These observations are briefly discussed in terms of outer membrane protein integration and of phage receptor interaction.     

Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA

Summary The purified bacteriophage replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of O and one molecule of P. The O-P oligomer is active in the in vitro replication of ori-containing DNA.Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the replication proteins and ori DNA. It was found that the P protein binds specifically to ori-containing plasmid DNA only in the presence of O protein. About 100 molecules of O and 10 molecules of P form a complex with the ori DNA. The DNA-O-P complex was shown to be active in an in vitro replication system.Since the physical interactions between ori and O and between P and the Escherichia coli dnaB replication protein are well documented, the evidence for a O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables to direct the host replication machinery to the replication of its own DNA.     

Directed integration of an F'' plasmid by integrative suppression: isolation of plaque forming lambda transducing phage for the dnaC gene.

Summary A new approach for isolation of a plaque forming specialized transducing phage is described. It consists of directed transposition of an F plasmid into the gal region of a dnaAts galE ^- Escherichia coli strain by integrative suppression and deletion of the chlD region in order to shorten the distance between the marker of interest on the F and the prophage serving to prepare an LFT¹ lysate.An F danC ⁺ thr ⁺ plasmid was used here and dthr and ddnaC phages were isolated. In addition, pdnaC was obtained from a double lysogen for ddnaC and b2.     

Construction and some properties of packageable plasmid F

Summary A derivative of plasmid F which is packageable in phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus Plasmid that carries the -lactamase gene, and a portion of guaA (B) transducing phage DNA carrying cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7×10⁷ daltons, about 84% size of phage genome, and can be packaged in coat when helper phage replicates in the plasmid-carrier cell. The package plasmid and the helper phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F^- cell and establishes a plasmid state with normal efficiency. In F⁺ or Hfr cells, the resident F factor hinders this process.