Nov 10, 1980 - JOURNAL OF VIROLOGY, Nov. 1981, p. 599-601. Vol. 40, No. 2. 0022-538X/81/1 10599-03$02.00/0. Coliphage 186 Infection Requires Host ...
JOURNAL OF VIROLOGY, Nov. 1981, p. 599-601 0022-538X/81/1 10599-03$02.00/0
Vol. 40, No. 2
Coliphage 186 Infection Requires Host Initiation Functions dnaA and dnaC IVAN HOOPERt AND J. BARRY EGAN* Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5001
Received 10 November 1980/Accepted 2 July 1981
We show that coliphage 186 infection is dependent upon host initiation functions, dnaA and dnaC, which differentiates the phage from X and P2. The possibility is therefore entertained that the delay in 186 replication seen after infection of UV-irradiated bacterial cells reflects the temporary unavailability of one or both these functions. Infections with P1 and Mu need host dnaC but not dnaA and show some sensitivity to preirradiation of the host but are not as sensitive as 186.
Bacteriophage 186 replication is delayed when the host cell is UV irradiated before infection (5). If this transient inhibition of 186 replication is not simply reflecting a dependency of 186 replication on concomitant host DNA replication (an alternative which itself is of intrinsic interest), then such behavior would appear to represent a case of UV irradiation inhibiting DNA synthesis in which the DNA has no UVinduced lesions. Its study, therefore, could afford us the opportunity to characterize aspects of UV inhibition of DNA synthesis other than the pyrimidine dimer. One working hypothesis that we are proposing is that the replication of the UV-damaged host DNA exhausts the supply of a molecule necessary for its continued replication, thereby leading to inhibition until supply of that molecule is regenerated; if another DNA molecule requiring the same host molecule for its replication is introduced into that environment, its replication will also be inhibited until the supply is restored. In this communication, we investigate which host replication functions are required for 186 infection, as one (or more) of these would be a candidate for the molecular entity referred to in the working hypothesis. We are particularly interested in those host replication functions not required by X or P2, for the latent periods of infections with these two phages are not influenced by prior UV irradiation of the host (5). Initially, therefore, we investigated the requirement of 186 for the initiation functions dnaA and dnaC. An isogenic set of strains was constructed by transduction into C600 thr leu thi lac tonA supE t Present address: Department of Agricultural Biochemistry, Waite Agricultural Research Institute, Urrbrae, South Australia.
(2). dnaAts508 (8) was cotransduced with asn+ to C600asn at an observed frequency of 23%, and dnaCts2 and dnaCts7 (4) were cotransduced with thyR (low thymine requirement) to C600thyA (high thymine requirement) at frequencies of 11 and 23%, respectively. The temperature sensitivity in DNA synthesis for the newly constructed strains was verified with pulse-labeling experiments, which showed the slow stop kinetics characteristic of DNA initiation mutants. After 186 infection of dnaAts508 and incubation at 30°C, progeny phage were released with a latent period of 60 min and burst size of 200, identical with the value obtained with the dnaA+ culture (Fig. la). At 41°C, the latent period for 186 infection of dnaA+ shortened to about 50 min and the burst size remained about 200, whereas infection of the dnaAts508 had a burst size of 1 (Fig. lb). Much the same pattern was observed with 186 infection of dnaCts2. At 30°C, a latent period of about 50 min and a burst size of about 200 were obtained for 186 infection of dnaC+ and dnaCts2 (Fig. lc). At 41°C, the burst size for 186 infection of dnaC+ was 70, whereas that of dnaCts2 was 1. Infection of another mutant, dnaCts7, gave a burst size of 3.5 (Fig. ld). Infections of the dnaA and dn,aC strains with P2 were also investigated, and we confirmed the results of Bowden et al. (3), who reported that dnaA and dnaC are not required by P2 (data not shown). Bowden et al. (3) used C strain derivatives, whereas the present work was with K-12 strain derivatives. As 186 required dnaA and dnaC for its replication whereas P2 did not, it was possible that either function could have been limiting after UV irradiation of the host. To assess the 599
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MINUTES AFTER INFECTION FIG. 1. 186 infection of dnaA and dnaC strains. Log-phase bacteria were grown with aeration at 30°C in LGC broth to 108 colony-forming units per ml. A 1-ml sample was incubated at the test temperature for 15 min, 5 x 107 186cItsp were added, and the mixture was incubated for 5 min before 10-fold dilution into 186 specific antiserum (K = 1.5 min-'). After a further 5-min incubation, during which free phage was reduced at least 50-fold, the mixture was diluted a further 100-fold (at least), and the incubation was continued. Samples were taken at various times, chloroformed, and PFU assayed on strain C600 and T agar. E907 dnaAts508 and E983 dnaCts2 were the mutant strains used. In each instance, the dna+ strains were temperatureinsensitive revertants. Symbols: 0, dna temperature-sensitive strain; 0, dna+ strain; X, E991 dnaCts7.
significance of each function in UV inhibition of DNA synthesis, it could be helpful to establish the UV capacities of phages that required dnaA but not dnaC, and vice versa. Touissant and Faelen (9) had previously reported the requirement of dnaC but not dnaA by Mu. We considered the dnaA result tentative, since in their experiments, Touissant and Faelen either infected the dnaAts strain at 30°C and transferred it ot 42°C or heat induced at 42°C a Mu lysogen of the dnaAts strain, and we had found that such regimes allowed a significant burst of 186 that could be eliminated by preincubation of the culture at the restrictive temperature before infection. When we infected dnaAts508 with Mu after a 15-min preincubation at 41°C, the burst size was 89, comparable with the burst size after infection of the dnaA+ control (Table 1), confirming the fact that Mu infection is independent of dnaA function. We also confirmed that Mu infection was dependent upon dnaC function (Table 1).
The dependency of P1 infection on host initiation functions was not known, although Abe (1) has reported that prophage P1 replication (as a plasmid) is independent of dnaA. When we investigated P1 infection, we found that it required dnaC but not dnaA (Table 1). Therefore, both Mu and P1 have a need of dnaC but not dnaA, and any lack of influence of the UV-irradiated cells on their latent periods would eliminate a role for dnaC in the UV inhibition of DNA synthesis. When investigated, the UV-irradiated host (45 J/m2) was found to extend the Mu latent period (from 58 to 80 min) but to have only a slight effect on the P1 latent period (extended from 45 to 55 min). Although these data do not indicate the nature of the inhibitory effect of UV irradiation on subsequent 186 infection, it has revealed the interesting fact that 186 requires the host initiation factors dnaA and dnaC. Whether this is a direct need, or reflects a need for concomitant host DNA replication, is a question currently
VOL. 40, 1981
NOTES
TABLE 1. Mu and P1 infection of dnaA and dnaC strains' Phage
Mu
1 Infected strain
dnaA508 dnaA+
~~~Temp (OC)
Infected strain
Tm
C
30
41
Temp ('C) 30 41
45 80
89 dnaC2 94 dnaC+
2.7 0.008 5.0 53
P1
dnaA46 706 576 dnaC2 31 0.07 dnaA+ 188 706 dnaC+ 33 682 aBacteria in LG broth (5) in log phase at 108 colonyforming units per ml at 30°C were incubated at the appropriate temperature with aeration for 15 min, phage Mucts6l (6) or Plkc (7) added at a multiplicity of 0.5, incubation was continued for either 100 min at 41°C or 180 min at 30°C, and samples were chloroformed before being assayed for PFU on strain C600 and LMC agar (L-10 mM MgCl2-2.5 mM CaCl2). The figures in the table represent burst sizes. dnaAts508 (JG173) (8) was used for Mu infection, and dnaAts46 (A3) (10) was used for P1 infection. dnaCts2 (E983; this study) was used for Mu and P1 infections. In each instance, the dna+ strains were temperature-insensitive revertants of the corresponding temperature-sensitive strains.
being investigated, as is the role this facet might play in the unique sensitivity of 186 replication to the environment of a UV-irradiated host cell.
601
We wish to acknowledge financial support from the Australian Research Grants Committee. LITERATURE CITED 1. Abe, M. 1974. The replication of prophage P1 DNA. Mol. Gen. Genet. 132:63-72. 2. Appleyard, R. K. 1954. Segregation of new lysogenic types during growth of the doubly lysogenic strain derived from Eshcerichia coli K-12. Genetics 39:440-452. 3. Bowden, D. W., R. S. Twersky, and R. Calendar. 1975. Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis. J. Bacteriol. 124:167-175. 4. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122. 5. Hooper, I., W. H. Woods, and J. B. Egan. 1981. Coliphage 186 replication is delayed when the host cell is UV irradiated before infection. J. Virol. 40:341-349. 6. Howe, J. 1973. Prophage deletion mapping of bacteriophage Mu-1. Virology 54:93-101. 7. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190206. 8. Monk, M., M. Peacey, and J. D. Gross. 1971. Repair of damage induced by ultraviolet light in DNA polymerase defective E. coli cells. J. Mol. Biol. 58:623-630. 9. Touissant, A., and M. Faelen. 1974. The dependence of temperature phage Mu-i upon replication functions of E. coli K12. Mol. Gen. Genet. 131:209-214. 10. Tresguerres, E. F., H. G. Nandadasa, and R. H. Pritchard. 1975. Suppression of initiation-negative strains of Escherichia coli by integration of the sex factor F. J. Bacteriol. 121:554-561.
