Journal of Molecular Biology
Volume 18, Issue 3, July 1966, Pages 429-447, IN1-IN2
Journal home page for Journal of Molecular Biology

The process of infection with bacteriophage ΦX174: X. Mutations in a ΦX lysis gene

https://doi.org/10.1016/S0022-2836(66)80035-9Get rights and content

The ability of conditional lethal mutants of phage ΦX174 to induce host cell lysis during infection under restrictive conditions has been studied. We have found amber (am) and temperature-sensitive (ts) mutants which present a variety of alterations in the normal lytic process. In particular, there is a class of am mutants which do not produce cell lysis but otherwise replicate normally in the restrictive host. These mutants constitute a single complementation group. The existence of these mutants implicates a phage-coded protein in the lytic process. This protein is not an essential structural component of the phage, since normal phage particles are produced in the absence of lysis.

The growth of a particular mutant of this type, ΦX am3, in the restrictive host, has been studied in detail. Phage maturation starts at approximately the same time and at the same rate as in a ΦX wild type (wt) infection. Maturation of ΦX am3, however, continues at this rate for about two hours. Burst sizes of 2 to 3 × 103 phage per cell are obtained. This is about ten times the normal ΦX wt phage yield. Only 1 to 2% of the phage are released spontaneously. The rest remain intracellular until released by artificial lysis. The turbidity of an infected culture continues to rise after infection. This effect primarily represents elongation of the infected cells, without increase in cell number. Cells infected by ΦX am3 are not able to form a colony and do not undergo more than a single division.

We have used ΦX am3 to study temporal exclusion in ΦX infection under conditions that are not complicated by lysis of the infected cells. ΦX wt is excluded by prior infection with ΦX am3. The superinfecting phage is blocked at a stage prior to synthesis of the molecule of the parental replicative form; its DNA remains an intact (infective) single strand.

We have also used ΦX am3 to study the effect of addition of chloramphenicol at various times during infection upon subsequent viral DNA synthesis. If chloramphenicol is added before initiation of progeny single-stranded DNA synthesis, the normal transition from replication of replicative form to single-stranded DNA formation does not occur; replication of the replicative form continues for at least 40 minutes. If chloramphenicol is added after the initiation of single-stranded DNA synthesis, such synthesis continues, at a lesser rate than normal, for 20 to 25 minutes; synthesis of replicative form is resumed for at least 40 to 50 minutes and its concentration may reach a level several-fold that normally observed.

References (25)

  • A. Burton et al.

    J. Mol. Biol.

    (1965)
  • D.T. Denhardt et al.

    J. Mol. Biol.

    (1965)
  • D.T. Denhardt et al.

    J. Mol. Biol.

    (1965)
  • C.E. Dowell et al.

    J. Mol. Biol.

    (1966)
  • R. Fujimura et al.

    Biophys. J.

    (1962)
  • A. Garen et al.

    J. Mol. Biol.

    (1965)
  • G.D. Guthrie et al.

    Biochim biophys. Acta

    (1963)
  • J.B. Hall et al.

    J. Mol. Biol.

    (1963)
  • P.H. Hofschneider et al.

    J. Mol. Biol.

    (1963)
  • C.A. Hutchison et al.

    J. Mol. Biol.

    (1963)
  • A. Markert et al.

    Virology

    (1965)
  • R.L. Sinsheimer

    J. Mol. Biol.

    (1959)
  • Cited by (144)

    • Viral replication modes in single-peak fitness landscapes: A dynamical systems analysis

      2019, Journal of Theoretical Biology
      Citation Excerpt :

      For DNA viruses, GR is the most likely mechanism of replication given their double-stranded nature, e.g., bacteriophage T2 (Luria, 1951). Exceptions maybe be single-stranded DNA viruses, such as bacteriophage ϕX174, that replicate via the SMR mode because it uses a rolling circle mechanism (Hutchison and Sinsheimer, 1966). At which point of the continuum between these two extreme modes of genome replication resides a particular virus has important evolutionary consequences.

    • Phage Lysis: Multiple Genes for Multiple Barriers

      2019, Advances in Virus Research
      Citation Excerpt :

      Gram-negative hosts also are prey to small single-strand nucleic acid phages, both ssDNA and ssRNA, including phages with grand roles in the history of molecular biology like ssDNA phage phiX174 and the ssRNA phages Qβ and MS2. All of these phages accomplish lysis by a single gene, each producing a single gene lysis protein (Sgl): E for phiX174 (Hutchison III and Sinsheimer, 1966), A2 for Qβ (Karnik and Billeter, 1983; Winter and Gold, 1983), and L for MS2 (Beremand and Blumenthal, 1979). E and L are small membrane proteins (91 and 75 aa, respectively) whereas A2 is the 45 kDa virion maturation protein that is present in one copy per virion but in Qβ, doubles as the Sgl.

    • Plackett-Burman randomization method for Bacterial Ghosts preparation form E. coli JM109

      2014, Saudi Pharmaceutical Journal
      Citation Excerpt :

      E lysis gene for BGs preparation has been well established. Simply, E lysis gene follows the Bacteriophage strategy for lysis of the cells and in fact, it is one of the Φx174 phage genes (Hutchinson and Sinsheimer, 1966; Haidinger et al., 2003). But, using such genes might have some risk factors in applications concerning the humans.

    • In vivo mutation analysis using the ΦX174 transgenic mouse and comparisons with other transgenes and endogenous genes

      2010, Mutation Research - Reviews in Mutation Research
      Citation Excerpt :

      Single-burst analysis provides additional information about each mutant isolated: an estimate of the number of mutant phage produced from the first transfected cell. A “burst” of phage occurs naturally when virus particles (phage) have multiplied inside the cell (Fig. 2) and a viral protein lyses the cell wall, releasing the burst of phage [41]. ΦX174 does not contain the GATC nucleotide sequence [4] that is necessary to initiate mismatch repair in E. coli[37] and, therefore, is not subject to mismatch repair.

    View all citing articles on Scopus

    The previous paper in this series was Dowell & Sinsheimer (1966).

    View full text