Akira Taketo

Synthesis of α3 phage DNA in replication mutants of Escherichia coli

Abstract Host functions for DNA replication of bacteriophage α3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna+ cells, conversion of phage α3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30–150 μg/ml of chloramphenicol, 200 μg/ml of rifampicin, 50 μg/ml of nalidixic acid, or 200 μg/ml of novobiocin. At 43°C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage α3 progeny RF was prevented by 50 μg/ml of mitomycin C (in hcr+ bacteria), 50 μg/ml of nalidixic acid or 200 μg/ml of novoviocin, but neither by 30 μg/ml of chloramphenicol nor by 200 μg/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for the progeny RF synthesis. Host factor dependence of α3 was relatively simple and, in contrast with phages oX174 and G4, α3 did not require dnaB and dnaC(D) activities.

Host genes involved in the replication of single-stranded DNA phage ϕK

SummaryUsing various replication mutants of E. coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined. Functional products of dnaE,-F,-G and -Z genes are required for the multiplication of ϕK, whereas dnaA,-B,-C(D), H,-I and -P are dispensable for viral replication. In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for ϕK. At the restrictive temperature, the yield of ϕK was markedly reduced in the ligts7 mutant and partially decreased in a polAts strain. The phage ϕK is thus less dependent on the host cells than ϕX174 and ϕA which require additionally the dnaB,-C(D) and -H functions. Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function. Although not much affected in polAts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative from in dna mutants of Escherichia coli

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.

Replication of ϕA and ϕX174 in Escherichia coli mutants thermosensitive in DNA synthesis

SummaryThermosensitive mutants of E. coli defective in DNA replication were tested for their capacity to support multiplication of ϕA and ϕX174. At the restrictive temperature, the viral growth was markedly affected in dnaH, dnaZ or ligts7 mutants. Even when these strains were transfected with RF1 molecules, the virus yield was still very low. The dnaI function was, however, dispensable for replication of ϕA and ϕX174. In addition, these viruses could multiply in dnaP or polAtsmutants at the high temperature.

Growth of single-stranded DNA phages in replication mutants of Escherichia coli

SummaryHost capacity for growth of single-stranded DNA phages was investigated with several replication mutants of E. coli. In dnaL708, dnaM709 and dnaS707 mutants, multiplication of ϕK was not restricted even at 42°C. In dnaM710 cells, however, growth of ϕK was severely affected at 42°C but not at 33°C. Upon infection of ϕK, parental replicative form was synthesized at the restrictive temperature, whereas subsequent step (replication of progeny replicative form) was blocked in the dnaZ strain. Growth of ϕX174 and α3, as tested by transfection, was also thermosensitive in the dnaM710 mutant but not in the dnaL708, dnaM709 and dnaS707 strains. In contrast with λ, microvirid phages could grow in E. coli cells bearing the groPC259, groPC756 or seg-2 mutation.

Host factor requirements and some properties of ΦXtB

SummaryReplication of ΦXtB, a capsid mutant of bacteriophage ΦX174, depends on the host functions directed by the E. coli genes dnaE, dnaF, dnaG, dnaZ, lig and rep. The cellular products of dnaA, dnaB, dnaC(D), dnaI, dnaP, polA, polB and xth genes are, however, dispensable for the viral growth. In these host factor requirements, ΦXtB resembles phages ΦK and St-1, rather than ΦX174. Host ranges of ΦXtB, St-1 and ΦK overlap considerably, and growth temperature of the three phages is somewhat higher than that of ΦX174. Furthermore, ΦXtB is, like ΦK, inactivated by antiserum against St-1. ΦXtB may thus fill an evolutionary gap between the ΦX174 group and the St-1 group.

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.

Effect of the dnaN Mutation on the Growth of Small DNA Phages

SummaryThe effect of the dnaN mutation on the growth of single-stranded DNA phages was studied by burst experiments. In HC138 dnaN cells exposed to 42.5° C at 5 min before infection, growth of spherical (microvirid or isometric) phages such as α3, πKh-1 and πX174 was partially reduced at the nonpermissive temperature. When infection was performed at 30 min after temperature shift-up, viral replication was completely inhibited at 42.5° C in the dnaN strain but not in a dna+ revertant. At 41° C, multiplication of filamentous (inovirid) phages M13 and fd was restricted specifically in HC138 F+dnaN bacteria. When dnaN cells lysogenic for λi21 were grown at 42.5° C for 60 min and then shifted down to 33° C, a burst of λi21 occurred with concomitant cellular lysis, manifesting induction of the prophage development.