Werner Arber

Host specificity of DNA produced by Escherichia coli: II. Control over acceptance of DNA from infecting phage λ*

DNA of λ·K (λ phage grown on E. coli strain K12) is shown to be degraded upon infection of the new host strains E. coli K12(P1) or E. coli B. This breakdown begins shortly after phage attachment and successful DNA injection. 32 P label from the λ·K DNA submitted to this degradation appears partly in acid-soluble components (organic and inorganic) and partly in acid-insoluble compounds. The host cell survives such an infection and permits diffusion of a fraction of the degradation products into the medium, while probably re-using another fraction. Genetic markers from λ·K are rescued in K12(P1) host cells infected with both restricted λ·K and unrestricted λ·K(Pl). Since DNA breakdown competes in time with the rescue, the probability of marker rescue is high if the unrestricted phage infects first and low if the restricted phage infects first. Only closely linked markers have a good chance to be rescued together. The host specificity imparted to phage DNA by the bacterial strain on which it was produced is thought to be responsible for its recognition as incompatible with a new host strain. Bacterial mutants are described which, despite the presence of prophage P1, accept infecting λ·K at relatively high rates.

Electron microscopical studies of phage multiplication: I. A method for quantitative analysis of particle suspensions

Abstract The agar-filtration method for preparing particle suspensions for the electron microscope is described and its application to particle counting is studied. The coefficient of variation of this method is found to be about 15%. The precision of the absolute determination of titers is, however, limited by the precision of the determination of the latex sphere diameter and is estimated to be 20 to 30%.

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

SummaryA cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20.DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.

HOST SPECIFICITY OF DNA PRODUCED BY ESCHERICHIA COLI V . THE ROLE OF METHIONINE IN THE PRODUCTION OF HOST SPECIFICITY.

Bacteriophage λ grown in auxotrophic met−, pro− or arg− strainsf of Escherichia coli K12 in the presence of the required amino acids show an efficiency of plating of approximately 1 on E. coli strains K12 and C. However, if met− cells are deprived of methionine during a portion of the latent period, the efficiency of plating of the progeny phage is lower on the host K12 than on strain C. Similar results are obtained with met− auxotrophs of strains B and K12(P1); deprivation of methionine during the latent period results in the production of phage with lower efficiency of plating on the host strain than on strain C. Such an effect is not observed following a similar starvation for proline or arginine. These results suggest that methionine is specifically required for the production of host specificity of DNA.

E. coli K-12 pel mutants, which block phage ? DNA injection, coincide with ptsM, which determines a component of a sugar transport system

SummaryEscherichia coli pel- mutants inhibit the penetration of bacteriophage lambda DNA into the cell. Using P1 mediated cotransduction, we mapped pel- mutations between markers fadD and eda in the interval of minute 40 of the revised E. coli K-12 map. This places pel in the same region as genes kdgR and ptsM. Mutations in kdgR usually do not alter the Pel phenotype, and vice versa. In contrast, about 30% of ptsM- mutants are also pel-, and all pel- mutants isolated are ptsM-. These results suggest that pel and ptsM are one and the same gene. This interpretation would identify the bacterial product required for injection of phage λ DNA as a component of the phosphoenolpyruvate-dependent phosphotransferase system specific for mannose, glucosamine, glucose and fructose. However, the experimental results do not exclude an alternative explanation: that pel and ptsM identify two closely linked genes which would be simulataneously affected at high frequency by a particular mutational event.

An Escherichia coli mutant which inhibits the injection of phage λ DNA

Abstract A new mutant of Escherichia coli K12, called pel − , inhibits the growth of phages λ, 434, and 82 but not of φ80. This inhibition is overcome by λhp mutants, some of which are temperature sensitive for growth in pel + and pel − bacteria. Phage λ adsorbs normally to the pel − host, but only 2–10% of the infected cells produce phage with a normal burst size or become lysogenic. The remainder of the cells survive the infection. The growth defect of λ cannot be complemented in trans upon simultaneous infection with λhp . When pel − strains lysogenic for λ are induced, 100% of the induced cells yield phage, and the burst size is normal in contrast to the small probability of phage growth after infection. After adsorption of λ to pel − cells, active phage do not elute spontaneously from the complex. Neither are adsorbed phage released in an inactivated form. Infection of pel − (P1) with 32 P-labeled unmodified λ does not lead to degradation of the phage DNA by P1-specific restriction, whereas the DNA of λhp is degraded. Electron micrographs of λ infected cells show phage particles with empty heads attached to the surface of pel + cells, while the particles attached to pel − cells appear to have full heads. We conclude that the pel − mutant allows adsorption of λ but that it inhibits the subsequent injection of the phage DNA.

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1.

SummaryThe genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.

Phage λ DNA injection into Escherichia coli pel− mutants is restored by mutations in phage genes V or H

Abstract Escherichia coli pel − mutants block the growth of phage λ by inhibiting DNA injection. λ hp mutants regain the ability to inject into pel − hosts. Most λ hp mutants are not temperature sensitive. All such mutants analyzed (15 independent mutants) lie within gene H . A few percent of the λ hp mutants are temperature sensitive; at 42° they grow neither on pel + nor on pel − hosts. Reversion studies suggest that the hp and the temperature-sensitive phenotypes are due to the same mutation. The λ hp ts phage particles are heat stable and inject their DNA at 42°. The thermolabile step appears to be expressed late in infection, during phage assembly. Genetic mapping places all λ hp ts mutants tested (13 independent mutants) in gene V . Our data indicate that the major λ phage tail protein V and the minor tail protein H play a role in DNA injection.

On the host-controlled modification of bacteriophage λ☆

Abstract Phage λ.C, grown on Escherichia coli strain C, is restricted on E. coli strains K12, K12(P1) and B251, and its DNA is broken down after injection into these bacterial strains. Phage λ.K or λ.K(P1)—that is, grown on K12 or K12(P1)—is accepted by C, but undergoes host controlled modification upon reproduction in C ( Weigle and Bertani, 1953 ). In a one-cycle growth of λ.K(P1) on E. coli C, parental DNA and host specificity undergo linked transfer into the progeny. Thus, host specificity is a property of the DNA molecule, as was previously shown for phage λ in another host system ( Arber and Dussoix, 1962 ). Host specificity is serially transferable, and the probability of transfer to the progeny is constant for any given DNA strand under given experimental conditions.

Multiple physical differences in the genome structure of functionally related bacteriophages P1 and P7

SummaryComparative restriction cleavage analysis of the genomes of bacteriophage P7, of several recombinant phages between P7 and P1, and of bacteriophage P1 allowed to draw PstI, BglII, BamHI and HindIII cleavage maps of all genomes studied. The data obtained complement Yun and Vapneks (1977) conclusions with regard to areas of major nonhomology based on electron microscopical heteroduplex analysis and they identify several additional minor differences between P1 and P7. The use of hybrid phage strains allowed to locate the genes for particular functions on the physical genome map.