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Multiplicity reactivation of ultraviolet-inactivated T1 can be detected using suitably irradiated bacteria as host cells. Under certain conditions the amount of multiplicity reactivation is independent of the dose on the bacteria, even when the dose is reduced to zero so that normal cells are infected. The results are consistent with the idea that in normal cells some other reactivation phenomenon masks multiplicity reactivation of T1.

When Shigella dysenteriae is lysogenized by the phage P1, it becomes immune to infection with “ordinary” T1 phage (“restricted T1” or “rT1”). A host-controlled variant of T1 (“unrestricted T1” or “uT1”) can multiply in the lysogenic S. dysenteriae. Three-factor crosses were done between suitably marked strains of rT1 and uT1 in both lysogenic and nonlysogenic S. dysenteriae. When lysogenic cells are infected with both rT1 and uT1, one finds some recombinants among the progeny, but few, if any phage bearing the genotype of the restricted parent. The proportion of recombinants in the total yield is about one-eighth to one-twelfth that in control crosses (in nonlysogenic hosts) and is independent of the order of addition of either parent and of the time elapsing between the addition of each parent. We infer, then, that (1) the genome of rT1 enters the lysogenic cells; (2) it is neither replicated nor is it destroyed; and (3) it can “mate” with genomes of uT1 which may be present. The proportions of the various recombinant classes are unusual. Complementary classes are not equal; certain markers may be recovered together with a high frequency, whereas certain others, more closely linked, are seldom found together. Certain “double crossover” classes are more frequent than certain “single crossover” classes. These observations can be explained in terms of a “copy choice” mechanism of recombination by assuming that the rT1 genome contains at least two “bad spots,” the location of which can be determined approximately and which force a “switch” in replication from the restricted genome to the unrestricted genome.

Early stages of invasion by T5 st can be studied through changes in the response of phage-bacterium complexes to a calcium-deficient buffer called challenge buffer . T5 st , as free phage, and uninfected Escherichia coli F are each stable in challenge buffer. Upon adsorption, phage-bacterium complexes remain resistant to challenge buffer until a certain event, called sensitization , occurs. The plaque-forming ability of sensitized complexes rapidly decays in challenge buffer at either 37.5° or 0°; decay can be arrested by simple addition of calcium to the medium. A second postadsorptive event, called desensitization , occurs in calcium-supplemented nutrient medium, but not in calcium-supplemented buffer; desensitized complexes are resistant to challenge buffer. A tentative interpretation of some of these phenomena is proposed.

The capacity of Escherichia coli B/r to yield phages T1 and T3 is more ultraviolet (UV)-sensitive than is the capacity to yield phage T2. The capacity to yield phage from UV-irradiated T1 or T3 is about ten times as UV-sensitive as the capacity for unirradiated phage. The capacity of UV-irradiated host cells for UV-irradiated T2 is the same as for normal T2. Ultraviolet-irradiated T1 is reactivated on UV-irradiated host cells. When unirradiated T1 is grown on UV-irradiated cells, the frequency of plaquemorphology mutants is the same as the spontaneous frequency. When UV-irradiated T1 is grown on UV-irradiated cells, there is about a sevenfold increase in mutant plaque frequency over the spontaneous frequency. Thirty phenotypically distinct plaque-morphology mutants of T1 have been found.

Mutational alterations of density in phage T5 are described. The changes of density are correlated with changes in temperature sensitivity of the phage. Genetic recombination has been observed between density markers and a plaque-type marker, but not between pairs of density mutants. No phenotypic mixing of density characters has been found.

Feiner, Rose R. (Columbia University, New York, N.Y.) and Solon A. Ellison . T1 bacteriophage production in Escherichia coli . J. Bacteriol. 85: 7–11. 1963.—Progeny released from complexes formed between unirradiated Escherichia coli strain Bs-1 and ultraviolet (UV)-irradiated phage T1 were equally infective for strains B and Bs-1, and were therefore not hostmodified. The capacity of Bs-1 for unirradiated or irradiated T1 is relatively insensitive to UV irradiation, as compared with that of strains B and B/r. In all three strains of E. coli , B, B/r, and Bs-1, the UV sensitivity of the capacity for phage T1 is independent of the UV sensitivity of colony-forming ability. These findings are discussed in relation to the partial genetic homology hypothesis.

When plated directly on Escherichia coli B, a UV-irradiated T1 population reveals two fractions of different sensitivities. The fraction with lower sensitivity (about 30% of the phage-bacterial complexes) is believed to result from a reactivation by host bacteria of UV lesions in the phage genome (host-cell reactivation = HCR). Using bacteria starved prior to phage infection, we obtain HCR in 100% of the phage-bacterial complexes. Also, synchronized bacteria all perform HCR during short periods before and after cell division, not during the remaining 70% of their division cycle. When DNA synthesis is stopped by thymine withdrawal in a thymine-requiring strain of E. coli, HCR occurs in every phage-bacterial complex. These findings explain the shape of the T1 survival curve obtained with direct plating of UV-irradiated phage. In a growing bacterial culture about 70% of the T1 particles presumably infect bacteria in which synthesis of bacterial DNA prevents HCR, whereas 30% infect bacteria without DNA synthesis and can undergo HCR. In bacteria that promote HCR, multiplicity reactivation (MR) of UV-irradiated T1 can be demonstrated. The efficiency of MR is the same as in bacteria that do not perform HCR. Multiplicities above two do not increase the chance of survival of T1 multicomplexes, a fact suggesting a limited participation of T1 in MR.

SummaryAddition of sulphhydril-compounds, freeze-drying and low temperature reduces inactivation by ionizing radiation of plaque-forming ability in T1-phage by various and nearly additive amounts: if combined these changes of circumambient conditions during irradiation lead to ‘factors of protection’ of p = 16 (as compared to irradiation in highly concentrated broth). The results fit a model of radiation effects as proposed by Alexander, Howard-Flanders et al. after its extension to provide for the effects of low temperature. Existing physico-chemical evidence in support of the model is discussed briefly. Replacing some of the thymine in the DNA of T1 by 5-bromouracil leads to a sensitization of inactivation by ionizing radiation which neither depends on nor changes the efficiency of the ‘protective measures’ mentioned above with one exception: when irradiation is done in sulphhydril-containing broth sensitization by 5-bromouracil vanishes completely.

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Lysis inhibition has been demonstrated with a small-plaque mutant of T5, designated M12. This appears to be the first example of the phenomenon outside the T-even system. M12 and T5 appear identical in host range, adsorption rate, serum neutralization, and injection rate, but differ in minimal latent period in single infection (45–50 minutes for M12 as against 37 minutes for T5) and in plating efficiency under various conditions. Evidence of lysis inhibition in multiply infected cultures was obtained with M12, but not T5, by observations on the clearing of turbid cultures, one-step growth experiments, and microscopic examination. The best demonstrations in one-step growth experiments were obtained when young bacterial cultures (Escherichia coli strain F, smooth derivative of FCb) were multiply infected with M12 in a single input (multiplicity of 6–8 adequate) and further incubated undiluted for 30–40 minutes, or diluted earlier into sonicated extracts of B 5 . The latter do not affect the lysis of cultures singly or multiply infected with T5 or singly infected with M12. One-step growth experiments suggest multistep release of phage in lysisinhibited cultures. A possible explanation, actual multiplication of the infected bacteria, received no support from microscopic studies.

The metabolism of nucleic acid in Escherichia coli infected with phage T5 has been studied using antibiotics, amino acid analogs, and auxotrophic bacterial strains. Comparison with other phage infections shows that the metabolism of E. coli infected with phage T5 is similar to that of E. coli infected with phage T2, T4, or T6.